Zebrafish in the Zon laboratory allow researchers to visualize drug responses at a single-cell resolution.
Zon Lab Publications
Featured Publications
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2018. Human tumor genomics and zebrafish modeling identify loss as a driver of mucosal melanoma. Science (New York, N.Y.). 362(6418):1055-1060. Pubmed: 30385465 DOI:10.1126/science.aau6509 Ablain J, Xu M, Rothschild H, Jordan RC, Mito JK, Daniels BH, Bell CF, Joseph NM, Wu H, Bastian BC, Zon LI, Yeh I. 2018. Human tumor genomics and zebrafish modeling identify loss as a driver of mucosal melanoma. Science (New York, N.Y.). 362(6418):1055-1060. Pubmed: 30385465 DOI:10.1126/science.aau6509 Melanomas originating from mucosal surfaces have low mutation burden, genomic instability, and poor prognosis. To identify potential driver genes, we sequenced hundreds of cancer-related genes in 43 human mucosal melanomas, cataloging point mutations, amplifications, and deletions. The gene, which encodes a negative regulator of mitogen-activated protein kinase (MAPK) signaling, was inactivated in 37% of the tumors. Four distinct genotypes were associated with loss. Using a rapid, tissue-specific CRISPR technique to model these genotypes in zebrafish, we found that functions as a tumor suppressor, particularly in the context of mutations. knockdown caused MAPK activation, increased cell proliferation, and conferred resistance to drugs inhibiting KIT tyrosine kinase activity. These findings provide a rationale for MAPK inhibition in SPRED1-deficient melanomas and introduce a zebrafish modeling approach that can be used more generally to dissect genetic interactions in cancer.Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. -
Mandelbaum J, Shestopalov IA, Henderson RE, Chau NG, Knoechel B, Wick MJ, Zon LI. 2018. Zebrafish blastomere screen identifies retinoic acid suppression of in adenoid cystic carcinoma. The Journal of experimental medicine. 215(10):2673-2685. Pubmed: 30209067 DOI:10.1084/jem.20180939 Mandelbaum J, Shestopalov IA, Henderson RE, Chau NG, Knoechel B, Wick MJ, Zon LI. 2018. Zebrafish blastomere screen identifies retinoic acid suppression of in adenoid cystic carcinoma. The Journal of experimental medicine. 215(10):2673-2685. Pubmed: 30209067 DOI:10.1084/jem.20180939 Pluripotent cells have been used to probe developmental pathways that are involved in genetic diseases and oncogenic events. To find new therapies that would target -driven tumors, we developed a pluripotent zebrafish blastomere culture system. We performed a chemical genetic screen and identified retinoic acid agonists as suppressors of expression. Retinoic acid treatment also decreased gene expression in human leukemia cells. Translocations that drive overexpression of the oncogenic transcription factor are molecular hallmarks of adenoid cystic carcinoma (ACC), a malignant salivary gland tumor with no effective therapy. Retinoic acid agonists inhibited tumor growth in vivo in ACC patient-derived xenograft models and decreased MYB binding at translocated enhancers, thereby potentially diminishing the MYB positive feedback loop driving ACC. Our findings establish the zebrafish pluripotent cell culture system as a method to identify modulators of tumor formation, particularly establishing retinoic acid as a potential new effective therapy for ACC.© 2018 Mandelbaum et al. -
Lahvic JL, Ammerman M, Li P, Blair MC, Stillman ER, Fast EM, Robertson AL, Christodoulou C, Perlin JR, Yang S, Chiang N, Norris PC, Daily ML, Redfield SE, Chan IT, Chatrizeh M, Chase ME, Weis O, Zhou Y, Serhan CN, Zon LI. 2018. Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132. Proceedings of the National Academy of Sciences of the United States of America. 115(37):9252-9257. Pubmed: 30139917 DOI:10.1073/pnas.1806077115 Lahvic JL, Ammerman M, Li P, Blair MC, Stillman ER, Fast EM, Robertson AL, Christodoulou C, Perlin JR, Yang S, Chiang N, Norris PC, Daily ML, Redfield SE, Chan IT, Chatrizeh M, Chase ME, Weis O, Zhou Y, Serhan CN, Zon LI. 2018. Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132. Proceedings of the National Academy of Sciences of the United States of America. 115(37):9252-9257. Pubmed: 30139917 DOI:10.1073/pnas.1806077115 Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based β-arrestin recruitment assay. Knockdown of zebrafish prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure-activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis. -
Kapp FG, Perlin JR, Hagedorn EJ, Gansner JM, Schwarz DE, O'Connell LA, Johnson NS, Amemiya C, Fisher DE, Wölfle U, Trompouki E, Niemeyer CM, Driever W, Zon LI. 2018. Protection from UV light is an evolutionarily conserved feature of the haematopoietic niche. Nature. 558(7710):445-448. Pubmed: 29899448 DOI:10.1038/s41586-018-0213-0 Kapp FG, Perlin JR, Hagedorn EJ, Gansner JM, Schwarz DE, O'Connell LA, Johnson NS, Amemiya C, Fisher DE, Wölfle U, Trompouki E, Niemeyer CM, Driever W, Zon LI. 2018. Protection from UV light is an evolutionarily conserved feature of the haematopoietic niche. Nature. 558(7710):445-448. Pubmed: 29899448 DOI:10.1038/s41586-018-0213-0 Haematopoietic stem and progenitor cells (HSPCs) require a specific microenvironment, the haematopoietic niche, which regulates HSPC behaviour. The location of this niche varies across species, but the evolutionary pressures that drive HSPCs to different microenvironments remain unknown. The niche is located in the bone marrow in adult mammals, whereas it is found in other locations in non-mammalian vertebrates, for example, in the kidney marrow in teleost fish. Here we show that a melanocyte umbrella above the kidney marrow protects HSPCs against ultraviolet light in zebrafish. Because mutants that lack melanocytes have normal steady-state haematopoiesis under standard laboratory conditions, we hypothesized that melanocytes above the stem cell niche protect HSPCs against ultraviolet-light-induced DNA damage. Indeed, after ultraviolet-light irradiation, unpigmented larvae show higher levels of DNA damage in HSPCs, as indicated by staining of cyclobutane pyrimidine dimers and have reduced numbers of HSPCs, as shown by cmyb (also known as myb) expression. The umbrella of melanocytes associated with the haematopoietic niche is highly evolutionarily conserved in aquatic animals, including the sea lamprey, a basal vertebrate. During the transition from an aquatic to a terrestrial environment, HSPCs relocated into the bone marrow, which is protected from ultraviolet light by the cortical bone around the marrow. Our studies reveal that melanocytes above the haematopoietic niche protect HSPCs from ultraviolet-light-induced DNA damage in aquatic vertebrates and suggest that during the transition to terrestrial life, ultraviolet light was an evolutionary pressure affecting the location of the haematopoietic niche. -
Blaser BW, Moore JL, Hagedorn EJ, Li B, Riquelme R, Lichtig A, Yang S, Zhou Y, Tamplin OJ, Binder V, Zon LI. 2017. CXCR1 remodels the vascular niche to promote hematopoietic stem and progenitor cell engraftment. The Journal of experimental medicine. 214(4):1011-1027. Pubmed: 28351983 DOI:10.1084/jem.20161616 Blaser BW, Moore JL, Hagedorn EJ, Li B, Riquelme R, Lichtig A, Yang S, Zhou Y, Tamplin OJ, Binder V, Zon LI. 2017. CXCR1 remodels the vascular niche to promote hematopoietic stem and progenitor cell engraftment. The Journal of experimental medicine. 214(4):1011-1027. Pubmed: 28351983 DOI:10.1084/jem.20161616 The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSPC) biology. Recent advances marking fluorescent HSPCs have allowed exquisite visualization of HSPCs in the caudal hematopoietic tissue (CHT) of the developing zebrafish. Here, we show that the chemokine and its receptor, , are expressed by zebrafish endothelial cells, and we identify signaling as a positive regulator of HSPC colonization. Single-cell tracking experiments demonstrated that this is a result of increases in HSPC-endothelial cell "cuddling," HSPC residency time within the CHT, and HSPC mitotic rate. Enhanced signaling was associated with an increase in the volume of the CHT and induction of expression. Finally, using parabiotic zebrafish, we show that acts HSPC nonautonomously to improve the efficiency of donor HSPC engraftment. This work identifies a mechanism by which the hematopoietic niche remodels to promote HSPC engraftment and suggests that signaling is a potential therapeutic target in patients undergoing hematopoietic stem cell transplantation.© 2017 Blaser et al. -
Tan JL, Fogley RD, Flynn RA, Ablain J, Yang S, Saint-André V, Fan ZP, Do BT, Laga AC, Fujinaga K, Santoriello C, Greer CB, Kim YJ, Clohessy JG, Bothmer A, Pandell N, Avagyan S, Brogie JE, van Rooijen E, Hagedorn EJ, Shyh-Chang N, White RM, Price DH, Pandolfi PP, Peterlin BM, Zhou Y, Kim TH, Asara JM, Chang HY, Young RA, Zon LI. 2016. Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma. Molecular cell. 62(1):34-46. Pubmed: 27058786 DOI:S1097-2765(16)00223-9 Tan JL, Fogley RD, Flynn RA, Ablain J, Yang S, Saint-André V, Fan ZP, Do BT, Laga AC, Fujinaga K, Santoriello C, Greer CB, Kim YJ, Clohessy JG, Bothmer A, Pandell N, Avagyan S, Brogie JE, van Rooijen E, Hagedorn EJ, Shyh-Chang N, White RM, Price DH, Pandolfi PP, Peterlin BM, Zhou Y, Kim TH, Asara JM, Chang HY, Young RA, Zon LI. 2016. Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma. Molecular cell. 62(1):34-46. Pubmed: 27058786 DOI:S1097-2765(16)00223-9 Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.Copyright © 2016 Elsevier Inc. All rights reserved. -
Kaufman CK, Mosimann C, Fan ZP, Yang S, Thomas AJ, Ablain J, Tan JL, Fogley RD, van Rooijen E, Hagedorn EJ, Ciarlo C, White RM, Matos DA, Puller AC, Santoriello C, Liao EC, Young RA, Zon LI. 2016. A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science (New York, N.Y.). 351(6272):aad2197. Pubmed: 26823433 DOI:10.1126/science.aad2197 Kaufman CK, Mosimann C, Fan ZP, Yang S, Thomas AJ, Ablain J, Tan JL, Fogley RD, van Rooijen E, Hagedorn EJ, Ciarlo C, White RM, Matos DA, Puller AC, Santoriello C, Liao EC, Young RA, Zon LI. 2016. A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science (New York, N.Y.). 351(6272):aad2197. Pubmed: 26823433 DOI:10.1126/science.aad2197 The "cancerized field" concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAF(V600E) mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAF(V600E)-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.Copyright © 2016, American Association for the Advancement of Science. -
Mosimann C, Panáková D, Werdich AA, Musso G, Burger A, Lawson KL, Carr LA, Nevis KR, Sabeh MK, Zhou Y, Davidson AJ, DiBiase A, Burns CE, Burns CG, MacRae CA, Zon LI. 2015. Chamber identity programs drive early functional partitioning of the heart. Nature communications. 6:8146. Pubmed: 26306682 DOI:10.1038/ncomms9146 Mosimann C, Panáková D, Werdich AA, Musso G, Burger A, Lawson KL, Carr LA, Nevis KR, Sabeh MK, Zhou Y, Davidson AJ, DiBiase A, Burns CE, Burns CG, MacRae CA, Zon LI. 2015. Chamber identity programs drive early functional partitioning of the heart. Nature communications. 6:8146. Pubmed: 26306682 DOI:10.1038/ncomms9146 The vertebrate heart muscle (myocardium) develops from the first heart field (FHF) and expands by adding second heart field (SHF) cells. While both lineages exist already in teleosts, the primordial contributions of FHF and SHF to heart structure and function remain incompletely understood. Here we delineate the functional contribution of the FHF and SHF to the zebrafish heart using the cis-regulatory elements of the draculin (drl) gene. The drl reporters initially delineate the lateral plate mesoderm, including heart progenitors. Subsequent myocardial drl reporter expression restricts to FHF descendants. We harnessed this unique feature to uncover that loss of tbx5a and pitx2 affect relative FHF versus SHF contributions to the heart. High-resolution physiology reveals distinctive electrical properties of each heart field territory that define a functional boundary within the single zebrafish ventricle. Our data establish that the transcriptional program driving cardiac septation regulates physiologic ventricle partitioning, which successively provides mechanical advantages of sequential contraction.
All Publications
2025
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Huisman BD, Michelson DA, Rubin SA, Kohlsaat K, Gomarga W, Fang Y, Lee JM, Del Nido P, Nathan M, Benoist C, Zon L, Mathis D. 2025. Cross-species analyses of thymic mimetic cells reveal evolutionarily ancient origins and both conserved and species-specific elements. Immunity. 58(1):108-123.e7. Pubmed: 39731911 DOI:S1074-7613(24)00540-5 Huisman BD, Michelson DA, Rubin SA, Kohlsaat K, Gomarga W, Fang Y, Lee JM, Del Nido P, Nathan M, Benoist C, Zon L, Mathis D. 2025. Cross-species analyses of thymic mimetic cells reveal evolutionarily ancient origins and both conserved and species-specific elements. Immunity. 58(1):108-123.e7. Pubmed: 39731911 DOI:S1074-7613(24)00540-5 Thymic mimetic cells are molecular hybrids between medullary-thymic-epithelial cells (mTECs) and diverse peripheral cell types. They are involved in eliminating autoreactive T cells and can perform supplementary functions reflective of their peripheral-cell counterparts. Current knowledge about mimetic cells derives largely from mouse models. To provide the high resolution that proved revelatory for mice, we performed single-cell RNA sequencing on purified mimetic-cell compartments from human pediatric donors. The single-cell profiles of individual donors were surprisingly similar, with diversification of neuroendocrine subtypes and expansion of the muscle subtype relative to mice. Informatic and imaging studies on the muscle-mTEC population highlighted a maturation trajectory suggestive of skeletal-muscle differentiation, some striated structures, and occasional cellular groupings reminiscent of neuromuscular junctions. We also profiled thymic mimetic cells from zebrafish. Integration of data from the three species identified species-specific adaptations but substantial interspecies conservation, highlighting the evolutionarily ancient nature of mimetic mTECs. Our findings provide a landscape view of human mimetic cells, with anticipated relevance in autoimmunity.Copyright © 2024 Elsevier Inc. All rights reserved. -
Jing R, Falchetti M, Han T, Najia M, Hensch LT, Meader E, Lummertz da Rocha E, Kononov M, Wang S, Bingham T, Li Z, Zhao Y, Frenis K, Kubaczka C, Yang S, Jha D, Rodrigues-Luiz GF, Rowe RG, Schlaeger TM, Maus MV, North TE, Zon LI, Daley GQ. 2025. Maturation and persistence of CAR T cells derived from human pluripotent stem cells via chemical inhibition of G9a/GLP. Cell stem cell. 32(1):71-85.e5. Pubmed: 39504968 DOI:S1934-5909(24)00366-7 Jing R, Falchetti M, Han T, Najia M, Hensch LT, Meader E, Lummertz da Rocha E, Kononov M, Wang S, Bingham T, Li Z, Zhao Y, Frenis K, Kubaczka C, Yang S, Jha D, Rodrigues-Luiz GF, Rowe RG, Schlaeger TM, Maus MV, North TE, Zon LI, Daley GQ. 2025. Maturation and persistence of CAR T cells derived from human pluripotent stem cells via chemical inhibition of G9a/GLP. Cell stem cell. 32(1):71-85.e5. Pubmed: 39504968 DOI:S1934-5909(24)00366-7 Elucidating mechanisms of T cell development can guide in vitro T cell differentiation from induced pluripotent stem cells (iPSCs) and facilitate off-the-shelf T cell-based immunotherapies. Using a stroma-free human iPSC-T cell differentiation platform, we screened for epigenetic modulators that influence T cell specification and identified the H3K9-directed histone methyltransferases G9a/GLP as repressors of T cell fate. We show that G9a/GLP inhibition during specific time windows of differentiation of hematopoietic stem and progenitor cells (HSPCs) skews cell fates toward lymphoid lineages. Inhibition of G9a/GLP promotes the production of lymphoid cells during zebrafish embryonic hematopoiesis, demonstrating the evolutionary conservation of G9a/GLP function. Importantly, chemical inhibition of G9a/GLP facilitates the generation of mature iPSC-T cells that bear transcriptional similarity to peripheral blood αβ T cells. When engineered to express chimeric antigen receptors, the epigenetically engineered iPSC-T cells exhibit enhanced effector functions in vitro and durable, persistent antitumor activity in a xenograft tumor-rechallenge model.Copyright © 2024 Elsevier Inc. All rights reserved. -
Jing R, Falchetti M, Han T, Najia M, Hensch LT, Meader E, Lummertz da Rocha E, Kononov M, Wang S, Bingham T, Li Z, Zhao Y, Frenis K, Kubaczka C, Yang S, Jha D, Rodrigues-Luiz GF, Rowe RG, Schlaeger TM, Maus MV, North TE, Zon LI, Daley GQ. 2025. Maturation and persistence of CAR T cells derived from human pluripotent stem cells via chemical inhibition of G9a/GLP. Cell stem cell. Pubmed: 39818202 DOI:10.1016/j.stem.2024.12.008 Jing R, Falchetti M, Han T, Najia M, Hensch LT, Meader E, Lummertz da Rocha E, Kononov M, Wang S, Bingham T, Li Z, Zhao Y, Frenis K, Kubaczka C, Yang S, Jha D, Rodrigues-Luiz GF, Rowe RG, Schlaeger TM, Maus MV, North TE, Zon LI, Daley GQ. 2025. Maturation and persistence of CAR T cells derived from human pluripotent stem cells via chemical inhibition of G9a/GLP. Cell stem cell. Pubmed: 39818202 DOI:10.1016/j.stem.2024.12.008 2024
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Roider E, Lakatos AIT, McConnell AM, Wang P, Mueller A, Kawakami A, Tsoi J, Szabolcs BL, Ascsillán AA, Suita Y, Igras V, Lo JA, Hsiao JJ, Lapides R, Pál DMP, Lengyel AS, Navarini A, Okazaki A, Iliopoulos O, Németh I, Graeber TG, Zon L, Giese RW, Kemeny LV, Fisher DE. 2024. MITF regulates IDH1, NNT, and a transcriptional program protecting melanoma from reactive oxygen species. Scientific reports. 14(1):21527. Pubmed: 39277608 DOI:10.1038/s41598-024-72031-9 Roider E, Lakatos AIT, McConnell AM, Wang P, Mueller A, Kawakami A, Tsoi J, Szabolcs BL, Ascsillán AA, Suita Y, Igras V, Lo JA, Hsiao JJ, Lapides R, Pál DMP, Lengyel AS, Navarini A, Okazaki A, Iliopoulos O, Németh I, Graeber TG, Zon L, Giese RW, Kemeny LV, Fisher DE. 2024. MITF regulates IDH1, NNT, and a transcriptional program protecting melanoma from reactive oxygen species. Scientific reports. 14(1):21527. Pubmed: 39277608 DOI:10.1038/s41598-024-72031-9 Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte function, development and plays a significant role in melanoma pathogenesis. MITF genomic amplification promotes melanoma development, and it can facilitate resistance to multiple therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo. Some of the MITF target genes involved, such as IDH1 and NNT, are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state.© 2024. The Author(s). -
Niu X, Melendez DL, Raj S, Cai J, Senadeera D, Mandelbaum J, Shestopalov IA, Martin SD, Zon LI, Schlaeger TM, Lai LP, McMahon AP, Craft AM, Galloway JL. 2024. A conserved transcription factor regulatory program promotes tendon fate. Developmental cell. 59(23):3106-3123.e12. Pubmed: 39216481 DOI:S1534-5807(24)00489-1 Niu X, Melendez DL, Raj S, Cai J, Senadeera D, Mandelbaum J, Shestopalov IA, Martin SD, Zon LI, Schlaeger TM, Lai LP, McMahon AP, Craft AM, Galloway JL. 2024. A conserved transcription factor regulatory program promotes tendon fate. Developmental cell. 59(23):3106-3123.e12. Pubmed: 39216481 DOI:S1534-5807(24)00489-1 Tendons, which transmit force from muscles to bones, are highly prone to injury. Understanding the mechanisms driving tendon fate would impact efforts to improve tendon healing, yet this knowledge is limited. To find direct regulators of tendon progenitor emergence, we performed a zebrafish high-throughput chemical screen. We established forskolin as a tenogenic inducer across vertebrates, functioning through Creb1a, which is required and sufficient for tendon fate. Putative enhancers containing cyclic AMP (cAMP) response elements (CREs) in humans, mice, and fish drove specific expression in zebrafish cranial and fin tendons. Analysis of these genomic regions identified motifs for early B cell factor (Ebf/EBF) transcription factors. Mutation of CRE or Ebf/EBF motifs significantly disrupted enhancer activity and specificity in tendons. Zebrafish ebf1a/ebf3a mutants displayed defects in tendon formation. Notably, Creb1a/CREB1 and Ebf1a/Ebf3a/EBF1 overexpression facilitated tenogenic induction in zebrafish and human pluripotent stem cells. Together, our work identifies the functional conservation of two transcription factors in promoting tendon fate.Copyright © 2024 Elsevier Inc. All rights reserved. -
Gunage R, Zon LI. 2024. Role of RNA modifications in blood development and regeneration. Experimental hematology. 138:104279. Pubmed: 39009277 DOI:S0301-472X(24)00138-3 Gunage R, Zon LI. 2024. Role of RNA modifications in blood development and regeneration. Experimental hematology. 138:104279. Pubmed: 39009277 DOI:S0301-472X(24)00138-3 Blood development and regeneration require rapid turnover of cells, and ribonucleic acid (RNA) modifications play a key role in it via regulating stemness and cell fate regulation. RNA modifications affect gene activity via posttranscriptional and translation-mediated mechanisms. Diverse molecular players involved in RNA-modification processes are abundantly expressed by hematopoietic stem cells and lineages. Close to 150 RNA chemical modifications have been reported, but only N6-methyl adenosine (mA), inosine (I), pseudouridine (Ψ), and m1A-a handful-have been studied in-cell fate regulation. The role of RNA modification in blood diseases and disorders is an emerging field and offers potential for therapeutic interventions. Knowledge of RNA-modification and enzymatic activities could be used to design therapies in the future. Here, we summarized the recent advances in RNA modification and the epitranscriptome field and discussed their regulation of blood development and regeneration.Published by Elsevier Inc. -
Noonan HR, Thornock AM, Barbano J, Xifaras ME, Baron CS, Yang S, Koczirka K, McConnell AM, Zon LI. 2024. A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma. eLife. 13. Pubmed: 38874379 DOI:10.7554/eLife.83527 Noonan HR, Thornock AM, Barbano J, Xifaras ME, Baron CS, Yang S, Koczirka K, McConnell AM, Zon LI. 2024. A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma. eLife. 13. Pubmed: 38874379 DOI:10.7554/eLife.83527 Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this TGFb Induced Enhancer driving green fluorescent protein (). was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of advanced melanomas. Single-cell RNA-sequencing revealed that melanoma cells down-regulated interferon response while up-regulating a novel set of chronic TGFb target genes. ChIP-sequencing demonstrated that AP-1 factor binding is required for activation of chronic TGFb response. Overexpression of , a chromatin remodeler associated with tumor spreading, showed activation of TGFb signaling in early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages localize to regions and preferentially phagocytose melanoma cells compared to melanoma cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors.© 2024, Noonan, Thornock et al. -
Feng H, Stachura DL, White RM, Gutierrez A, Zhang L, Sanda T, Jette CA, Testa JR, Neuberg DS, Langenau DM, Kutok JL, Zon LI, Traver D, Fleming MD, Kanki JP, Look AT. 2024. T-Lymphoblastic Lymphoma Cells Express High Levels of BCL2, S1P1, and ICAM1, Leading to a Blockade of Tumor Cell Intravasation. Cancer cell. 42(6):1130-1131. Pubmed: 38861925 DOI:S1535-6108(24)00187-9 Feng H, Stachura DL, White RM, Gutierrez A, Zhang L, Sanda T, Jette CA, Testa JR, Neuberg DS, Langenau DM, Kutok JL, Zon LI, Traver D, Fleming MD, Kanki JP, Look AT. 2024. T-Lymphoblastic Lymphoma Cells Express High Levels of BCL2, S1P1, and ICAM1, Leading to a Blockade of Tumor Cell Intravasation. Cancer cell. 42(6):1130-1131. Pubmed: 38861925 DOI:S1535-6108(24)00187-9 -
Robertson AL, Yue L, Choudhuri A, Kubaczka C, Wattrus SJ, Mandelbaum J, Avagyan S, Yang S, Freeman RJ, Chan V, Blair MC, Daley GQ, Zon LI. 2024. Hematopoietic stem cell division is governed by distinct RUNX1 binding partners. bioRxiv : the preprint server for biology. Pubmed: 38895208 DOI:10.1101/2024.06.07.596542 Robertson AL, Yue L, Choudhuri A, Kubaczka C, Wattrus SJ, Mandelbaum J, Avagyan S, Yang S, Freeman RJ, Chan V, Blair MC, Daley GQ, Zon LI. 2024. Hematopoietic stem cell division is governed by distinct RUNX1 binding partners. bioRxiv : the preprint server for biology. Pubmed: 38895208 DOI:10.1101/2024.06.07.596542 A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases. -
Liu W, Ding Y, Shen Z, Xu C, Yi W, Wang D, Zhou Y, Zon LI, Liu JX. 2024. BF170 hydrochloride enhances the emergence of hematopoietic stem and progenitor cells. Development (Cambridge, England). 151(13). Pubmed: 38940293 DOI:10.1242/dev.202476 Liu W, Ding Y, Shen Z, Xu C, Yi W, Wang D, Zhou Y, Zon LI, Liu JX. 2024. BF170 hydrochloride enhances the emergence of hematopoietic stem and progenitor cells. Development (Cambridge, England). 151(13). Pubmed: 38940293 DOI:10.1242/dev.202476 Generation of hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo, especially the generation of safe therapeutic HSPCs, still remains inefficient. In this study, we have identified compound BF170 hydrochloride as a previously unreported pro-hematopoiesis molecule, using the differentiation assays of primary zebrafish blastomere cell culture and mouse embryoid bodies (EBs), and we demonstrate that BF170 hydrochloride promoted definitive hematopoiesis in vivo. During zebrafish definitive hematopoiesis, BF170 hydrochloride increases blood flow, expands hemogenic endothelium (HE) cells and promotes HSPC emergence. Mechanistically, the primary cilia-Ca2+-Notch/NO signaling pathway, which is downstream of the blood flow, mediated the effects of BF170 hydrochloride on HSPC induction in vivo. Our findings, for the first time, reveal that BF170 hydrochloride is a compound that enhances HSPC induction and may be applied to the ex vivo expansion of HSPCs.© 2024. Published by The Company of Biologists Ltd. -
Babu S, Chen J, Robitschek E, Baron CS, McConnell A, Wu C, Dedeilia A, Sade-Feldman M, Modhurima R, Manos MP, Chen KY, Cox AM, Ludwig CG, Yang J, Kellis M, Buchbinder EI, Hacohen N, Boland GM, Abraham BJ, Liu D, Zon LI, Insco ML. 2024. Specific oncogene activation of the cell of origin in mucosal melanoma. bioRxiv : the preprint server for biology. Pubmed: 38712250 DOI:10.1101/2024.04.22.590595 Babu S, Chen J, Robitschek E, Baron CS, McConnell A, Wu C, Dedeilia A, Sade-Feldman M, Modhurima R, Manos MP, Chen KY, Cox AM, Ludwig CG, Yang J, Kellis M, Buchbinder EI, Hacohen N, Boland GM, Abraham BJ, Liu D, Zon LI, Insco ML. 2024. Specific oncogene activation of the cell of origin in mucosal melanoma. bioRxiv : the preprint server for biology. Pubmed: 38712250 DOI:10.1101/2024.04.22.590595 Mucosal melanoma (MM) is a deadly cancer derived from mucosal melanocytes. To test the consequences of MM genetics, we developed a zebrafish model in which all melanocytes experienced CCND1 expression and loss of PTEN and TP53. Surprisingly, melanoma only developed from melanocytes lining internal organs, analogous to the location of patient MM. We found that zebrafish MMs had a unique chromatin landscape from cutaneous melanoma. Internal melanocytes could be labeled using a MM-specific transcriptional enhancer. Normal zebrafish internal melanocytes shared a gene expression signature with MMs. Patient and zebrafish MMs have increased migratory neural crest gene and decreased antigen presentation gene expression, consistent with the increased metastatic behavior and decreased immunotherapy sensitivity of MM. Our work suggests the cell state of the originating melanocyte influences the behavior of derived melanomas. Our animal model phenotypically and transcriptionally mimics patient tumors, allowing this model to be used for MM therapeutic discovery. -
Baron CS, Mitchell O, Avagyan S, Menard R, Yang S, Robertson AL, Potluri R, Shendure J, Madelaine R, McKenna A, Zon LI. 2024. Leukemia-derived apelin selects endothelial niche clones to promote tumorigenesis. bioRxiv : the preprint server for biology. Pubmed: 39314385 DOI:10.1101/2024.09.09.612077 Baron CS, Mitchell O, Avagyan S, Menard R, Yang S, Robertson AL, Potluri R, Shendure J, Madelaine R, McKenna A, Zon LI. 2024. Leukemia-derived apelin selects endothelial niche clones to promote tumorigenesis. bioRxiv : the preprint server for biology. Pubmed: 39314385 DOI:10.1101/2024.09.09.612077 Hematopoietic stem cells are regulated by endothelial and mesenchymal stromal cells in the marrow niche1-3. Leukemogenesis was long believed to be solely driven by genetic perturbations in hematopoietic cells but introduction of genetic mutations in the microenvironment demonstrated the ability of niche cells to drive disease progression4-8. The mechanisms by which the stem cell niche induces leukemia remain poorly understood. Here, using cellular barcoding in zebrafish, we found that clones of niche endothelial and stromal cells are significantly expanded in leukemic marrows. The pro-angiogenic peptide apelin secreted by leukemic cells induced sinusoidal endothelial cell clonal selection and transcriptional reprogramming towards an angiogenic state to promote leukemogenesis in vivo. Overexpression of apelin in normal hematopoietic stem cells led to clonal amplification of the niche endothelial cells and promotes clonal dominance of blood cells. Knock-out of apelin in leukemic zebrafish resulted in a significant reduction in disease progression. Our results demonstrate that leukemic cells remodel the clonal and transcriptional landscape of the marrow niche to promote leukemogenesis and provide a potential therapeutic opportunity for anti-apelin treatment. -
Langenau DM, Keefe MD, Storer NY, Guyon JR, Kutok JL, Le X, Goessling W, Neuberg DS, Kunkel LM, Zon LI. 2024. Corrigendium: Effects of RAS on the genesis of embryonal rhabdomyosarcoma. Genes & development. 38(5-6):289. Pubmed: 38631822 DOI:10.1101/gad.351747.124 Langenau DM, Keefe MD, Storer NY, Guyon JR, Kutok JL, Le X, Goessling W, Neuberg DS, Kunkel LM, Zon LI. 2024. Corrigendium: Effects of RAS on the genesis of embryonal rhabdomyosarcoma. Genes & development. 38(5-6):289. Pubmed: 38631822 DOI:10.1101/gad.351747.124 -
Bornhorst D, Gheller B, Zon LI. 2024. Colorimetric Barcoding to Track, Isolate, and Analyze Hematopoietic Stem Cell Clones. Methods in molecular biology (Clifton, N.J.). 2707:265-277. Pubmed: 37668919 DOI:10.1007/978-1-0716-3401-1_18 Bornhorst D, Gheller B, Zon LI. 2024. Colorimetric Barcoding to Track, Isolate, and Analyze Hematopoietic Stem Cell Clones. Methods in molecular biology (Clifton, N.J.). 2707:265-277. Pubmed: 37668919 DOI:10.1007/978-1-0716-3401-1_18 In zebrafish, hematopoietic stem cells (HSCs) are born in the developing aorta during embryogenesis. From the definitive wave of hematopoiesis onward, blood homeostasis relies on self-renewal and differentiation of progeny of existing HSCs, or clones, rather than de novo generation. Here, we describe an approach to quantify the number and size of HSC clones at various times throughout the lifespan of the animal using a fluorescent, multicolor labeling strategy. The system is based on combining the multicolor Zebrabow system with an inducible, early lateral plate mesoderm and hematopoietic lineage specific cre driver (draculin (drl)). The cre driver can be temporally controlled and activated in early hematopoiesis to introduce a color barcoding unique to each HSC and subsequently inherited by their daughter cells. Clonal diversity and dominance can be investigated in normal development and blood disease progression, such as blood cancers. This adoptable method allows researchers to obtain quantitative insight into clonality-defining events and their contribution to adult hematopoiesis.© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature. -
Ludin A, Stirtz GL, Tal A, Nirmal AJ, Besson N, Jones SM, Pfaff KL, Manos M, Liu S, Barrera I, Gong Q, Rodrigues CP, Sahu A, Jerison E, Alessi JV, Ricciuti B, Richardson DS, Weiss JD, Moreau HM, Stanhope ME, Afeyan AB, Sefton J, McCall WD, Formato E, Yang S, Zhou Y, van Konijnenburg DPH, Cole HL, Cordova M, Deng L, Rajadhyaksha M, Quake SR, Awad MM, Chen F, Sorger PK, Hodi FS, Rodig SJ, Murphy GF, Zon LI. 2024. Craters on the melanoma surface facilitate tumor-immune interactions and demonstrate pathologic response to checkpoint blockade in humans. bioRxiv : the preprint server for biology. Pubmed: 39345527 DOI:10.1101/2024.09.18.613595 Ludin A, Stirtz GL, Tal A, Nirmal AJ, Besson N, Jones SM, Pfaff KL, Manos M, Liu S, Barrera I, Gong Q, Rodrigues CP, Sahu A, Jerison E, Alessi JV, Ricciuti B, Richardson DS, Weiss JD, Moreau HM, Stanhope ME, Afeyan AB, Sefton J, McCall WD, Formato E, Yang S, Zhou Y, van Konijnenburg DPH, Cole HL, Cordova M, Deng L, Rajadhyaksha M, Quake SR, Awad MM, Chen F, Sorger PK, Hodi FS, Rodig SJ, Murphy GF, Zon LI. 2024. Craters on the melanoma surface facilitate tumor-immune interactions and demonstrate pathologic response to checkpoint blockade in humans. bioRxiv : the preprint server for biology. Pubmed: 39345527 DOI:10.1101/2024.09.18.613595 Immunotherapy leads to cancer eradication despite the tumor's immunosuppressive environment. Here, we used extended long-term in-vivo imaging and high-resolution spatial transcriptomics of endogenous melanoma in zebrafish, and multiplex imaging of human melanoma, to identify domains that facilitate immune response during immunotherapy. We identified crater-shaped pockets at the margins of zebrafish and human melanoma, rich with beta-2 microglobulin (B2M) and antigen recognition molecules. The craters harbor the highest density of CD8 T cells in the tumor. In zebrafish, CD8 T cells formed prolonged interactions with melanoma cells within craters, characteristic of antigen recognition. Following immunostimulatory treatment, the craters enlarged and became the major site of activated CD8 T cell accumulation and tumor killing that was B2M dependent. In humans, craters predicted immune response to ICB therapy, showing response better than high T cell infiltration. This marks craters as potential new diagnostic tool for immunotherapy success and targets to enhance ICB response. -
McConnell AM, Chassé MH, Noonan HR, Mito JK, Barbano J, Weiskopf E, Gosselink IF, Prasad M, Yang S, Abarzua P, Lian CG, Murphy GF, Trapnell C, Zon LI. 2024. An attractor state zone precedes neural crest fate in melanoma initiation. bioRxiv : the preprint server for biology. Pubmed: 39484503 DOI:10.1101/2024.10.22.618007 McConnell AM, Chassé MH, Noonan HR, Mito JK, Barbano J, Weiskopf E, Gosselink IF, Prasad M, Yang S, Abarzua P, Lian CG, Murphy GF, Trapnell C, Zon LI. 2024. An attractor state zone precedes neural crest fate in melanoma initiation. bioRxiv : the preprint server for biology. Pubmed: 39484503 DOI:10.1101/2024.10.22.618007 The field cancerization theory suggests that a group of cells containing oncogenic mutations are predisposed to transformation. We previously identified single cells in zebrafish that reactivate an embryonic neural crest state before initiating melanoma. Here we show that single cells reactivate the neural crest fate from within large fields of adjacent abnormal melanocytes, which we term the "cancer precursor zone." These cancer precursor zone melanocytes have an aberrant morphology, dysplastic nuclei, and altered gene expression. Using single cell RNA-seq and ATAC-seq, we defined a distinct transcriptional cell attractor state for cancer precursor zones and validated the stage-specific gene expression initiation signatures in human melanoma. We identify the cancer precursor zone driver, ID1, which binds to TCF12 and inhibits downstream targets important for the maintenance of melanocyte morphology and cell cycle control. Examination of patient samples revealed precursor melanocytes expressing ID1, often surrounding invasive melanoma, indicating a role for ID1 in early melanomagenesis. This work reveals a surprising field effect of melanoma initiation in which tumors arise from within a zone of morphologically distinct, but clinically covert, precursors with altered transcriptional fate. Our studies identify novel targets that could improve early diagnosis and prevention of melanoma. -
Kim JW, Fedorov EA, Zon LI. 2024. G-CSF-induced hematopoietic stem cell mobilization from the embryonic hematopoietic niche does not require neutrophils and macrophages. Experimental hematology. 131:104147. Pubmed: 38160994 DOI:S0301-472X(23)01806-4 Kim JW, Fedorov EA, Zon LI. 2024. G-CSF-induced hematopoietic stem cell mobilization from the embryonic hematopoietic niche does not require neutrophils and macrophages. Experimental hematology. 131:104147. Pubmed: 38160994 DOI:S0301-472X(23)01806-4 Hematopoietic stem cell transplantation requires the collection of hematopoietic cells from patients or stem cell donors. Granulocyte colony-stimulating factor (G-CSF) is widely used in the clinic to mobilize hematopoietic stem and progenitor cells (HSPCs) from the adult bone marrow niche into circulation, allowing a collection of HSPCs from the blood. The mechanism by which G-CSF acts to mobilize HSPCs is unclear, with some studies showing a direct stimulation of stem cells and others suggesting that myeloid cells are required. In this study, we developed a heat-inducible G-CSF transgenic zebrafish line to study HSPC mobilization in vivo. Live imaging of HSPCs after G-CSF induction revealed an increase in circulating HSPCs, demonstrating a successful HSPC mobilization. These mobilized HSPCs went on to prematurely colonize the kidney marrow, the adult zebrafish hematopoietic niche. We eliminated neutrophils or macrophages using a nitroreductase-based cell ablation system and found that G-CSF still mobilizes HSPCs from the niche. Our findings indicate that neutrophils and macrophages are not required for G-CSF-induced HSPC mobilization from the embryonic hematopoietic niche.Copyright © 2024 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved. -
Pessoa Rodrigues C, Collins JM, Yang S, Martinez C, Kim JW, Lama C, Nam AS, Alt C, Lin C, Zon LI. 2024. Transcripts of repetitive DNA elements signal to block phagocytosis of hematopoietic stem cells. Science (New York, N.Y.). 385(6714):eadn1629. Pubmed: 39264994 DOI:10.1126/science.adn1629 Pessoa Rodrigues C, Collins JM, Yang S, Martinez C, Kim JW, Lama C, Nam AS, Alt C, Lin C, Zon LI. 2024. Transcripts of repetitive DNA elements signal to block phagocytosis of hematopoietic stem cells. Science (New York, N.Y.). 385(6714):eadn1629. Pubmed: 39264994 DOI:10.1126/science.adn1629 Macrophages maintain hematopoietic stem cell (HSC) quality by assessing cell surface Calreticulin (Calr), an "eat-me" signal induced by reactive oxygen species (ROS). Using zebrafish genetics, we identified Beta-2-microglobulin (B2m) as a crucial "don't eat-me" signal on blood stem cells. A chemical screen revealed inducers of surface Calr that promoted HSC proliferation without triggering ROS or macrophage clearance. Whole-genome CRISPR-Cas9 screening showed that Toll-like receptor 3 (Tlr3) signaling regulated expression. Targeting or reduced the HSC clonality. Elevated B2m levels correlated with high expression of repetitive element (RE) transcripts. Overall, our data suggest that RE-associated double-stranded RNA could interact with TLR3 to stimulate surface expression of B2m on hematopoietic stem and progenitor cells. These findings suggest that the balance of Calr and B2m regulates macrophage-HSC interactions and defines hematopoietic clonality. 2023
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Sporrij A, Choudhuri A, Prasad M, Muhire B, Fast EM, Manning ME, Weiss JD, Koh M, Yang S, Kingston RE, Tolstorukov MY, Clevers H, Zon LI. 2023. PGE alters chromatin through H2A.Z-variant enhancer nucleosome modification to promote hematopoietic stem cell fate. Proceedings of the National Academy of Sciences of the United States of America. 120(19):e2220613120. Pubmed: 37126722 DOI:10.1073/pnas.2220613120 Sporrij A, Choudhuri A, Prasad M, Muhire B, Fast EM, Manning ME, Weiss JD, Koh M, Yang S, Kingston RE, Tolstorukov MY, Clevers H, Zon LI. 2023. PGE alters chromatin through H2A.Z-variant enhancer nucleosome modification to promote hematopoietic stem cell fate. Proceedings of the National Academy of Sciences of the United States of America. 120(19):e2220613120. Pubmed: 37126722 DOI:10.1073/pnas.2220613120 Prostaglandin E2 (PGE) and 16,16-dimethyl-PGE (dmPGE) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies [C. Cutler , 3074-3081(2013); W. Goessling , 445-458 (2011); W. Goessling , 1136-1147 (2009)]. Here, we report that PGE-induced changes in chromatin at enhancer regions through histone-variant H2A.Z permit acute inflammatory gene induction to promote HSPC fate. We found that dmPGE-inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes permissive of CREB transcription factor (TF) binding. CREB binding to enhancer nucleosomes following dmPGE stimulation is concomitant with deposition of histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone-variant nucleosomes retained within inducible enhancers facilitate TF binding. Histone-variant acetylation by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism through which inflammatory mediators, such as dmPGE, lead to acute transcriptional changes and modify HSPC behavior to improve stem cell transplantation. -
Hagedorn EJ, Perlin JR, Freeman RJ, Wattrus SJ, Han T, Mao C, Kim JW, Fernández-Maestre I, Daily ML, D'Amato C, Fairchild MJ, Riquelme R, Li B, Ragoonanan DAVE, Enkhbayar K, Henault EL, Wang HG, Redfield SE, Collins SH, Lichtig A, Yang S, Zhou Y, Kunar B, Gomez-Salinero JM, Dinh TT, Pan J, Holler K, Feldman HA, Butcher EC, van Oudenaarden A, Rafii S, Junker JP, Zon LI. 2023. Transcription factor induction of vascular blood stem cell niches in vivo. Developmental cell. 58(12):1037-1051.e4. Pubmed: 37119815 DOI:S1534-5807(23)00160-0 Hagedorn EJ, Perlin JR, Freeman RJ, Wattrus SJ, Han T, Mao C, Kim JW, Fernández-Maestre I, Daily ML, D'Amato C, Fairchild MJ, Riquelme R, Li B, Ragoonanan DAVE, Enkhbayar K, Henault EL, Wang HG, Redfield SE, Collins SH, Lichtig A, Yang S, Zhou Y, Kunar B, Gomez-Salinero JM, Dinh TT, Pan J, Holler K, Feldman HA, Butcher EC, van Oudenaarden A, Rafii S, Junker JP, Zon LI. 2023. Transcription factor induction of vascular blood stem cell niches in vivo. Developmental cell. 58(12):1037-1051.e4. Pubmed: 37119815 DOI:S1534-5807(23)00160-0 The hematopoietic niche is a supportive microenvironment composed of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses in zebrafish, we define a conserved gene expression signature and cis-regulatory landscape that are unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code that involves members of the Ets, Sox, and nuclear hormone receptor families and is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance, and division of HSPCs in vivo. These studies set forth an approach for generating synthetic HSPC niches, in vitro or in vivo, and for effective therapies to modulate the endogenous niche.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved. -
Avagyan S, Zon LI. 2023. Clonal hematopoiesis and inflammation - the perpetual cycle. Trends in cell biology. 33(8):695-707. Pubmed: 36593155 DOI:S0962-8924(22)00275-6 Avagyan S, Zon LI. 2023. Clonal hematopoiesis and inflammation - the perpetual cycle. Trends in cell biology. 33(8):695-707. Pubmed: 36593155 DOI:S0962-8924(22)00275-6 Acquired genetic or cytogenetic alterations in a blood stem cell that confer clonal fitness promote its relative expansion leading to clonal hematopoiesis (CH). Despite a largely intact hematopoietic output, CH is associated with a heightened risk of progression to hematologic malignancies and with non-hematologic health manifestations, including cardiovascular disease and overall mortality. We focus on the evidence for the role of inflammation in establishing, maintaining and reciprocally being affected by CH. We describe the known pro-inflammatory signals associated with CH and preclinical studies that elucidated the cellular mechanisms involved. We review the evolving literature on early-onset CH in germline predisposition conditions and the possible role of immune dysregulation in this context.Copyright © 2022 Elsevier Ltd. All rights reserved. -
Tavakoli S, Garcia V, Gähwiler E, Adatto I, Rangan A, Messemer KA, Kakhki SA, Yang S, Chan VS, Manning ME, Fotowat H, Zhou Y, Wagers AJ, Zon LI. 2023. Transplantation-based screen identifies inducers of muscle progenitor cell engraftment across vertebrate species. Cell reports. 42(4):112365. Pubmed: 37018075 DOI:S2211-1247(23)00376-5 Tavakoli S, Garcia V, Gähwiler E, Adatto I, Rangan A, Messemer KA, Kakhki SA, Yang S, Chan VS, Manning ME, Fotowat H, Zhou Y, Wagers AJ, Zon LI. 2023. Transplantation-based screen identifies inducers of muscle progenitor cell engraftment across vertebrate species. Cell reports. 42(4):112365. Pubmed: 37018075 DOI:S2211-1247(23)00376-5 Stem cell transplantation presents a potentially curative strategy for genetic disorders of skeletal muscle, but this approach is limited by the deleterious effects of cell expansion in vitro and consequent poor engraftment efficiency. In an effort to overcome this limitation, we sought to identify molecular signals that enhance the myogenic activity of cultured muscle progenitors. Here, we report the development and application of a cross-species small-molecule screening platform employing zebrafish and mice, which enables rapid, direct evaluation of the effects of chemical compounds on the engraftment of transplanted muscle precursor cells. Using this system, we screened a library of bioactive lipids to discriminate those that could increase myogenic engraftment in vivo in zebrafish and mice. This effort identified two lipids, lysophosphatidic acid and niflumic acid, both linked to the activation of intracellular calcium-ion flux, which showed conserved, dose-dependent, and synergistic effects in promoting muscle engraftment across these vertebrate species.Published by Elsevier Inc. -
Binder V, Li W, Faisal M, Oyman K, Calkins DL, Shaffer J, Teets EM, Sher S, Magnotte A, Belardo A, Deruelle W, Gregory TC, Orwick S, Hagedorn EJ, Perlin JR, Avagyan S, Lichtig A, Barrett F, Ammerman M, Yang S, Zhou Y, Carson WE, Shive HR, Blachly JS, Lapalombella R, Zon LI, Blaser BW. 2023. Microenvironmental control of hematopoietic stem cell fate via CXCL8 and protein kinase C. Cell reports. 42(5):112528. Pubmed: 37209097 DOI:S2211-1247(23)00539-9 Binder V, Li W, Faisal M, Oyman K, Calkins DL, Shaffer J, Teets EM, Sher S, Magnotte A, Belardo A, Deruelle W, Gregory TC, Orwick S, Hagedorn EJ, Perlin JR, Avagyan S, Lichtig A, Barrett F, Ammerman M, Yang S, Zhou Y, Carson WE, Shive HR, Blachly JS, Lapalombella R, Zon LI, Blaser BW. 2023. Microenvironmental control of hematopoietic stem cell fate via CXCL8 and protein kinase C. Cell reports. 42(5):112528. Pubmed: 37209097 DOI:S2211-1247(23)00539-9 Altered hematopoietic stem cell (HSC) fate underlies primary blood disorders but microenvironmental factors controlling this are poorly understood. Genetically barcoded genome editing of synthetic target arrays for lineage tracing (GESTALT) zebrafish were used to screen for factors expressed by the sinusoidal vascular niche that alter the phylogenetic distribution of the HSC pool under native conditions. Dysregulated expression of protein kinase C delta (PKC-δ, encoded by prkcda) increases the number of HSC clones by up to 80% and expands polyclonal populations of immature neutrophil and erythroid precursors. PKC agonists such as cxcl8 augment HSC competition for residency within the niche and expand defined niche populations. CXCL8 induces association of PKC-δ with the focal adhesion complex, activating extracellular signal-regulated kinase (ERK) signaling and expression of niche factors in human endothelial cells. Our findings demonstrate the existence of reserve capacity within the niche that is controlled by CXCL8 and PKC and has significant impact on HSC phylogenetic and phenotypic fate.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved. -
Oksuz O, Henninger JE, Warneford-Thomson R, Zheng MM, Erb H, Vancura A, Overholt KJ, Hawken SW, Banani SF, Lauman R, Reich LN, Robertson AL, Hannett NM, Lee TI, Zon LI, Bonasio R, Young RA. 2023. Transcription factors interact with RNA to regulate genes. Molecular cell. 83(14):2449-2463.e13. Pubmed: 37402367 DOI:S1097-2765(23)00434-3 Oksuz O, Henninger JE, Warneford-Thomson R, Zheng MM, Erb H, Vancura A, Overholt KJ, Hawken SW, Banani SF, Lauman R, Reich LN, Robertson AL, Hannett NM, Lee TI, Zon LI, Bonasio R, Young RA. 2023. Transcription factors interact with RNA to regulate genes. Molecular cell. 83(14):2449-2463.e13. Pubmed: 37402367 DOI:S1097-2765(23)00434-3 Transcription factors (TFs) orchestrate the gene expression programs that define each cell's identity. The canonical TF accomplishes this with two domains, one that binds specific DNA sequences and the other that binds protein coactivators or corepressors. We find that at least half of TFs also bind RNA, doing so through a previously unrecognized domain with sequence and functional features analogous to the arginine-rich motif of the HIV transcriptional activator Tat. RNA binding contributes to TF function by promoting the dynamic association between DNA, RNA, and TF on chromatin. TF-RNA interactions are a conserved feature important for vertebrate development and disrupted in disease. We propose that the ability to bind DNA, RNA, and protein is a general property of many TFs and is fundamental to their gene regulatory function.Copyright © 2023 Elsevier Inc. All rights reserved. -
Roider E, Lakatos AIT, McConnell AM, Wang P, Mueller A, Kawakami A, Tsoi J, Szabolcs BL, Ascsillán AA, Suita Y, Igras V, Lo JA, Hsiao JJ, Lapides R, Pál DMP, Lengyel AS, Navarini A, Okazaki A, Iliopoulos O, Németh I, Graeber TG, Zon L, Giese RW, Kemeny LV, Fisher DE. 2023. MITF regulates IDH1 and NNT and drives a transcriptional program protecting cutaneous melanoma from reactive oxygen species. bioRxiv : the preprint server for biology. Pubmed: 38014031 DOI:10.1101/2023.11.10.564582 Roider E, Lakatos AIT, McConnell AM, Wang P, Mueller A, Kawakami A, Tsoi J, Szabolcs BL, Ascsillán AA, Suita Y, Igras V, Lo JA, Hsiao JJ, Lapides R, Pál DMP, Lengyel AS, Navarini A, Okazaki A, Iliopoulos O, Németh I, Graeber TG, Zon L, Giese RW, Kemeny LV, Fisher DE. 2023. MITF regulates IDH1 and NNT and drives a transcriptional program protecting cutaneous melanoma from reactive oxygen species. bioRxiv : the preprint server for biology. Pubmed: 38014031 DOI:10.1101/2023.11.10.564582 Array -
Insco ML, Abraham BJ, Dubbury SJ, Kaltheuner IH, Dust S, Wu C, Chen KY, Liu D, Bellaousov S, Cox AM, Martin BJE, Zhang T, Ludwig CG, Fabo T, Modhurima R, Esgdaille DE, Henriques T, Brown KM, Chanock SJ, Geyer M, Adelman K, Sharp PA, Young RA, Boutz PL, Zon LI. 2023. Oncogenic mutations impede nuclear RNA surveillance. Science (New York, N.Y.). 380(6642):eabn7625. Pubmed: 37079685 DOI:10.1126/science.abn7625 Insco ML, Abraham BJ, Dubbury SJ, Kaltheuner IH, Dust S, Wu C, Chen KY, Liu D, Bellaousov S, Cox AM, Martin BJE, Zhang T, Ludwig CG, Fabo T, Modhurima R, Esgdaille DE, Henriques T, Brown KM, Chanock SJ, Geyer M, Adelman K, Sharp PA, Young RA, Boutz PL, Zon LI. 2023. Oncogenic mutations impede nuclear RNA surveillance. Science (New York, N.Y.). 380(6642):eabn7625. Pubmed: 37079685 DOI:10.1126/science.abn7625 RNA surveillance pathways detect and degrade defective transcripts to ensure RNA fidelity. We found that disrupted nuclear RNA surveillance is oncogenic. Cyclin-dependent kinase 13 () is mutated in melanoma, and patient-mutated accelerates zebrafish melanoma. mutation causes aberrant RNA stabilization. CDK13 is required for ZC3H14 phosphorylation, which is necessary and sufficient to promote nuclear RNA degradation. Mutant CDK13 fails to activate nuclear RNA surveillance, causing aberrant protein-coding transcripts to be stabilized and translated. Forced aberrant RNA expression accelerates melanoma in zebrafish. We found recurrent mutations in genes encoding nuclear RNA surveillance components in many malignancies, establishing nuclear RNA surveillance as a tumor-suppressive pathway. Activating nuclear RNA surveillance is crucial to avoid accumulation of aberrant RNAs and their ensuing consequences in development and disease. -
Song J, Gooding AR, Hemphill WO, Love BD, Robertson A, Yao L, Zon LI, North TE, Kasinath V, Cech TR. 2023. Structural basis for inactivation of PRC2 by G-quadruplex RNA. Science (New York, N.Y.). 381(6664):1331-1337. Pubmed: 37733873 DOI:10.1126/science.adh0059 Song J, Gooding AR, Hemphill WO, Love BD, Robertson A, Yao L, Zon LI, North TE, Kasinath V, Cech TR. 2023. Structural basis for inactivation of PRC2 by G-quadruplex RNA. Science (New York, N.Y.). 381(6664):1331-1337. Pubmed: 37733873 DOI:10.1126/science.adh0059 Polycomb repressive complex 2 (PRC2) silences genes through trimethylation of histone H3K27. PRC2 associates with numerous precursor messenger RNAs (pre-mRNAs) and long noncoding RNAs (lncRNAs) with a binding preference for G-quadruplex RNA. In this work, we present a 3.3-Å-resolution cryo-electron microscopy structure of PRC2 bound to a G-quadruplex RNA. Notably, RNA mediates the dimerization of PRC2 by binding both protomers and inducing a protein interface composed of two copies of the catalytic subunit EZH2, thereby blocking nucleosome DNA interaction and histone H3 tail accessibility. Furthermore, an RNA-binding loop of EZH2 facilitates the handoff between RNA and DNA, another activity implicated in PRC2 regulation by RNA. We identified a gain-of-function mutation in this loop that activates PRC2 in zebrafish. Our results reveal mechanisms for RNA-mediated regulation of a chromatin-modifying enzyme. -
LeBleu VS, Dai J, Tsutakawa S, MacDonald BA, Alge JL, Sund M, Xie L, Sugimoto H, Tainer J, Zon LI, Kalluri R. 2023. Identification of unique α4 chain structure and conserved antiangiogenic activity of α3NC1 type IV collagen in zebrafish. Developmental dynamics : an official publication of the American Association of Anatomists. 252(7):1046-1060. Pubmed: 37002899 DOI:10.1002/dvdy.590 LeBleu VS, Dai J, Tsutakawa S, MacDonald BA, Alge JL, Sund M, Xie L, Sugimoto H, Tainer J, Zon LI, Kalluri R. 2023. Identification of unique α4 chain structure and conserved antiangiogenic activity of α3NC1 type IV collagen in zebrafish. Developmental dynamics : an official publication of the American Association of Anatomists. 252(7):1046-1060. Pubmed: 37002899 DOI:10.1002/dvdy.590 Array© 2023 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy. 2022
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Wattrus SJ, Smith ML, Rodrigues CP, Hagedorn EJ, Kim JW, Budnik B, Zon LI. 2022. Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality. Science (New York, N.Y.). 377(6613):1413-1419. Pubmed: 36137040 DOI:10.1126/science.abo4837 Wattrus SJ, Smith ML, Rodrigues CP, Hagedorn EJ, Kim JW, Budnik B, Zon LI. 2022. Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality. Science (New York, N.Y.). 377(6613):1413-1419. Pubmed: 36137040 DOI:10.1126/science.abo4837 Tissue-specific stem cells persist for a lifetime and can differentiate to maintain homeostasis or transform to initiate cancer. Despite their importance, there are no described quality assurance mechanisms for newly formed stem cells. We observed intimate and specific interactions between macrophages and nascent blood stem cells in zebrafish embryos. Macrophage interactions frequently led to either removal of cytoplasmic material and stem cell division or complete engulfment and stem cell death. Stressed stem cells were marked by surface Calreticulin, which stimulated macrophage interactions. Using cellular barcoding, we found that Calreticulin knock-down or embryonic macrophage depletion reduced the number of stem cell clones that established adult hematopoiesis. Our work supports a model in which embryonic macrophages determine hematopoietic clonality by monitoring stem cell quality. -
Adatto I, Lawrence C, Krug L, Zon LI. 2022. The effects of intensive feeding on reproductive performance in laboratory zebrafish (Danio rerio). PloS one. 17(11):e0278302. Pubmed: 36445925 DOI:10.1371/journal.pone.0278302 Adatto I, Lawrence C, Krug L, Zon LI. 2022. The effects of intensive feeding on reproductive performance in laboratory zebrafish (Danio rerio). PloS one. 17(11):e0278302. Pubmed: 36445925 DOI:10.1371/journal.pone.0278302 The zebrafish (Danio rerio) is among the most widely used model animals in scientific research. Historically, these fish have been reared in the laboratory using simple methods developed by home aquarists. For laboratories with high demand for breeding and generation turn-over, however, there has been a shift away from this approach towards one that leverages techniques, tools, and feeds from commercial aquaculture to help accelerate growth rates and decrease generation times. While these advances have improved efficiency, the effects of feeding zebrafish diets that are designed to grow disparately related cold-water fish species to market size quickly are not well-understood. To explore the impacts that intensive feeding protocols may have on this species, groups of zebrafish larvae from two different wild-type lines were stocked into treatment tanks at a standard density of 10 fish per liter and were administered either a "high" or "low" food diet for a maximum of 63 days. During their growth phase, the "high" food diet group received at least 8x more rotifers and at least 2x more Artemia than the "low" food diet group. Growth, survival, and reproductive performance (fecundity and viability) were measured in these fish and in their offspring. We found that fish that were fed more grew more rapidly and were able to reproduce earlier than fish that were fed less, but they were also more likely to produce higher proportions of non-viable embryos.Copyright: © 2022 Adatto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. -
Rubin SA, Baron CS, Pessoa Rodrigues C, Duran M, Corbin AF, Yang SP, Trapnell C, Zon LI. 2022. Single-cell analyses reveal early thymic progenitors and pre-B cells in zebrafish. The Journal of experimental medicine. 219(9). Pubmed: 35938989 DOI:10.1084/jem.20220038 Rubin SA, Baron CS, Pessoa Rodrigues C, Duran M, Corbin AF, Yang SP, Trapnell C, Zon LI. 2022. Single-cell analyses reveal early thymic progenitors and pre-B cells in zebrafish. The Journal of experimental medicine. 219(9). Pubmed: 35938989 DOI:10.1084/jem.20220038 The zebrafish has proven to be a valuable model organism for studying hematopoiesis, but relatively little is known about zebrafish immune cell development and functional diversity. Elucidating key aspects of zebrafish lymphocyte development and exploring the breadth of effector functions would provide valuable insight into the evolution of adaptive immunity. We performed single-cell RNA sequencing on ∼70,000 cells from the zebrafish marrow and thymus to establish a gene expression map of zebrafish immune cell development. We uncovered rich cellular diversity in the juvenile and adult zebrafish thymus, elucidated B- and T-cell developmental trajectories, and transcriptionally characterized subsets of hematopoietic stem and progenitor cells and early thymic progenitors. Our analysis permitted the identification of two dendritic-like cell populations and provided evidence in support of the existence of a pre-B cell state. Our results provide critical insights into the landscape of zebrafish immunology and offer a foundation for cellular and genetic studies.© 2022 Rubin et al. -
Sankaran VG, Weissman JS, Zon LI. 2022. Cellular barcoding to decipher clonal dynamics in disease. Science (New York, N.Y.). 378(6616):eabm5874. Pubmed: 36227997 DOI:10.1126/science.abm5874 Sankaran VG, Weissman JS, Zon LI. 2022. Cellular barcoding to decipher clonal dynamics in disease. Science (New York, N.Y.). 378(6616):eabm5874. Pubmed: 36227997 DOI:10.1126/science.abm5874 Cellular barcodes are distinct DNA sequences that enable one to track specific cells across time or space. Recent advances in our ability to detect natural or synthetic cellular barcodes, paired with single-cell readouts of cell state, have markedly increased our knowledge of clonal dynamics and genealogies of the cells that compose a variety of tissues and organs. These advances hold promise to redefine our view of human disease. Here, we provide an overview of cellular barcoding approaches, discuss applications to gain new insights into disease mechanisms, and provide an outlook on future applications. We discuss unanticipated insights gained through barcoding in studies of cancer and blood cell production and describe how barcoding can be applied to a growing array of medical fields, particularly with the increasing recognition of clonal contributions in human diseases. -
Ablain J, Al Mahi A, Rothschild H, Prasad M, Aires S, Yang S, Dokukin ME, Xu S, Dang M, Sokolov I, Lian CG, Zon LI. 2022. Loss of NECTIN1 triggers melanoma dissemination upon local IGF1 depletion. Nature genetics. 54(12):1839-1852. Pubmed: 36229674 DOI:10.1038/s41588-022-01191-z Ablain J, Al Mahi A, Rothschild H, Prasad M, Aires S, Yang S, Dokukin ME, Xu S, Dang M, Sokolov I, Lian CG, Zon LI. 2022. Loss of NECTIN1 triggers melanoma dissemination upon local IGF1 depletion. Nature genetics. 54(12):1839-1852. Pubmed: 36229674 DOI:10.1038/s41588-022-01191-z Cancer genetics has uncovered many tumor-suppressor and oncogenic pathways, but few alterations have revealed mechanisms involved in tumor spreading. Here, we examined the role of the third most significant chromosomal deletion in human melanoma that inactivates the adherens junction gene NECTIN1 in 55% of cases. We found that NECTIN1 loss stimulates melanoma cell migration in vitro and spreading in vivo in both zebrafish and human tumors specifically in response to decreased IGF1 signaling. In human melanoma biopsy specimens, adherens junctions were seen exclusively in areas with low IGF1 levels, but not in NECTIN1-deficient tumors. Our study establishes NECTIN1 as a major determinant of melanoma dissemination and uncovers a genetic control of the response to microenvironmental signals.© 2022. The Author(s). -
Mito JK, Weber MC, Corbin A, Murphy GF, Zon LI. 2022. Modeling Spitz melanoma in zebrafish using sequential mutagenesis. Disease models & mechanisms. 15(8). Pubmed: 36017742 DOI:10.1242/dmm.049452 Mito JK, Weber MC, Corbin A, Murphy GF, Zon LI. 2022. Modeling Spitz melanoma in zebrafish using sequential mutagenesis. Disease models & mechanisms. 15(8). Pubmed: 36017742 DOI:10.1242/dmm.049452 Spitz neoplasms are a diverse group of molecularly and histologically defined melanocytic tumors with varying biologic potentials. The precise classification of Spitz neoplasms can be challenging. Recent studies have revealed recurrent fusions involving multiple kinases in a large proportion of Spitz tumors. In this study, we generated a transgenic zebrafish model of Spitz melanoma using a previously identified ZCCHC8-ROS1 fusion gene. Animals developed grossly apparent melanocytic proliferations as early as 3 weeks of age and overt melanoma as early as 5 weeks. By 7 weeks, ZCCHC8-ROS1 induced a histologic spectrum of neoplasms ranging from hyperpigmented patches to melanoma. Given the swift onset of these tumors during development, we extended this approach into adult fish using a recently described electroporation technique. Tissue-specific expression of ZCCHC8-ROS1 in adults led to melanocyte expansion without overt progression to melanoma. Subsequent electroporation with tissue-specific CRISPR, targeting only tp53 was sufficient to induce transformation to melanoma. Our model exhibits the use of sequential mutagenesis in the adult zebrafish, and demonstrates that ZCCHC8-ROS1 induces a spectrum of melanocytic lesions that closely mimics human Spitz neoplasms.© 2022. Published by The Company of Biologists Ltd. 2021
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He S, Zimmerman MW, Layden HM, Berezovskaya A, Etchin J, Martel MW, Thurston G, Jing CB, van Rooijen E, Kaufman CK, Rodig SJ, Zon LI, Patton EE, Mansour MR, Look AT. 2021. Synergistic melanoma cell death mediated by inhibition of both MCL1 and BCL2 in high-risk tumors driven by NF1/PTEN loss. Oncogene. 40(38):5718-5729. Pubmed: 34331013 DOI:10.1038/s41388-021-01926-y He S, Zimmerman MW, Layden HM, Berezovskaya A, Etchin J, Martel MW, Thurston G, Jing CB, van Rooijen E, Kaufman CK, Rodig SJ, Zon LI, Patton EE, Mansour MR, Look AT. 2021. Synergistic melanoma cell death mediated by inhibition of both MCL1 and BCL2 in high-risk tumors driven by NF1/PTEN loss. Oncogene. 40(38):5718-5729. Pubmed: 34331013 DOI:10.1038/s41388-021-01926-y Melanomas driven by loss of the NF1 tumor suppressor have a high risk of treatment failure and effective therapies have not been developed. Here we show that loss-of-function mutations of nf1 and pten result in aggressive melanomas in zebrafish, representing the first animal model of NF1-mutant melanomas harboring PTEN loss. MEK or PI3K inhibitors show little activity when given alone due to cross-talk between the pathways, and high toxicity when given together. The mTOR inhibitors, sirolimus, everolimus, and temsirolimus, were the most active single agents tested, potently induced tumor-suppressive autophagy, but not apoptosis. Because addition of the BCL2 inhibitor venetoclax resulted in compensatory upregulation of MCL1, we established a three-drug combination composed of sirolimus, venetoclax, and the MCL1 inhibitor S63845. This well-tolerated drug combination potently and synergistically induces apoptosis in both zebrafish and human NF1/PTEN-deficient melanoma cells, providing preclinical evidence justifying an early-stage clinical trial in patients with NF1/PTEN-deficient melanoma.© 2021. The Author(s). -
García-Castillo J, Alcaraz-Pérez F, Martínez-Balsalobre E, García-Moreno D, Rossmann MP, Fernández-Lajarín M, Bernabé-García M, Pérez-Oliva AB, Rodríguez-Cortez VC, Bueno C, Adatto I, Agarwal S, Menéndez P, Zon LI, Mulero V, Cayuela ML. 2021. Telomerase RNA recruits RNA polymerase II to target gene promoters to enhance myelopoiesis. Proceedings of the National Academy of Sciences of the United States of America. 118(32). Pubmed: 34353901 DOI:10.1073/pnas.2015528118 García-Castillo J, Alcaraz-Pérez F, Martínez-Balsalobre E, García-Moreno D, Rossmann MP, Fernández-Lajarín M, Bernabé-García M, Pérez-Oliva AB, Rodríguez-Cortez VC, Bueno C, Adatto I, Agarwal S, Menéndez P, Zon LI, Mulero V, Cayuela ML. 2021. Telomerase RNA recruits RNA polymerase II to target gene promoters to enhance myelopoiesis. Proceedings of the National Academy of Sciences of the United States of America. 118(32). Pubmed: 34353901 DOI:10.1073/pnas.2015528118 Dyskeratosis congenita (DC) is a rare inherited bone marrow failure and cancer predisposition syndrome caused by mutations in telomerase or telomeric proteins. Here, we report that zebrafish telomerase RNA () binds to specific DNA sequences of master myeloid genes and controls their expression by recruiting RNA Polymerase II (Pol II). Zebrafish harboring the CR4-CR5 domain mutation found in DC patients hardly interacted with Pol II and failed to regulate myeloid gene expression in vivo and to increase their transcription rates in vitro. Similarly, regulated myeloid gene expression and Pol II promoter occupancy in human myeloid progenitor cells. Strikingly, induced pluripotent stem cells derived from DC patients with a mutation in the CR4-CR5 domain showed impaired myelopoiesis, while those with mutated telomerase catalytic subunit differentiated normally. Our findings show that acts as a transcription factor, revealing a target for therapeutic intervention in DC patients. -
Lévesque JP, Purton LE, Hidalgo A, Zon LI, Katayama Y, Scadden DT, Bowman TV, Stanley ER, Lucas D, Pinho S. 2021. In memory of Paul Sylvain Frenette, a pioneering explorer of the hematopoietic stem cell niche who left far too early. Experimental hematology. Pubmed: 34403758 DOI:10.1016/j.exphem.2021.08.002 Lévesque JP, Purton LE, Hidalgo A, Zon LI, Katayama Y, Scadden DT, Bowman TV, Stanley ER, Lucas D, Pinho S. 2021. In memory of Paul Sylvain Frenette, a pioneering explorer of the hematopoietic stem cell niche who left far too early. Experimental hematology. Pubmed: 34403758 DOI:10.1016/j.exphem.2021.08.002 -
McConnell AM, Zon LI. 2021. Dissecting melanocytes to predict melanoma. Nature cell biology. 23(9):930-931. Pubmed: 34475537 DOI:10.1038/s41556-021-00748-0 McConnell AM, Zon LI. 2021. Dissecting melanocytes to predict melanoma. Nature cell biology. 23(9):930-931. Pubmed: 34475537 DOI:10.1038/s41556-021-00748-0 With advanced high-throughput technologies, scientists can now use transcriptional signatures to study melanocytes as they become cancer. A new study identifies transcriptional programs at single-cell resolution across platforms and species, which enables prediction of melanoma prognosis and response to immune-checkpoint inhibitor therapy. -
Avagyan S, Weber MC, Ma S, Prasad M, Mannherz WP, Yang S, Buenrostro JD, Zon LI. 2021. Single-cell ATAC-seq reveals GATA2-dependent priming defect in myeloid and a maturation bottleneck in lymphoid lineages. Blood advances. 5(13):2673-2686. Pubmed: 34170284 DOI:10.1182/bloodadvances.2020002992 Avagyan S, Weber MC, Ma S, Prasad M, Mannherz WP, Yang S, Buenrostro JD, Zon LI. 2021. Single-cell ATAC-seq reveals GATA2-dependent priming defect in myeloid and a maturation bottleneck in lymphoid lineages. Blood advances. 5(13):2673-2686. Pubmed: 34170284 DOI:10.1182/bloodadvances.2020002992 Germline heterozygous mutations in GATA2 are associated with a syndrome characterized by cytopenias, atypical infections, and increased risk of hematologic malignancies. Here, we generated a zebrafish mutant of gata2b that recapitulated the myelomonocytopenia and B-cell lymphopenia of GATA2 deficiency syndrome. Using single-cell assay for transposase accessible chromatin with sequencing of marrow cells, we showed that loss of gata2b led to contrasting alterations in chromosome accessibility in early myeloid and lymphoid progenitors, associated with defects in gene expression. Within the myeloid lineage in gata2b mutant zebrafish, we identified an attenuated myeloid differentiation with reduced transcriptional priming and skewing away from the monocytic program. In contrast, in early lymphoid progenitors, gata2b loss led to accumulation of B-lymphoid transcription factor accessibility coupled with increased expression of the B-cell lineage-specification program. However, gata2b mutant zebrafish had incomplete B-cell lymphopoiesis with loss of lineage-specific transcription factor accessibility in differentiating B cells, in the context of aberrantly reduced oxidative metabolic pathways. Our results establish that transcriptional events in early progenitors driven by Gata2 are required to complete normal differentiation.© 2021 by The American Society of Hematology. -
Allouche J, Rachmin I, Adhikari K, Pardo LM, Lee JH, McConnell AM, Kato S, Fan S, Kawakami A, Suita Y, Wakamatsu K, Igras V, Zhang J, Navarro PP, Lugo CM, Noonan HR, Christie KA, Itin K, Mujahid N, Lo JA, Won CH, Evans CL, Weng QY, Wang H, Osseiran S, Lovas A, Németh I, Cozzio A, Navarini AA, Hsiao JJ, Nguyen N, Kemény LV, Iliopoulos O, Berking C, Ruzicka T, Gonzalez-José R, Bortolini MC, Canizales-Quinteros S, Acuna-Alonso V, Gallo C, Poletti G, Bedoya G, Rothhammer F, Ito S, Schiaffino MV, Chao LH, Kleinstiver BP, Tishkoff S, Zon LI, Nijsten T, Ruiz-Linares A, Fisher DE, Roider E. 2021. NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism. Cell. 184(16):4268-4283.e20. Pubmed: 34233163 DOI:S0092-8674(21)00757-1 Allouche J, Rachmin I, Adhikari K, Pardo LM, Lee JH, McConnell AM, Kato S, Fan S, Kawakami A, Suita Y, Wakamatsu K, Igras V, Zhang J, Navarro PP, Lugo CM, Noonan HR, Christie KA, Itin K, Mujahid N, Lo JA, Won CH, Evans CL, Weng QY, Wang H, Osseiran S, Lovas A, Németh I, Cozzio A, Navarini AA, Hsiao JJ, Nguyen N, Kemény LV, Iliopoulos O, Berking C, Ruzicka T, Gonzalez-José R, Bortolini MC, Canizales-Quinteros S, Acuna-Alonso V, Gallo C, Poletti G, Bedoya G, Rothhammer F, Ito S, Schiaffino MV, Chao LH, Kleinstiver BP, Tishkoff S, Zon LI, Nijsten T, Ruiz-Linares A, Fisher DE, Roider E. 2021. NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism. Cell. 184(16):4268-4283.e20. Pubmed: 34233163 DOI:S0092-8674(21)00757-1 Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.Copyright © 2021 Elsevier Inc. All rights reserved. -
Avagyan S, Henninger JE, Mannherz WP, Mistry M, Yoon J, Yang S, Weber MC, Moore JL, Zon LI. 2021. Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis. Science (New York, N.Y.). 374(6568):768-772. Pubmed: 34735227 DOI:10.1126/science.aba9304 Avagyan S, Henninger JE, Mannherz WP, Mistry M, Yoon J, Yang S, Weber MC, Moore JL, Zon LI. 2021. Resistance to inflammation underlies enhanced fitness in clonal hematopoiesis. Science (New York, N.Y.). 374(6568):768-772. Pubmed: 34735227 DOI:10.1126/science.aba9304 Clonal hematopoiesis results from enhanced fitness of a mutant hematopoietic stem and progenitor cell (HSPC), but how such clones expand is unclear. We developed a technique that combines mosaic mutagenesis with color labeling of HSPCs to study how acquired mutations affect clonal fitness in a native environment. Mutations in clonal hematopoiesis–associated genes such as promoted clonal dominance. Single-cell transcriptional analysis revealed that mutations stimulated expression of proinflammatory genes in mature myeloid cells and anti-inflammatory genes in progenitor cells of the mutant clone. Biallelic loss of one such immunomodulator, , abrogated the ability of mutant clones to establish clonal dominance. These results support a model where clonal fitness of mutant clones is driven by enhanced resistance to inflammatory signals from their mutant mature cell progeny. -
Sporrij A, Zon LI. 2021. Nucleotide stress responses in neural crest cell fate and melanoma. Cell cycle (Georgetown, Tex.). 20(15):1455-1467. Pubmed: 34281491 DOI:10.1080/15384101.2021.1947567 Sporrij A, Zon LI. 2021. Nucleotide stress responses in neural crest cell fate and melanoma. Cell cycle (Georgetown, Tex.). 20(15):1455-1467. Pubmed: 34281491 DOI:10.1080/15384101.2021.1947567 Melanoma is the deadliest form of skin cancer. While clinical developments have significantly improved patient prognosis, effective treatment is often obstructed by limited response rates, intrinsic or acquired resistance to therapy, and adverse events. Melanoma initiation and progression are associated with transcriptional reprogramming of melanocytes to a cell state that resembles the lineage from which the cells are specified during development, that is the neural crest. Convergence to a neural crest cell (NCC)-like state revealed the therapeutic potential of targeting developmental pathways for the treatment of melanoma. Neural crest cells have a unique sensitivity to metabolic dysregulation, especially nucleotide depletion. Mutations in the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) particularly affect neural crest-derived tissues and cause Miller syndrome, a genetic disorder characterized by craniofacial malformations in patients. The developmental susceptibility of the neural crest to nucleotide deficiency is conserved in melanoma and provides a metabolic vulnerability that can be exploited for therapeutic purposes. We review the current knowledge on nucleotide stress responses in neural crest and melanoma and discuss how the recent scientific advances that have improved our understanding of transcriptional regulation during nucleotide depletion can impact melanoma treatment. -
Ablain J, Liu S, Moriceau G, Lo RS, Zon LI. 2021. SPRED1 deletion confers resistance to MAPK inhibition in melanoma. The Journal of experimental medicine. 218(3). Pubmed: 33306107 DOI:10.1084/jem.20201097 Ablain J, Liu S, Moriceau G, Lo RS, Zon LI. 2021. SPRED1 deletion confers resistance to MAPK inhibition in melanoma. The Journal of experimental medicine. 218(3). Pubmed: 33306107 DOI:10.1084/jem.20201097 Functional evaluation of genetic lesions can discover a role in cancer initiation and progression and help develop novel therapeutic strategies. We previously identified the negative MAPK regulator SPRED1 as a novel tumor suppressor in KIT-driven melanoma. Here, we show that SPRED1 is also frequently deleted in human melanoma driven by mutant BRAF. We found that SPRED1 inactivation in human melanoma cell lines and primary zebrafish melanoma conferred resistance to BRAFV600E inhibition in vitro and in vivo. Mechanistically, SPRED1 loss promoted melanoma cell proliferation under mutant BRAF inhibition by reactivating MAPK activity. Consistently, biallelic deletion of SPRED1 was observed in a patient whose melanoma acquired resistance to MAPK-targeted therapy. These studies combining work in human cells and in vivo modeling in zebrafish demonstrate a new mechanism of resistance to BRAFV600E inhibition in melanoma.© 2020 Ablain et al. -
Zon LI, Boisvert JD. 2021. A uniform format for manuscript submission. Cell. 184(7):1654-1656. Pubmed: 33798436 DOI:S0092-8674(21)00073-8 Zon LI, Boisvert JD. 2021. A uniform format for manuscript submission. Cell. 184(7):1654-1656. Pubmed: 33798436 DOI:S0092-8674(21)00073-8 Many scientists spend unnecessary time reformatting papers to submit them to different journals. We propose a uniform submission format that we hope journals will include in their options for submission. Widespread adoption of this uniform submission format could shorten the submission and publishing process, freeing up time for research.Copyright © 2021 Elsevier Inc. All rights reserved. -
Rossmann MP, Dubois SM, Agarwal S, Zon LI. 2021. Mitochondrial function in development and disease. Disease models & mechanisms. 14(6). Pubmed: 34114603 DOI:10.1242/dmm.048912 Rossmann MP, Dubois SM, Agarwal S, Zon LI. 2021. Mitochondrial function in development and disease. Disease models & mechanisms. 14(6). Pubmed: 34114603 DOI:10.1242/dmm.048912 Mitochondria are organelles with vital functions in almost all eukaryotic cells. Often described as the cellular 'powerhouses' due to their essential role in aerobic oxidative phosphorylation, mitochondria perform many other essential functions beyond energy production. As signaling organelles, mitochondria communicate with the nucleus and other organelles to help maintain cellular homeostasis, allow cellular adaptation to diverse stresses, and help steer cell fate decisions during development. Mitochondria have taken center stage in the research of normal and pathological processes, including normal tissue homeostasis and metabolism, neurodegeneration, immunity and infectious diseases. The central role that mitochondria assume within cells is evidenced by the broad impact of mitochondrial diseases, caused by defects in either mitochondrial or nuclear genes encoding for mitochondrial proteins, on different organ systems. In this Review, we will provide the reader with a foundation of the mitochondrial 'hardware', the mitochondrion itself, with its specific dynamics, quality control mechanisms and cross-organelle communication, including its roles as a driver of an innate immune response, all with a focus on development, disease and aging. We will further discuss how mitochondrial DNA is inherited, how its mutation affects cell and organismal fitness, and current therapeutic approaches for mitochondrial diseases in both model organisms and humans.© 2021. Published by The Company of Biologists Ltd. -
Fazio M, van Rooijen E, Mito JK, Modhurima R, Weiskopf E, Yang S, Zon LI. 2021. Recurrent co-alteration of HDGF and SETDB1 on chromosome 1q drives cutaneous melanoma progression and poor prognosis. Pigment cell & melanoma research. 34(3):641-647. Pubmed: 33064882 DOI:10.1111/pcmr.12937 Fazio M, van Rooijen E, Mito JK, Modhurima R, Weiskopf E, Yang S, Zon LI. 2021. Recurrent co-alteration of HDGF and SETDB1 on chromosome 1q drives cutaneous melanoma progression and poor prognosis. Pigment cell & melanoma research. 34(3):641-647. Pubmed: 33064882 DOI:10.1111/pcmr.12937 A progressive increase in copy number variation (CNV) characterizes the natural history of cutaneous melanoma progression toward later disease stages, but our understanding of genetic drivers underlying chromosomal arm-level CNVs remains limited. To identify candidate progression drivers, we mined the TCGA SKCM dataset and identified HDGF as a recurrently amplified gene whose high mRNA expression correlates with poor patient survival. Using melanocyte-specific overexpression in the zebrafish BRAF -driven MiniCoopR melanoma model, we show that HDGF accelerates melanoma development in vivo. Transcriptional analysis of HDGF compared to control EGFP tumors showed the activation of endothelial/angiogenic pathways. We validated this observation using an endothelial kdrl:mCherry reporter line which showed HDGF to increases tumor vasculature. HDGF is frequently co-altered with the established melanoma driver SETDB1. Both genes are located on chromosome 1q, and their co-amplification is observed in up to 13% of metastatic melanoma. TCGA patients with both genes amplified and/or overexpressed have a worse melanoma specific survival. We tested co-expression of HDGF and SETDB1 in the MiniCoopR model, which resulted in faster and more aggressive melanoma development than either gene individually. Our work identifies the co-amplification of HDGF and SETDB1 as a functional driver of melanoma progression and poor patient prognosis.© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. -
Wattrus SJ, Zon LI. 2021. Blood in the water: recent uses of zebrafish to study myeloid biology. Current opinion in hematology. 28(1):43-49. Pubmed: 33264224 DOI:10.1097/MOH.0000000000000627 Wattrus SJ, Zon LI. 2021. Blood in the water: recent uses of zebrafish to study myeloid biology. Current opinion in hematology. 28(1):43-49. Pubmed: 33264224 DOI:10.1097/MOH.0000000000000627 Array -
Choudhuri A, Han T, Zon LI. 2021. From development toward therapeutics, a collaborative effort on blood progenitors. Stem cell reports. 16(7):1674-1685. Pubmed: 34115985 DOI:S2213-6711(21)00261-7 Choudhuri A, Han T, Zon LI. 2021. From development toward therapeutics, a collaborative effort on blood progenitors. Stem cell reports. 16(7):1674-1685. Pubmed: 34115985 DOI:S2213-6711(21)00261-7 The National Heart, Lung, and Blood Institute Progenitor Cell Translational Consortium Blood Progenitor Meeting was hosted virtually on November 5, 2020, with 93 attendees across 20 research groups. The purpose of this meeting was to exchange recent findings, discuss current efforts, and identify prospective opportunities in the field of hematopoietic stem and progenitor cell research and therapeutic discovery.Copyright © 2021. -
Rossmann MP, Zon LI. 2021. 'Enhancing' red cell fate through epigenetic mechanisms. Current opinion in hematology. 28(3):129-137. Pubmed: 33741760 DOI:10.1097/MOH.0000000000000654 Rossmann MP, Zon LI. 2021. 'Enhancing' red cell fate through epigenetic mechanisms. Current opinion in hematology. 28(3):129-137. Pubmed: 33741760 DOI:10.1097/MOH.0000000000000654 ArrayCopyright © 2021 Wolters Kluwer Health, Inc. All rights reserved. -
Fazio M, van Rooijen E, Dang M, van de Hoek G, Ablain J, Mito JK, Yang S, Thomas A, Michael J, Fabo T, Modhurima R, Pessina P, Kaufman CK, Zhou Y, White RM, Zon LI. 2021. SATB2 induction of a neural crest mesenchyme-like program drives melanoma invasion and drug resistance. eLife. 10. Pubmed: 33527896 DOI:10.7554/eLife.64370 Fazio M, van Rooijen E, Dang M, van de Hoek G, Ablain J, Mito JK, Yang S, Thomas A, Michael J, Fabo T, Modhurima R, Pessina P, Kaufman CK, Zhou Y, White RM, Zon LI. 2021. SATB2 induction of a neural crest mesenchyme-like program drives melanoma invasion and drug resistance. eLife. 10. Pubmed: 33527896 DOI:10.7554/eLife.64370 Recent genomic and scRNA-seq analyses of melanoma demonstrated a lack of recurrent genetic drivers of metastasis, while identifying common transcriptional states correlating with invasion or drug resistance. To test whether transcriptional adaptation can drive melanoma progression, we made use of a zebrafish mitfa:; model, in which malignant progression is characterized by minimal genetic evolution. We undertook an overexpression-screen of 80 epigenetic/transcriptional regulators and found neural crest-mesenchyme developmental regulator SATB2 to accelerate aggressive melanoma development. Its overexpression induces invadopodia formation and invasion in zebrafish tumors and human melanoma cell lines. SATB2 binds and activates neural crest-regulators, including and . The transcriptional program induced by SATB2 overlaps with known MITFAXL and AQP1NGFR1 drug-resistant states and functionally drives enhanced tumor propagation and resistance to Vemurafenib in vivo. In summary, we show that melanoma transcriptional rewiring by SATB2 to a neural crest mesenchyme-like program can drive invasion and drug resistance in autochthonous tumors.© 2021, Fazio et al. -
Patton EE, Mueller KL, Adams DJ, Anandasabapathy N, Aplin AE, Bertolotto C, Bosenberg M, Ceol CJ, Burd CE, Chi P, Herlyn M, Holmen SL, Karreth FA, Kaufman CK, Khan S, Kobold S, Leucci E, Levy C, Lombard DB, Lund AW, Marie KL, Marine JC, Marais R, McMahon M, Robles-Espinoza CD, Ronai ZA, Samuels Y, Soengas MS, Villanueva J, Weeraratna AT, White RM, Yeh I, Zhu J, Zon LI, Hurlbert MS, Merlino G. 2021. Melanoma models for the next generation of therapies. Cancer cell. 39(5):610-631. Pubmed: 33545064 DOI:S1535-6108(21)00055-6 Patton EE, Mueller KL, Adams DJ, Anandasabapathy N, Aplin AE, Bertolotto C, Bosenberg M, Ceol CJ, Burd CE, Chi P, Herlyn M, Holmen SL, Karreth FA, Kaufman CK, Khan S, Kobold S, Leucci E, Levy C, Lombard DB, Lund AW, Marie KL, Marine JC, Marais R, McMahon M, Robles-Espinoza CD, Ronai ZA, Samuels Y, Soengas MS, Villanueva J, Weeraratna AT, White RM, Yeh I, Zhu J, Zon LI, Hurlbert MS, Merlino G. 2021. Melanoma models for the next generation of therapies. Cancer cell. 39(5):610-631. Pubmed: 33545064 DOI:S1535-6108(21)00055-6 There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.Copyright © 2021 Elsevier Inc. All rights reserved. -
Rossmann MP, Hoi K, Chan V, Abraham BJ, Yang S, Mullahoo J, Papanastasiou M, Wang Y, Elia I, Perlin JR, Hagedorn EJ, Hetzel S, Weigert R, Vyas S, Nag PP, Sullivan LB, Warren CR, Dorjsuren B, Greig EC, Adatto I, Cowan CA, Schreiber SL, Young RA, Meissner A, Haigis MC, Hekimi S, Carr SA, Zon LI. 2021. Cell-specific transcriptional control of mitochondrial metabolism by TIF1γ drives erythropoiesis. Science (New York, N.Y.). 372(6543):716-721. Pubmed: 33986176 DOI:10.1126/science.aaz2740 Rossmann MP, Hoi K, Chan V, Abraham BJ, Yang S, Mullahoo J, Papanastasiou M, Wang Y, Elia I, Perlin JR, Hagedorn EJ, Hetzel S, Weigert R, Vyas S, Nag PP, Sullivan LB, Warren CR, Dorjsuren B, Greig EC, Adatto I, Cowan CA, Schreiber SL, Young RA, Meissner A, Haigis MC, Hekimi S, Carr SA, Zon LI. 2021. Cell-specific transcriptional control of mitochondrial metabolism by TIF1γ drives erythropoiesis. Science (New York, N.Y.). 372(6543):716-721. Pubmed: 33986176 DOI:10.1126/science.aaz2740 Transcription and metabolism both influence cell function, but dedicated transcriptional control of metabolic pathways that regulate cell fate has rarely been defined. We discovered, using a chemical suppressor screen, that inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) rescues erythroid differentiation in bloodless zebrafish mutant embryos defective for transcriptional intermediary factor 1 gamma (). This rescue depends on the functional link of DHODH to mitochondrial respiration. The transcription elongation factor TIF1γ directly controls coenzyme Q (CoQ) synthesis gene expression. Upon loss, CoQ levels are reduced, and a high succinate/α-ketoglutarate ratio leads to increased histone methylation. A CoQ analog rescues 's bloodless phenotype. These results demonstrate that mitochondrial metabolism is a key output of a lineage transcription factor that drives cell fate decisions in the early blood lineage.Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. -
Fast EM, Sporrij A, Manning M, Rocha EL, Yang S, Zhou Y, Guo J, Baryawno N, Barkas N, Scadden D, Camargo F, Zon LI. 2021. External signals regulate continuous transcriptional states in hematopoietic stem cells. eLife. 10. Pubmed: 34939923 DOI:10.7554/eLife.66512 Fast EM, Sporrij A, Manning M, Rocha EL, Yang S, Zhou Y, Guo J, Baryawno N, Barkas N, Scadden D, Camargo F, Zon LI. 2021. External signals regulate continuous transcriptional states in hematopoietic stem cells. eLife. 10. Pubmed: 34939923 DOI:10.7554/eLife.66512 Hematopoietic stem cells (HSCs) must ensure adequate blood cell production following distinct external stressors. A comprehensive understanding of in vivo heterogeneity and specificity of HSC responses to external stimuli is currently lacking. We performed single-cell RNA sequencing (scRNA-Seq) on functionally validated mouse HSCs and LSK (Lin-, c-Kit+, Sca1+) progenitors after in vivo pharmacological perturbation of niche signals interferon, granulocyte colony-stimulating factor (G-CSF), and prostaglandin. We identified six HSC states that are characterized by enrichment but not exclusive expression of marker genes. External signals induced rapid transitions between HSC states but transcriptional response varied both between external stimulants and within the HSC population for a given perturbation. In contrast to LSK progenitors, HSCs were characterized by a greater link between molecular signatures at baseline and in response to external stressors. Chromatin analysis of unperturbed HSCs and LSKs by scATAC-Seq suggested some HSC-specific, cell intrinsic predispositions to niche signals. We compiled a comprehensive resource of HSC- and LSK progenitor-specific chromatin and transcriptional features that represent determinants of signal receptiveness and regenerative potential during stress hematopoiesis.© 2021, Fast et al. -
Khajavi M, Zhou Y, Schiffer AJ, Bazinet L, Birsner AE, Zon L, D'Amato RJ. 2021. Identification of Basp1 as a novel angiogenesis-regulating gene by multi-model system studies. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 35(5):e21404. Pubmed: 33899275 DOI:10.1096/fj.202001936RRR Khajavi M, Zhou Y, Schiffer AJ, Bazinet L, Birsner AE, Zon L, D'Amato RJ. 2021. Identification of Basp1 as a novel angiogenesis-regulating gene by multi-model system studies. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 35(5):e21404. Pubmed: 33899275 DOI:10.1096/fj.202001936RRR We have previously used the genetic diversity available in common inbred mouse strains to identify quantitative trait loci (QTLs) responsible for the differences in angiogenic response using the corneal micropocket neovascularization (CoNV) assay. Employing a mouse genome-wide association study (GWAS) approach, the region on chromosome 15 containing Basp1 was identified as being significantly associated with angiogenesis in inbred strains. Here, we developed a unique strategy to determine and verify the role of BASP1 in angiogenic pathways. Basp1 expression in cornea had a strong correlation with a haplotype shared by mouse strains with varied angiogenic phenotypes. In addition, inhibition of BASP1 demonstrated a dosage-dependent effect in both primary mouse brain endothelial and human microvascular endothelial cell (HMVEC) migration. To investigate its role in vivo, we knocked out basp1 in transgenic kdrl:zsGreen zebrafish embryos using a widely adopted CRISPR-Cas9 system. These embryos had severely disrupted vessel formation compared to control siblings. We further show that basp1 promotes angiogenesis by upregulating β-catenin gene and the Dll4/Notch1 signaling pathway. These results, to the best of our knowledge, provide the first in vivo evidence to indicate the role of Basp1 as an angiogenesis-regulating gene and opens the potential therapeutic avenues for a wide variety of systemic angiogenesis-dependent diseases.© 2021 Federation of American Societies for Experimental Biology. -
Hanna GJ, ONeill A, Cutler JM, Flynn M, Vijaykumar T, Clark JR, Wirth LJ, Lorch JH, Park JC, Mito JK, Lohr JG, Kaufman J, Burr NS, Zon LI, Haddad RI. 2021. A phase II trial of all-trans retinoic acid (ATRA) in advanced adenoid cystic carcinoma. Oral oncology. 119:105366. Pubmed: 34091189 DOI:S1368-8375(21)00189-5 Hanna GJ, ONeill A, Cutler JM, Flynn M, Vijaykumar T, Clark JR, Wirth LJ, Lorch JH, Park JC, Mito JK, Lohr JG, Kaufman J, Burr NS, Zon LI, Haddad RI. 2021. A phase II trial of all-trans retinoic acid (ATRA) in advanced adenoid cystic carcinoma. Oral oncology. 119:105366. Pubmed: 34091189 DOI:S1368-8375(21)00189-5 ArrayCopyright © 2021 Elsevier Ltd. All rights reserved. -
Patton EE, Zon LI, Langenau DM. 2021. Zebrafish disease models in drug discovery: from preclinical modelling to clinical trials. Nature reviews. Drug discovery. 20(8):611-628. Pubmed: 34117457 DOI:10.1038/s41573-021-00210-8 Patton EE, Zon LI, Langenau DM. 2021. Zebrafish disease models in drug discovery: from preclinical modelling to clinical trials. Nature reviews. Drug discovery. 20(8):611-628. Pubmed: 34117457 DOI:10.1038/s41573-021-00210-8 Numerous drug treatments that have recently entered the clinic or clinical trials have their genesis in zebrafish. Zebrafish are well established for their contribution to developmental biology and have now emerged as a powerful preclinical model for human disease, as their disease characteristics, aetiology and progression, and molecular mechanisms are clinically relevant and highly conserved. Zebrafish respond to small molecules and drug treatments at physiologically relevant dose ranges and, when combined with cell-specific or tissue-specific reporters and gene editing technologies, drug activity can be studied at single-cell resolution within the complexity of a whole animal, across tissues and over an extended timescale. These features enable high-throughput and high-content phenotypic drug screening, repurposing of available drugs for personalized and compassionate use, and even the development of new drug classes. Often, drugs and drug leads explored in zebrafish have an inter-organ mechanism of action and would otherwise not be identified through targeted screening approaches. Here, we discuss how zebrafish is an important model for drug discovery, the process of how these discoveries emerge and future opportunities for maximizing zebrafish potential in medical discoveries.© 2021. Springer Nature Limited. 2020
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Zhang B, Ma S, Rachmin I, He M, Baral P, Choi S, Gonçalves WA, Shwartz Y, Fast EM, Su Y, Zon LI, Regev A, Buenrostro JD, Cunha TM, Chiu IM, Fisher DE, Hsu YC. 2020. Hyperactivation of sympathetic nerves drives depletion of melanocyte stem cells. Nature. 577(7792):676-681. Pubmed: 31969699 DOI:10.1038/s41586-020-1935-3 Zhang B, Ma S, Rachmin I, He M, Baral P, Choi S, Gonçalves WA, Shwartz Y, Fast EM, Su Y, Zon LI, Regev A, Buenrostro JD, Cunha TM, Chiu IM, Fisher DE, Hsu YC. 2020. Hyperactivation of sympathetic nerves drives depletion of melanocyte stem cells. Nature. 577(7792):676-681. Pubmed: 31969699 DOI:10.1038/s41586-020-1935-3 Empirical and anecdotal evidence has associated stress with accelerated hair greying (formation of unpigmented hairs), but so far there has been little scientific validation of this link. Here we report that, in mice, acute stress leads to hair greying through the fast depletion of melanocyte stem cells. Using a combination of adrenalectomy, denervation, chemogenetics, cell ablation and knockout of the adrenergic receptor specifically in melanocyte stem cells, we find that the stress-induced loss of melanocyte stem cells is independent of immune attack or adrenal stress hormones. Instead, hair greying results from activation of the sympathetic nerves that innervate the melanocyte stem-cell niche. Under conditions of stress, the activation of these sympathetic nerves leads to burst release of the neurotransmitter noradrenaline (also known as norepinephrine). This causes quiescent melanocyte stem cells to proliferate rapidly, and is followed by their differentiation, migration and permanent depletion from the niche. Transient suppression of the proliferation of melanocyte stem cells prevents stress-induced hair greying. Our study demonstrates that neuronal activity that is induced by acute stress can drive a rapid and permanent loss of somatic stem cells, and illustrates an example in which the maintenance of somatic stem cells is directly influenced by the overall physiological state of the organism. -
Santoriello C, Sporrij A, Yang S, Flynn RA, Henriques T, Dorjsuren B, Custo Greig E, McCall W, Stanhope ME, Fazio M, Superdock M, Lichtig A, Adatto I, Abraham BJ, Kalocsay M, Jurynec M, Zhou Y, Adelman K, Calo E, Zon LI. 2020. RNA helicase DDX21 mediates nucleotide stress responses in neural crest and melanoma cells. Nature cell biology. 22(4):372-379. Pubmed: 32231306 DOI:10.1038/s41556-020-0493-0 Santoriello C, Sporrij A, Yang S, Flynn RA, Henriques T, Dorjsuren B, Custo Greig E, McCall W, Stanhope ME, Fazio M, Superdock M, Lichtig A, Adatto I, Abraham BJ, Kalocsay M, Jurynec M, Zhou Y, Adelman K, Calo E, Zon LI. 2020. RNA helicase DDX21 mediates nucleotide stress responses in neural crest and melanoma cells. Nature cell biology. 22(4):372-379. Pubmed: 32231306 DOI:10.1038/s41556-020-0493-0 The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(aln) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress. -
Kato S, Weng QY, Insco ML, Chen KY, Muralidhar S, Pozniak J, Diaz JMS, Drier Y, Nguyen N, Lo JA, van Rooijen E, Kemeny LV, Zhan Y, Feng Y, Silkworth W, Powell CT, Liau BB, Xiong Y, Jin J, Newton-Bishop J, Zon LI, Bernstein BE, Fisher DE. 2020. Gain-of-Function Genetic Alterations of G9a Drive Oncogenesis. Cancer discovery. 10(7):980-997. Pubmed: 32269030 DOI:10.1158/2159-8290.CD-19-0532 Kato S, Weng QY, Insco ML, Chen KY, Muralidhar S, Pozniak J, Diaz JMS, Drier Y, Nguyen N, Lo JA, van Rooijen E, Kemeny LV, Zhan Y, Feng Y, Silkworth W, Powell CT, Liau BB, Xiong Y, Jin J, Newton-Bishop J, Zon LI, Bernstein BE, Fisher DE. 2020. Gain-of-Function Genetic Alterations of G9a Drive Oncogenesis. Cancer discovery. 10(7):980-997. Pubmed: 32269030 DOI:10.1158/2159-8290.CD-19-0532 Epigenetic regulators, when genomically altered, may become driver oncogenes that mediate otherwise unexplained pro-oncogenic changes lacking a clear genetic stimulus, such as activation of the WNT/β-catenin pathway in melanoma. This study identifies previously unrecognized recurrent activating mutations in the G9a histone methyltransferase gene, as well as G9a genomic copy gains in approximately 26% of human melanomas, which collectively drive tumor growth and an immunologically sterile microenvironment beyond melanoma. Furthermore, the WNT pathway is identified as a key tumorigenic target of G9a gain-of-function, via suppression of the WNT antagonist DKK1. Importantly, genetic or pharmacologic suppression of mutated or amplified G9a using multiple and models demonstrates that G9a is a druggable target for therapeutic intervention in melanoma and other cancers harboring G9a genomic aberrations. SIGNIFICANCE: Oncogenic G9a abnormalities drive tumorigenesis and the "cold" immune microenvironment by activating WNT signaling through DKK1 repression. These results reveal a key druggable mechanism for tumor development and identify strategies to restore "hot" tumor immune microenvironments..©2020 American Association for Cancer Research. -
Fazio M, Ablain J, Chuan Y, Langenau DM, Zon LI. 2020. Zebrafish patient avatars in cancer biology and precision cancer therapy. Nature reviews. Cancer. 20(5):263-273. Pubmed: 32251397 DOI:10.1038/s41568-020-0252-3 Fazio M, Ablain J, Chuan Y, Langenau DM, Zon LI. 2020. Zebrafish patient avatars in cancer biology and precision cancer therapy. Nature reviews. Cancer. 20(5):263-273. Pubmed: 32251397 DOI:10.1038/s41568-020-0252-3 In precision oncology, two major strategies are being pursued for predicting clinically relevant tumour behaviours, such as treatment response and emergence of drug resistance: inference based on genomic, transcriptomic, epigenomic and/or proteomic analysis of patient samples, and phenotypic assays in personalized cancer avatars. The latter approach has historically relied on in vivo mouse xenografts and in vitro organoids or 2D cell cultures. Recent progress in rapid combinatorial genetic modelling, the development of a genetically immunocompromised strain for xenotransplantation of human patient samples in adult zebrafish and the first clinical trial using xenotransplantation in zebrafish larvae for phenotypic testing of drug response bring this tiny vertebrate to the forefront of the precision medicine arena. In this Review, we discuss advances in transgenic and transplantation-based zebrafish cancer avatars, and how these models compare with and complement mouse xenografts and human organoids. We also outline the unique opportunities that these different models present for prediction studies and current challenges they face for future clinical deployment. -
Chen MJ, Lummertz da Rocha E, Cahan P, Kubaczka C, Hunter P, Sousa P, Mullin NK, Fujiwara Y, Nguyen M, Tan Y, Landry S, Han A, Yang S, Lu YF, Jha DK, Vo LT, Zhou Y, North TE, Zon LI, Daley GQ, Schlaeger TM. 2020. Transcriptome Dynamics of Hematopoietic Stem Cell Formation Revealed Using a Combinatorial Runx1 and Ly6a Reporter System. Stem cell reports. 14(5):956-971. Pubmed: 32302558 DOI:S2213-6711(20)30110-7 Chen MJ, Lummertz da Rocha E, Cahan P, Kubaczka C, Hunter P, Sousa P, Mullin NK, Fujiwara Y, Nguyen M, Tan Y, Landry S, Han A, Yang S, Lu YF, Jha DK, Vo LT, Zhou Y, North TE, Zon LI, Daley GQ, Schlaeger TM. 2020. Transcriptome Dynamics of Hematopoietic Stem Cell Formation Revealed Using a Combinatorial Runx1 and Ly6a Reporter System. Stem cell reports. 14(5):956-971. Pubmed: 32302558 DOI:S2213-6711(20)30110-7 Studies of hematopoietic stem cell (HSC) development from pre-HSC-producing hemogenic endothelial cells (HECs) are hampered by the rarity of these cells and the presence of other cell types with overlapping marker expression profiles. We generated a Tg(Runx1-mKO2; Ly6a-GFP) dual reporter mouse to visualize hematopoietic commitment and study pre-HSC emergence and maturation. Runx1-mKO2 marked all intra-arterial HECs and hematopoietic cluster cells (HCCs), including pre-HSCs, myeloid- and lymphoid progenitors, and HSCs themselves. However, HSC and lymphoid potential were almost exclusively found in reporter double-positive (DP) cells. Robust HSC activity was first detected in DP cells of the placenta, reflecting the importance of this niche for (pre-)HSC maturation and expansion before the fetal liver stage. A time course analysis by single-cell RNA sequencing revealed that as pre-HSCs mature into fetal liver stage HSCs, they show signs of interferon exposure, exhibit signatures of multi-lineage differentiation gene expression, and develop a prolonged cell cycle reminiscent of quiescent adult HSCs.Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved. -
Miyata M, Gillemans N, Hockman D, Demmers JAA, Cheng JF, Hou J, Salminen M, Fisher CA, Taylor S, Gibbons RJ, Ganis JJ, Zon LI, Grosveld F, Mulugeta E, Sauka-Spengler T, Higgs DR, Philipsen S. 2020. An evolutionarily ancient mechanism for regulation of hemoglobin expression in vertebrate red cells. Blood. 136(3):269-278. Pubmed: 32396940 DOI:10.1182/blood.2020004826 Miyata M, Gillemans N, Hockman D, Demmers JAA, Cheng JF, Hou J, Salminen M, Fisher CA, Taylor S, Gibbons RJ, Ganis JJ, Zon LI, Grosveld F, Mulugeta E, Sauka-Spengler T, Higgs DR, Philipsen S. 2020. An evolutionarily ancient mechanism for regulation of hemoglobin expression in vertebrate red cells. Blood. 136(3):269-278. Pubmed: 32396940 DOI:10.1182/blood.2020004826 The oxygen transport function of hemoglobin (HB) is thought to have arisen ∼500 million years ago, roughly coinciding with the divergence between jawless (Agnatha) and jawed (Gnathostomata) vertebrates. Intriguingly, extant HBs of jawless and jawed vertebrates were shown to have evolved twice, and independently, from different ancestral globin proteins. This raises the question of whether erythroid-specific expression of HB also evolved twice independently. In all jawed vertebrates studied to date, one of the HB gene clusters is linked to the widely expressed NPRL3 gene. Here we show that the nprl3-linked hb locus of a jawless vertebrate, the river lamprey (Lampetra fluviatilis), shares a range of structural and functional properties with the equivalent jawed vertebrate HB locus. Functional analysis demonstrates that an erythroid-specific enhancer is located in intron 7 of lamprey nprl3, which corresponds to the NPRL3 intron 7 MCS-R1 enhancer of jawed vertebrates. Collectively, our findings signify the presence of an nprl3-linked multiglobin gene locus, which contains a remote enhancer that drives globin expression in erythroid cells, before the divergence of jawless and jawed vertebrates. Different globin genes from this ancestral cluster evolved in the current NPRL3-linked HB genes in jawless and jawed vertebrates. This provides an explanation of the enigma of how, in different species, globin genes linked to the same adjacent gene could undergo convergent evolution.© 2020 by The American Society of Hematology. -
Choi J, Zhang T, Vu A, Ablain J, Makowski MM, Colli LM, Xu M, Hennessey RC, Yin J, Rothschild H, Gräwe C, Kovacs MA, Funderburk KM, Brossard M, Taylor J, Pasaniuc B, Chari R, Chanock SJ, Hoggart CJ, Demenais F, Barrett JH, Law MH, Iles MM, Yu K, Vermeulen M, Zon LI, Brown KM. 2020. Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma. Nature communications. 11(1):2718. Pubmed: 32483191 DOI:10.1038/s41467-020-16590-1 Choi J, Zhang T, Vu A, Ablain J, Makowski MM, Colli LM, Xu M, Hennessey RC, Yin J, Rothschild H, Gräwe C, Kovacs MA, Funderburk KM, Brossard M, Taylor J, Pasaniuc B, Chari R, Chanock SJ, Hoggart CJ, Demenais F, Barrett JH, Law MH, Iles MM, Yu K, Vermeulen M, Zon LI, Brown KM. 2020. Massively parallel reporter assays of melanoma risk variants identify MX2 as a gene promoting melanoma. Nature communications. 11(1):2718. Pubmed: 32483191 DOI:10.1038/s41467-020-16590-1 Genome-wide association studies (GWAS) have identified ~20 melanoma susceptibility loci, most of which are not functionally characterized. Here we report an approach integrating massively-parallel reporter assays (MPRA) with cell-type-specific epigenome and expression quantitative trait loci (eQTL) to identify susceptibility genes/variants from multiple GWAS loci. From 832 high-LD variants, we identify 39 candidate functional variants from 14 loci displaying allelic transcriptional activity, a subset of which corroborates four colocalizing melanocyte cis-eQTL genes. Among these, we further characterize the locus encompassing the HIV-1 restriction gene, MX2 (Chr21q22.3), and validate a functional intronic variant, rs398206. rs398206 mediates the binding of the transcription factor, YY1, to increase MX2 levels, consistent with the cis-eQTL of MX2 in primary human melanocytes. Melanocyte-specific expression of human MX2 in a zebrafish model demonstrates accelerated melanoma formation in a BRAF background. Our integrative approach streamlines GWAS follow-up studies and highlights a pleiotropic function of MX2 in melanoma susceptibility. -
Harris JM, Wang AY, Boulanger-Weill J, Santoriello C, Foianini S, Lichtman JW, Zon LI, Arlotta P. 2020. Long-Range Optogenetic Control of Axon Guidance Overcomes Developmental Boundaries and Defects. Developmental cell. 53(5):577-588.e7. Pubmed: 32516597 DOI:S1534-5807(20)30398-1 Harris JM, Wang AY, Boulanger-Weill J, Santoriello C, Foianini S, Lichtman JW, Zon LI, Arlotta P. 2020. Long-Range Optogenetic Control of Axon Guidance Overcomes Developmental Boundaries and Defects. Developmental cell. 53(5):577-588.e7. Pubmed: 32516597 DOI:S1534-5807(20)30398-1 Axons connect neurons together, establishing the wiring architecture of neuronal networks. Axonal connectivity is largely built during embryonic development through highly constrained processes of axon guidance, which have been extensively studied. However, the inability to control axon guidance, and thus neuronal network architecture, has limited investigation of how axonal connections influence subsequent development and function of neuronal networks. Here, we use zebrafish motor neurons expressing a photoactivatable Rac1 to co-opt endogenous growth cone guidance machinery to precisely and non-invasively direct axon growth using light. Axons can be guided over large distances, within complex environments of living organisms, overriding competing endogenous signals and redirecting axons across potent repulsive barriers to construct novel circuitry. Notably, genetic axon guidance defects can be rescued, restoring functional connectivity. These data demonstrate that intrinsic growth cone guidance machinery can be co-opted to non-invasively build new connectivity, allowing investigation of neural network dynamics in intact living organisms.Copyright © 2020 Elsevier Inc. All rights reserved. -
Johansson JA, Marie KL, Lu Y, Brombin A, Santoriello C, Zeng Z, Zich J, Gautier P, von Kriegsheim A, Brunsdon H, Wheeler AP, Dreger M, Houston DR, Dooley CM, Sims AH, Busch-Nentwich EM, Zon LI, Illingworth RS, Patton EE. 2020. PRL3-DDX21 Transcriptional Control of Endolysosomal Genes Restricts Melanocyte Stem Cell Differentiation. Developmental cell. 54(3):317-332.e9. Pubmed: 32652076 DOI:S1534-5807(20)30463-9 Johansson JA, Marie KL, Lu Y, Brombin A, Santoriello C, Zeng Z, Zich J, Gautier P, von Kriegsheim A, Brunsdon H, Wheeler AP, Dreger M, Houston DR, Dooley CM, Sims AH, Busch-Nentwich EM, Zon LI, Illingworth RS, Patton EE. 2020. PRL3-DDX21 Transcriptional Control of Endolysosomal Genes Restricts Melanocyte Stem Cell Differentiation. Developmental cell. 54(3):317-332.e9. Pubmed: 32652076 DOI:S1534-5807(20)30463-9 Melanocytes, replenished throughout life by melanocyte stem cells (MSCs), play a critical role in pigmentation and melanoma. Here, we reveal a function for the metastasis-associated phosphatase of regenerating liver 3 (PRL3) in MSC regeneration. We show that PRL3 binds to the RNA helicase DDX21, thereby restricting productive transcription by RNAPII at master transcription factor (MITF)-regulated endolysosomal vesicle genes. In zebrafish, this mechanism controls premature melanoblast expansion and differentiation from MSCs. In melanoma patients, restricted transcription of this endolysosomal vesicle pathway is a hallmark of PRL3-high melanomas. Our work presents the conceptual advance that PRL3-mediated control of transcriptional elongation is a differentiation checkpoint mechanism for activated MSCs and has clinical relevance for the activity of PRL3 in regenerating tissue and cancer.Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved. -
Minnoye L, Taskiran II, Mauduit D, Fazio M, Van Aerschot L, Hulselmans G, Christiaens V, Makhzami S, Seltenhammer M, Karras P, Primot A, Cadieu E, van Rooijen E, Marine JC, Egidy G, Ghanem GE, Zon L, Wouters J, Aerts S. 2020. Cross-species analysis of enhancer logic using deep learning. Genome research. 30(12):1815-1834. Pubmed: 32732264 DOI:10.1101/gr.260844.120 Minnoye L, Taskiran II, Mauduit D, Fazio M, Van Aerschot L, Hulselmans G, Christiaens V, Makhzami S, Seltenhammer M, Karras P, Primot A, Cadieu E, van Rooijen E, Marine JC, Egidy G, Ghanem GE, Zon L, Wouters J, Aerts S. 2020. Cross-species analysis of enhancer logic using deep learning. Genome research. 30(12):1815-1834. Pubmed: 32732264 DOI:10.1101/gr.260844.120 Deciphering the genomic regulatory code of enhancers is a key challenge in biology because this code underlies cellular identity. A better understanding of how enhancers work will improve the interpretation of noncoding genome variation and empower the generation of cell type-specific drivers for gene therapy. Here, we explore the combination of deep learning and cross-species chromatin accessibility profiling to build explainable enhancer models. We apply this strategy to decipher the enhancer code in melanoma, a relevant case study owing to the presence of distinct melanoma cell states. We trained and validated a deep learning model, called DeepMEL, using chromatin accessibility data of 26 melanoma samples across six different species. We show the accuracy of DeepMEL predictions on the CAGI5 challenge, where it significantly outperforms existing models on the melanoma enhancer of Next, we exploit DeepMEL to analyze enhancer architectures and identify accurate transcription factor binding sites for the core regulatory complexes in the two different melanoma states, with distinct roles for each transcription factor, in terms of nucleosome displacement or enhancer activation. Finally, DeepMEL identifies orthologous enhancers across distantly related species, where sequence alignment fails, and the model highlights specific nucleotide substitutions that underlie enhancer turnover. DeepMEL can be used from the Kipoi database to predict and optimize candidate enhancers and to prioritize enhancer mutations. In addition, our computational strategy can be applied to other cancer or normal cell types.© 2020 Minnoye et al.; Published by Cold Spring Harbor Laboratory Press. -
Wattrus SJ, Zon LI. 2020. A Transgenic System for Rapid Magnetic Enrichment of Rare Embryonic Cells. Zebrafish. 17(5):354-357. Pubmed: 32749922 DOI:10.1089/zeb.2020.1904 Wattrus SJ, Zon LI. 2020. A Transgenic System for Rapid Magnetic Enrichment of Rare Embryonic Cells. Zebrafish. 17(5):354-357. Pubmed: 32749922 DOI:10.1089/zeb.2020.1904 Collecting large numbers of rare cells for high-throughput molecular analysis remains a technical challenge, primarily due to limitations in existing technologies. In developmental biology this has impeded single-cell analysis of primordial organs, which derive from few cells. In this study, we share novel transgenic lines for rapid cell enrichment from zebrafish embryos using human surface antigens for immunological binding and magnetic sorting. As proof of principle, we tagged, enriched, and performed single-cell RNA sequencing on nascent hematopoietic stem/progenitor cells and endothelial cells from early embryos. Our method is a quick, efficient, and cost-effective approach to a previously intractable problem. -
Hsu J, Huang HT, Lee CT, Choudhuri A, Wilson NK, Abraham BJ, Moignard V, Kucinski I, Yu S, Hyde RK, Tober J, Cai X, Li Y, Guo Y, Yang S, Superdock M, Trompouki E, Calero-Nieto FJ, Ghamari A, Jiang J, Gao P, Gao L, Nguyen V, Robertson AL, Durand EM, Kathrein KL, Aifantis I, Gerber SA, Tong W, Tan K, Cantor AB, Zhou Y, Liu PP, Young RA, Göttgens B, Speck NA, Zon LI. 2020. CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation. Proceedings of the National Academy of Sciences of the United States of America. 117(38):23626-23635. Pubmed: 32883883 DOI:10.1073/pnas.2003228117 Hsu J, Huang HT, Lee CT, Choudhuri A, Wilson NK, Abraham BJ, Moignard V, Kucinski I, Yu S, Hyde RK, Tober J, Cai X, Li Y, Guo Y, Yang S, Superdock M, Trompouki E, Calero-Nieto FJ, Ghamari A, Jiang J, Gao P, Gao L, Nguyen V, Robertson AL, Durand EM, Kathrein KL, Aifantis I, Gerber SA, Tong W, Tan K, Cantor AB, Zhou Y, Liu PP, Young RA, Göttgens B, Speck NA, Zon LI. 2020. CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation. Proceedings of the National Academy of Sciences of the United States of America. 117(38):23626-23635. Pubmed: 32883883 DOI:10.1073/pnas.2003228117 Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation. -
Taylor AM, Macari ER, Chan IT, Blair MC, Doulatov S, Vo LT, Raiser DM, Siva K, Basak A, Pirouz M, Shah AN, McGrath K, Humphries JM, Stillman E, Alter BP, Calo E, Gregory RI, Sankaran VG, Flygare J, Ebert BL, Zhou Y, Daley GQ, Zon LI. 2020. Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia. Science translational medicine. 12(566). Pubmed: 33087503 DOI:10.1126/scitranslmed.abb5831 Taylor AM, Macari ER, Chan IT, Blair MC, Doulatov S, Vo LT, Raiser DM, Siva K, Basak A, Pirouz M, Shah AN, McGrath K, Humphries JM, Stillman E, Alter BP, Calo E, Gregory RI, Sankaran VG, Flygare J, Ebert BL, Zhou Y, Daley GQ, Zon LI. 2020. Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia. Science translational medicine. 12(566). Pubmed: 33087503 DOI:10.1126/scitranslmed.abb5831 Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 () mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34 hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34 cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. -
Zon L, Gomes AP, Cance WG, Ribas A, Tuveson D, Postel-Vinay S, Massard C, Barlési F. 2020. Impact of COVID-19 Pandemic on Cancer Research. Cancer cell. 38(5):591-593. Pubmed: 33086030 DOI:S1535-6108(20)30538-9 Zon L, Gomes AP, Cance WG, Ribas A, Tuveson D, Postel-Vinay S, Massard C, Barlési F. 2020. Impact of COVID-19 Pandemic on Cancer Research. Cancer cell. 38(5):591-593. Pubmed: 33086030 DOI:S1535-6108(20)30538-9 The COVID-19 pandemic is profoundly changing cancer researchers and cancer research. Leaders from different fields and at different career stages share their perspectives.Copyright © 2020 Elsevier Inc. All rights reserved. -
Choudhuri A, Trompouki E, Abraham BJ, Colli LM, Kock KH, Mallard W, Yang ML, Vinjamur DS, Ghamari A, Sporrij A, Hoi K, Hummel B, Boatman S, Chan V, Tseng S, Nandakumar SK, Yang S, Lichtig A, Superdock M, Grimes SN, Bowman TV, Zhou Y, Takahashi S, Joehanes R, Cantor AB, Bauer DE, Ganesh SK, Rinn J, Albert PS, Bulyk ML, Chanock SJ, Young RA, Zon LI. 2020. Common variants in signaling transcription-factor-binding sites drive phenotypic variability in red blood cell traits. Nature genetics. 52(12):1333-1345. Pubmed: 33230299 DOI:10.1038/s41588-020-00738-2 Choudhuri A, Trompouki E, Abraham BJ, Colli LM, Kock KH, Mallard W, Yang ML, Vinjamur DS, Ghamari A, Sporrij A, Hoi K, Hummel B, Boatman S, Chan V, Tseng S, Nandakumar SK, Yang S, Lichtig A, Superdock M, Grimes SN, Bowman TV, Zhou Y, Takahashi S, Joehanes R, Cantor AB, Bauer DE, Ganesh SK, Rinn J, Albert PS, Bulyk ML, Chanock SJ, Young RA, Zon LI. 2020. Common variants in signaling transcription-factor-binding sites drive phenotypic variability in red blood cell traits. Nature genetics. 52(12):1333-1345. Pubmed: 33230299 DOI:10.1038/s41588-020-00738-2 Genome-wide association studies identify genomic variants associated with human traits and diseases. Most trait-associated variants are located within cell-type-specific enhancers, but the molecular mechanisms governing phenotypic variation are less well understood. Here, we show that many enhancer variants associated with red blood cell (RBC) traits map to enhancers that are co-bound by lineage-specific master transcription factors (MTFs) and signaling transcription factors (STFs) responsive to extracellular signals. The majority of enhancer variants reside on STF and not MTF motifs, perturbing DNA binding by various STFs (BMP/TGF-β-directed SMADs or WNT-induced TCFs) and affecting target gene expression. Analyses of engineered human blood cells and expression quantitative trait loci verify that disrupted STF binding leads to altered gene expression. Our results propose that the majority of the RBC-trait-associated variants that reside on transcription-factor-binding sequences fall in STF target sequences, suggesting that the phenotypic variation of RBC traits could stem from altered responsiveness to extracellular stimuli. -
Justice MJ, Hmeljak J, Sankaran VG, Socolovsky M, Zon LI. 2020. From blood development to disease: a paradigm for clinical translation. Disease models & mechanisms. 13(1). Pubmed: 31836582 DOI:10.1242/dmm.043661 Justice MJ, Hmeljak J, Sankaran VG, Socolovsky M, Zon LI. 2020. From blood development to disease: a paradigm for clinical translation. Disease models & mechanisms. 13(1). Pubmed: 31836582 DOI:10.1242/dmm.043661 Translating basic research to the clinic is a primary aim of Disease Models & Mechanisms, and the recent successes in hematopoiesis research provide a blueprint of how fundamental biological research can provide solutions to important clinical problems. These advances were the main motivation for choosing hematopoiesis disorders as the focus of our inaugural meeting, 'Blood Disorders: Models, Mechanisms and Therapies', which was held in early October 2019. This Editorial discusses the reasons for and the challenges of interdisciplinary research in hematopoiesis, provides examples of how research in model systems is a key translational step towards effective treatments for blood disorders and summarizes what the community believes are the key exciting developments and challenges in this field.© 2020. Published by The Company of Biologists Ltd. -
Cassar S, Adatto I, Freeman JL, Gamse JT, Iturria I, Lawrence C, Muriana A, Peterson RT, Van Cruchten S, Zon LI. 2020. Use of Zebrafish in Drug Discovery Toxicology. Chemical research in toxicology. 33(1):95-118. Pubmed: 31625720 DOI:10.1021/acs.chemrestox.9b00335 Cassar S, Adatto I, Freeman JL, Gamse JT, Iturria I, Lawrence C, Muriana A, Peterson RT, Van Cruchten S, Zon LI. 2020. Use of Zebrafish in Drug Discovery Toxicology. Chemical research in toxicology. 33(1):95-118. Pubmed: 31625720 DOI:10.1021/acs.chemrestox.9b00335 Unpredicted human safety events in clinical trials for new drugs are costly in terms of human health and money. The drug discovery industry attempts to minimize those events with diligent preclinical safety testing. Current standard practices are good at preventing toxic compounds from being tested in the clinic; however, false negative preclinical toxicity results are still a reality. Continual improvement must be pursued in the preclinical realm. Higher-quality therapies can be brought forward with more information about potential toxicities and associated mechanisms. The zebrafish model is a bridge between in vitro assays and mammalian in vivo studies. This model is powerful in its breadth of application and tractability for research. In the past two decades, our understanding of disease biology and drug toxicity has grown significantly owing to thousands of studies on this tiny vertebrate. This Review summarizes challenges and strengths of the model, discusses the 3Rs value that it can deliver, highlights translatable and untranslatable biology, and brings together reports from recent studies with zebrafish focusing on new drug discovery toxicology. -
Makarova N, Kalaparthi V, Wang A, Williams C, Dokukin ME, Kaufman CK, Zon L, Sokolov I. 2020. Difference in biophysical properties of cancer-initiating cells in melanoma mutated zebrafish. Journal of the mechanical behavior of biomedical materials. 107:103746. Pubmed: 32364948 DOI:S1751-6161(20)30300-3 Makarova N, Kalaparthi V, Wang A, Williams C, Dokukin ME, Kaufman CK, Zon L, Sokolov I. 2020. Difference in biophysical properties of cancer-initiating cells in melanoma mutated zebrafish. Journal of the mechanical behavior of biomedical materials. 107:103746. Pubmed: 32364948 DOI:S1751-6161(20)30300-3 Despite sharing oncogenetic mutations, only a small number of cells within a given tissue will undergo malignant transformation. Biochemical and physical factors responsible for this cancer-initiation process are not well understood. Here we study biophysical differences of pre-melanoma and melanoma cells in a BRAF/P53 zebrafish model. The AFM indentation technique was used to study the cancer-initiating cells while the surrounding melanocytes were the control. We observed a statistically significant decrease in the modulus of elasticity (the effective Young's modulus) of cancer-initiating cells compared to the surrounding melanocytes. No significant differences in the pericellular coat surrounding cells were observed. These results contribute to a better understanding of the factors responsible for the initiation of cancer.Copyright © 2020 Elsevier Ltd. All rights reserved. 2019
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Jurynec MJ, Bai X, Bisgrove BW, Jackson H, Nechiporuk A, Palu RAS, Grunwald HA, Su YC, Hoshijima K, Yost HJ, Zon LI, Grunwald DJ. 2019. The Paf1 complex and P-TEFb have reciprocal and antagonist roles in maintaining multipotent neural crest progenitors. Development (Cambridge, England). 146(24). Pubmed: 31784460 DOI:10.1242/dev.180133 Jurynec MJ, Bai X, Bisgrove BW, Jackson H, Nechiporuk A, Palu RAS, Grunwald HA, Su YC, Hoshijima K, Yost HJ, Zon LI, Grunwald DJ. 2019. The Paf1 complex and P-TEFb have reciprocal and antagonist roles in maintaining multipotent neural crest progenitors. Development (Cambridge, England). 146(24). Pubmed: 31784460 DOI:10.1242/dev.180133 Multipotent progenitor populations are necessary for generating diverse tissue types during embryogenesis. We show the RNA polymerase-associated factor 1 complex (Paf1C) is required to maintain multipotent progenitors of the neural crest (NC) lineage in zebrafish. Mutations affecting each Paf1C component result in near-identical NC phenotypes; mutant embryos carrying a null mutation in were analyzed in detail. In the absence of zygotic function, definitive premigratory NC progenitors arise but fail to maintain expression of the specification gene. The mutant NC progenitors migrate aberrantly and fail to differentiate appropriately. Blood and germ cell progenitor development is affected similarly. Development of mutant NC could be rescued by additional loss of positive transcription elongation factor b (P-TEFb) activity, a key factor in promoting transcription elongation. Consistent with the interpretation that inhibiting/delaying expression of some genes is essential for maintaining progenitors, mutant embryos lacking the CDK9 kinase component of P-TEFb exhibit a surfeit of NC progenitors and their derivatives. We propose Paf1C and P-TEFb act antagonistically to regulate the timing of the expression of genes needed for NC development.© 2019. Published by The Company of Biologists Ltd. -
Li P, Lahvic JL, Binder V, Pugach EK, Riley EB, Tamplin OJ, Panigrahy D, Bowman TV, Barrett FG, Heffner GC, McKinney-Freeman S, Schlaeger TM, Daley GQ, Zeldin DC, Zon LI. 2019. Author Correction: Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment. Nature. 573(7772):E1. Pubmed: 31435017 DOI:10.1038/s41586-019-1489-4 Li P, Lahvic JL, Binder V, Pugach EK, Riley EB, Tamplin OJ, Panigrahy D, Bowman TV, Barrett FG, Heffner GC, McKinney-Freeman S, Schlaeger TM, Daley GQ, Zeldin DC, Zon LI. 2019. Author Correction: Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment. Nature. 573(7772):E1. Pubmed: 31435017 DOI:10.1038/s41586-019-1489-4 An Amendment to this paper has been published and can be accessed via a link at the top of the paper. -
Morad G, Carman CV, Hagedorn EJ, Perlin JR, Zon LI, Mustafaoglu N, Park TE, Ingber DE, Daisy CC, Moses MA. 2019. Tumor-Derived Extracellular Vesicles Breach the Intact Blood-Brain Barrier Transcytosis. ACS nano. 13(12):13853-13865. Pubmed: 31479239 DOI:10.1021/acsnano.9b04397 Morad G, Carman CV, Hagedorn EJ, Perlin JR, Zon LI, Mustafaoglu N, Park TE, Ingber DE, Daisy CC, Moses MA. 2019. Tumor-Derived Extracellular Vesicles Breach the Intact Blood-Brain Barrier Transcytosis. ACS nano. 13(12):13853-13865. Pubmed: 31479239 DOI:10.1021/acsnano.9b04397 The restrictive nature of the blood-brain barrier (BBB) creates a major challenge for brain drug delivery with current nanomedicines lacking the ability to cross the BBB. Extracellular vesicles (EVs) have been shown to contribute to the progression of a variety of brain diseases including metastatic brain cancer and have been suggested as promising therapeutics and drug delivery vehicles. However, the ability of native tumor-derived EVs to breach the BBB and the mechanism(s) involved in this process remain unknown. Here, we demonstrate that tumor-derived EVs can breach the intact BBB , and by using state-of-the-art and models of the BBB, we have identified transcytosis as the mechanism underlying this process. Moreover, high spatiotemporal resolution microscopy demonstrated that the endothelial recycling endocytic pathway is involved in this transcellular transport. We further identify and characterize the mechanism by which tumor-derived EVs circumvent the low physiologic rate of transcytosis in the BBB by decreasing the brain endothelial expression of rab7 and increasing the efficiency of their transport. These findings identify previously unknown mechanisms by which tumor-derived EVs breach an intact BBB during the course of brain metastasis and can be leveraged to guide and inform the development of drug delivery approaches to deliver therapeutic cargoes across the BBB for treatment of a variety of brain diseases including, but not limited to, brain malignancies. -
Gómez-Abenza E, Ibáñez-Molero S, García-Moreno D, Fuentes I, Zon LI, Mione MC, Cayuela ML, Gabellini C, Mulero V. 2019. Zebrafish modeling reveals that SPINT1 regulates the aggressiveness of skin cutaneous melanoma and its crosstalk with tumor immune microenvironment. Journal of experimental & clinical cancer research : CR. 38(1):405. Pubmed: 31519199 DOI:10.1186/s13046-019-1389-3 Gómez-Abenza E, Ibáñez-Molero S, García-Moreno D, Fuentes I, Zon LI, Mione MC, Cayuela ML, Gabellini C, Mulero V. 2019. Zebrafish modeling reveals that SPINT1 regulates the aggressiveness of skin cutaneous melanoma and its crosstalk with tumor immune microenvironment. Journal of experimental & clinical cancer research : CR. 38(1):405. Pubmed: 31519199 DOI:10.1186/s13046-019-1389-3 Array -
Lo WL, Shah NH, Rubin SA, Zhang W, Horkova V, Fallahee IR, Stepanek O, Zon LI, Kuriyan J, Weiss A. 2019. Slow phosphorylation of a tyrosine residue in LAT optimizes T cell ligand discrimination. Nature immunology. 20(11):1481-1493. Pubmed: 31611699 DOI:10.1038/s41590-019-0502-2 Lo WL, Shah NH, Rubin SA, Zhang W, Horkova V, Fallahee IR, Stepanek O, Zon LI, Kuriyan J, Weiss A. 2019. Slow phosphorylation of a tyrosine residue in LAT optimizes T cell ligand discrimination. Nature immunology. 20(11):1481-1493. Pubmed: 31611699 DOI:10.1038/s41590-019-0502-2 Self-non-self discrimination is central to T cell-mediated immunity. The kinetic proofreading model can explain T cell antigen receptor (TCR) ligand discrimination; however, the rate-limiting steps have not been identified. Here, we show that tyrosine phosphorylation of the T cell adapter protein LAT at position Y132 is a critical kinetic bottleneck for ligand discrimination. LAT phosphorylation at Y132, mediated by the kinase ZAP-70, leads to the recruitment and activation of phospholipase C-γ1 (PLC-γ1), an important effector molecule for T cell activation. The slow phosphorylation of Y132, relative to other phosphosites on LAT, is governed by a preceding glycine residue (G131) but can be accelerated by substituting this glycine with aspartate or glutamate. Acceleration of Y132 phosphorylation increases the speed and magnitude of PLC-γ1 activation and enhances T cell sensitivity to weaker stimuli, including weak agonists and self-peptides. These observations suggest that the slow phosphorylation of Y132 acts as a proofreading step to facilitate T cell ligand discrimination. -
Chaturantabut S, Shwartz A, Evason KJ, Cox AG, Labella K, Schepers AG, Yang S, Acuña M, Houvras Y, Mancio-Silva L, Romano S, Gorelick DA, Cohen DE, Zon LI, Bhatia SN, North TE, Goessling W. 2019. Estrogen Activation of G-Protein-Coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-Kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes. Gastroenterology. 156(6):1788-1804.e13. Pubmed: 30641053 DOI:S0016-5085(19)30031-9 Chaturantabut S, Shwartz A, Evason KJ, Cox AG, Labella K, Schepers AG, Yang S, Acuña M, Houvras Y, Mancio-Silva L, Romano S, Gorelick DA, Cohen DE, Zon LI, Bhatia SN, North TE, Goessling W. 2019. Estrogen Activation of G-Protein-Coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-Kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes. Gastroenterology. 156(6):1788-1804.e13. Pubmed: 30641053 DOI:S0016-5085(19)30031-9 ArrayCopyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved. -
Tyrkalska SD, Pérez-Oliva AB, Rodríguez-Ruiz L, Martínez-Morcillo FJ, Alcaraz-Pérez F, Martínez-Navarro FJ, Lachaud C, Ahmed N, Schroeder T, Pardo-Sánchez I, Candel S, López-Muñoz A, Choudhuri A, Rossmann MP, Zon LI, Cayuela ML, García-Moreno D, Mulero V. 2019. Inflammasome Regulates Hematopoiesis through Cleavage of the Master Erythroid Transcription Factor GATA1. Immunity. 51(1):50-63.e5. Pubmed: 31174991 DOI:S1074-7613(19)30225-0 Tyrkalska SD, Pérez-Oliva AB, Rodríguez-Ruiz L, Martínez-Morcillo FJ, Alcaraz-Pérez F, Martínez-Navarro FJ, Lachaud C, Ahmed N, Schroeder T, Pardo-Sánchez I, Candel S, López-Muñoz A, Choudhuri A, Rossmann MP, Zon LI, Cayuela ML, García-Moreno D, Mulero V. 2019. Inflammasome Regulates Hematopoiesis through Cleavage of the Master Erythroid Transcription Factor GATA1. Immunity. 51(1):50-63.e5. Pubmed: 31174991 DOI:S1074-7613(19)30225-0 Chronic inflammatory diseases are associated with altered hematopoiesis that could result in neutrophilia and anemia. Here we report that genetic or chemical manipulation of different inflammasome components altered the differentiation of hematopoietic stem and progenitor cells (HSPC) in zebrafish. Although the inflammasome was dispensable for the emergence of HSPC, it was intrinsically required for their myeloid differentiation. In addition, Gata1 transcript and protein amounts increased in inflammasome-deficient larvae, enforcing erythropoiesis and inhibiting myelopoiesis. This mechanism is evolutionarily conserved, since pharmacological inhibition of the inflammasome altered erythroid differentiation of human erythroleukemic K562 cells. In addition, caspase-1 inhibition rapidly upregulated GATA1 protein in mouse HSPC promoting their erythroid differentiation. Importantly, pharmacological inhibition of the inflammasome rescued zebrafish disease models of neutrophilic inflammation and anemia. These results indicate that the inflammasome plays a major role in the pathogenesis of neutrophilia and anemia of chronic diseases and reveal druggable targets for therapeutic interventions.Copyright © 2019 Elsevier Inc. All rights reserved. -
Zon L. 2019. Improving the visibility of developmental biology: time for induction and specification. Development (Cambridge, England). 146(2). Pubmed: 30651297 DOI:10.1242/dev.174631 Zon L. 2019. Improving the visibility of developmental biology: time for induction and specification. Development (Cambridge, England). 146(2). Pubmed: 30651297 DOI:10.1242/dev.174631 Developmental biology is a prominent field that has captured the imagination of many scientists. Over the years, research in the area has seen a steady number of amazing accomplishments, with peaks in activity following the development and application of new technologies. Although the field continues to flourish and produce excellent work, I have recently noticed difficulty with its perception and visibility. Having joined the developmental biology community during the early 1990s, and contributing since as a stem cell researcher, cancer biologist and an MD, I have a unique perspective on these challenges. Here, I discuss these issues and challenges and offer potential solutions for a field that is very important to me.© 2019. Published by The Company of Biologists Ltd. -
Gu Q, Yang X, Lv J, Zhang J, Xia B, Kim JD, Wang R, Xiong F, Meng S, Clements TP, Tandon B, Wagner DS, Diaz MF, Wenzel PL, Miller YI, Traver D, Cooke JP, Li W, Zon LI, Chen K, Bai Y, Fang L. 2019. AIBP-mediated cholesterol efflux instructs hematopoietic stem and progenitor cell fate. Science (New York, N.Y.). 363(6431):1085-1088. Pubmed: 30705153 DOI:10.1126/science.aav1749 Gu Q, Yang X, Lv J, Zhang J, Xia B, Kim JD, Wang R, Xiong F, Meng S, Clements TP, Tandon B, Wagner DS, Diaz MF, Wenzel PL, Miller YI, Traver D, Cooke JP, Li W, Zon LI, Chen K, Bai Y, Fang L. 2019. AIBP-mediated cholesterol efflux instructs hematopoietic stem and progenitor cell fate. Science (New York, N.Y.). 363(6431):1085-1088. Pubmed: 30705153 DOI:10.1126/science.aav1749 Hypercholesterolemia, the driving force of atherosclerosis, accelerates the expansion and mobilization of hematopoietic stem and progenitor cells (HSPCs). The molecular determinants connecting hypercholesterolemia with hematopoiesis are unclear. Here, we report that a somite-derived prohematopoietic cue, AIBP, orchestrates HSPC emergence from the hemogenic endothelium, a type of specialized endothelium manifesting hematopoietic potential. Mechanistically, AIBP-mediated cholesterol efflux activates endothelial Srebp2, the master transcription factor for cholesterol biosynthesis, which in turn transactivates Notch and promotes HSPC emergence. Srebp2 inhibition impairs hypercholesterolemia-induced HSPC expansion. Srebp2 activation and Notch up-regulation are associated with HSPC expansion in hypercholesterolemic human subjects. Genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq), RNA sequencing (RNA-seq), and assay for transposase-accessible chromatin using sequencing (ATAC-seq) indicate that Srebp2 transregulates Notch pathway genes required for hematopoiesis. Our studies outline an AIBP-regulated Srebp2-dependent paradigm for HSPC emergence in development and HPSC expansion in atherosclerotic cardiovascular disease.Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. -
Cagan RL, Zon LI, White RM. 2019. Modeling Cancer with Flies and Fish. Developmental cell. 49(3):317-324. Pubmed: 31063751 DOI:S1534-5807(19)30283-7 Cagan RL, Zon LI, White RM. 2019. Modeling Cancer with Flies and Fish. Developmental cell. 49(3):317-324. Pubmed: 31063751 DOI:S1534-5807(19)30283-7 Cancer has joined heart disease as the leading source of mortality in the US. In an era of organoids, patient-derived xenografts, and organs on a chip, model organisms continue to thrive with a combination of powerful genetic tools, rapid pace of discovery, and affordability. Model organisms enable the analysis of both the tumor and its associated microenvironment, aspects that are particularly relevant to our understanding of metastasis and drug resistance. In this Perspective, we explore some of the strengths of fruit flies and zebrafish for addressing fundamental cancer questions and how these two organisms can contribute to identifying promising therapeutic candidates.Copyright © 2019 Elsevier Inc. All rights reserved. -
McConnell AM, Mito JK, Ablain J, Dang M, Formichella L, Fisher DE, Zon LI. 2019. Neural crest state activation in NRAS driven melanoma, but not in NRAS-driven melanocyte expansion. Developmental biology. 449(2):107-114. Pubmed: 29883661 DOI:S0012-1606(17)30855-2 McConnell AM, Mito JK, Ablain J, Dang M, Formichella L, Fisher DE, Zon LI. 2019. Neural crest state activation in NRAS driven melanoma, but not in NRAS-driven melanocyte expansion. Developmental biology. 449(2):107-114. Pubmed: 29883661 DOI:S0012-1606(17)30855-2 NRAS mutations are frequently found in many deadly malignancies and are the second most common oncogene driving malignant melanoma. Here, we generate a rapid transient transgenic zebrafish model of NRAS-mutant melanoma. These fish develop extensive melanocytic proliferation in approximately 4 weeks. The majority of these lesions do not engraft upon transplantation and lack overt histologic features of malignancy. Our previous work demonstrated that activation of a neural crest cell transcriptional program is a key initiating event in zebrafish BRAF/p53-driven melanomas using the fluorescent reporter crestin:EGFP. By 8-12 weeks of age, some lesions progress to malignant melanoma and have cytologic atypia, destructive tissue invasion, and express neural crest progenitor markers, including crestin:EGFP. Our studies demonstrate that NRAS induces extensive melanocyte expansion, which arise during zebrafish development and lack a transformed phenotype. These early lesions are highly predisposed to reactivate a neural crest progenitor fate and form malignant melanomas.Copyright © 2018 Elsevier Inc. All rights reserved. 2018
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Rost MS, Shestopalov I, Liu Y, Vo AH, Richter CE, Emly SM, Barrett FG, Stachura DL, Holinstat M, Zon LI, Shavit JA. 2018. Nfe2 is dispensable for early but required for adult thrombocyte formation and function in zebrafish. Blood advances. 2(23):3418-3427. Pubmed: 30504234 DOI:10.1182/bloodadvances.2018021865 Rost MS, Shestopalov I, Liu Y, Vo AH, Richter CE, Emly SM, Barrett FG, Stachura DL, Holinstat M, Zon LI, Shavit JA. 2018. Nfe2 is dispensable for early but required for adult thrombocyte formation and function in zebrafish. Blood advances. 2(23):3418-3427. Pubmed: 30504234 DOI:10.1182/bloodadvances.2018021865 The NFE2 transcription factor is expressed in multiple hematopoietic lineages with a well-defined role in regulating megakaryocyte biogenesis and platelet production in mammals. Mice deficient in NFE2 develop severe thrombocytopenia with lethality resulting from neonatal hemorrhage. Recent data in mammals reveal potential differences in embryonic and adult thrombopoiesis. Multiple studies in zebrafish have revealed mechanistic insights into hematopoiesis, although thrombopoiesis has been less studied. Rather than platelets, zebrafish possess thrombocytes, which are nucleated cells with similar functional properties. Using transcription activator-like effector nucleases to generate mutations in , we show that unlike mammals, zebrafish survive to adulthood in the absence of Nfe2. Despite developing severe thrombocytopenia, homozygous mutants do not display overt hemorrhage or reduced survival. Surprisingly, quantification of circulating thrombocytes in mutant 6-day-old larvae revealed no significant differences from wild-type siblings. Both wild-type and null larvae formed thrombocyte-rich clots in response to endothelial injury. In addition, ex vivo thrombocytic colony formation was intact in mutants, and adult kidney marrow displayed expansion of hematopoietic progenitors. These data suggest that loss of Nfe2 results in a late block in adult thrombopoiesis, with secondary expansion of precursors: features consistent with mammals. Overall, our data suggest parallels with erythropoiesis, including distinct primitive and definitive pathways of development and potential for a previously unknown Nfe2-independent pathway of embryonic thrombopoiesis. Long-term homozygous mutant survival will facilitate in-depth study of Nfe2 deficiency in vivo, and further investigation could lead to alternative methodologies for the enhancement of platelet production.© 2018 by The American Society of Hematology. -
Bennett AH, O'Donohue MF, Gundry SR, Chan AT, Widrick J, Draper I, Chakraborty A, Zhou Y, Zon LI, Gleizes PE, Beggs AH, Gupta VA. 2018. RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes. PLoS genetics. 14(3):e1007226. Pubmed: 29518074 DOI:10.1371/journal.pgen.1007226 Bennett AH, O'Donohue MF, Gundry SR, Chan AT, Widrick J, Draper I, Chakraborty A, Zhou Y, Zon LI, Gleizes PE, Beggs AH, Gupta VA. 2018. RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes. PLoS genetics. 14(3):e1007226. Pubmed: 29518074 DOI:10.1371/journal.pgen.1007226 Gene expression in a tissue-specific context depends on the combined efforts of epigenetic, transcriptional and post-transcriptional processes that lead to the production of specific proteins that are important determinants of cellular identity. Ribosomes are a central component of the protein biosynthesis machinery in cells; however, their regulatory roles in the translational control of gene expression in skeletal muscle remain to be defined. In a genetic screen to identify critical regulators of myogenesis, we identified a DEAD-Box RNA helicase, DDX27, that is required for skeletal muscle growth and regeneration. We demonstrate that DDX27 regulates ribosomal RNA (rRNA) maturation, and thereby the ribosome biogenesis and the translation of specific transcripts during myogenesis. These findings provide insight into the translational regulation of gene expression in myogenesis and suggest novel functions for ribosomes in regulating gene expression in skeletal muscles. -
Yu Y, Schleich K, Yue B, Ji S, Lohneis P, Kemper K, Silvis MR, Qutob N, van Rooijen E, Werner-Klein M, Li L, Dhawan D, Meierjohann S, Reimann M, Elkahloun A, Treitschke S, Dörken B, Speck C, Mallette FA, Zon LI, Holmen SL, Peeper DS, Samuels Y, Schmitt CA, Lee S. 2018. Targeting the Senescence-Overriding Cooperative Activity of Structurally Unrelated H3K9 Demethylases in Melanoma. Cancer cell. 33(4):785. Pubmed: 29634951 DOI:S1535-6108(18)30113-2 Yu Y, Schleich K, Yue B, Ji S, Lohneis P, Kemper K, Silvis MR, Qutob N, van Rooijen E, Werner-Klein M, Li L, Dhawan D, Meierjohann S, Reimann M, Elkahloun A, Treitschke S, Dörken B, Speck C, Mallette FA, Zon LI, Holmen SL, Peeper DS, Samuels Y, Schmitt CA, Lee S. 2018. Targeting the Senescence-Overriding Cooperative Activity of Structurally Unrelated H3K9 Demethylases in Melanoma. Cancer cell. 33(4):785. Pubmed: 29634951 DOI:S1535-6108(18)30113-2 -
Uenishi GI, Jung HS, Kumar A, Park MA, Hadland BK, McLeod E, Raymond M, Moskvin O, Zimmerman CE, Theisen DJ, Swanson S, J Tamplin O, Zon LI, Thomson JA, Bernstein ID, Slukvin II. 2018. NOTCH signaling specifies arterial-type definitive hemogenic endothelium from human pluripotent stem cells. Nature communications. 9(1):1828. Pubmed: 29739946 DOI:10.1038/s41467-018-04134-7 Uenishi GI, Jung HS, Kumar A, Park MA, Hadland BK, McLeod E, Raymond M, Moskvin O, Zimmerman CE, Theisen DJ, Swanson S, J Tamplin O, Zon LI, Thomson JA, Bernstein ID, Slukvin II. 2018. NOTCH signaling specifies arterial-type definitive hemogenic endothelium from human pluripotent stem cells. Nature communications. 9(1):1828. Pubmed: 29739946 DOI:10.1038/s41467-018-04134-7 NOTCH signaling is required for the arterial specification and formation of hematopoietic stem cells (HSCs) and lympho-myeloid progenitors in the embryonic aorta-gonad-mesonephros region and extraembryonic vasculature from a distinct lineage of vascular endothelial cells with hemogenic potential. However, the role of NOTCH signaling in hemogenic endothelium (HE) specification from human pluripotent stem cell (hPSC) has not been studied. Here, using a chemically defined hPSC differentiation system combined with the use of DLL1-Fc and DAPT to manipulate NOTCH, we discover that NOTCH activation in hPSC-derived immature HE progenitors leads to formation of CD144CD43CD73DLL4Runx1 + 23-GFP arterial-type HE, which requires NOTCH signaling to undergo endothelial-to-hematopoietic transition and produce definitive lympho-myeloid and erythroid cells. These findings demonstrate that NOTCH-mediated arterialization of HE is an essential prerequisite for establishing definitive lympho-myeloid program and suggest that exploring molecular pathways that lead to arterial specification may aid in vitro approaches to enhance definitive hematopoiesis from hPSCs. -
Yu Y, Schleich K, Yue B, Ji S, Lohneis P, Kemper K, Silvis MR, Qutob N, van Rooijen E, Werner-Klein M, Li L, Dhawan D, Meierjohann S, Reimann M, Elkahloun A, Treitschke S, Dörken B, Speck C, Mallette FA, Zon LI, Holmen SL, Peeper DS, Samuels Y, Schmitt CA, Lee S. 2018. Targeting the Senescence-Overriding Cooperative Activity of Structurally Unrelated H3K9 Demethylases in Melanoma. Cancer cell. 33(2):322-336.e8. Pubmed: 29438700 DOI:S1535-6108(18)30002-3 Yu Y, Schleich K, Yue B, Ji S, Lohneis P, Kemper K, Silvis MR, Qutob N, van Rooijen E, Werner-Klein M, Li L, Dhawan D, Meierjohann S, Reimann M, Elkahloun A, Treitschke S, Dörken B, Speck C, Mallette FA, Zon LI, Holmen SL, Peeper DS, Samuels Y, Schmitt CA, Lee S. 2018. Targeting the Senescence-Overriding Cooperative Activity of Structurally Unrelated H3K9 Demethylases in Melanoma. Cancer cell. 33(2):322-336.e8. Pubmed: 29438700 DOI:S1535-6108(18)30002-3 Oncogene-induced senescence, e.g., in melanocytic nevi, terminates the expansion of pre-malignant cells via transcriptional silencing of proliferation-related genes due to decoration of their promoters with repressive trimethylated histone H3 lysine 9 (H3K9) marks. We show here that structurally distinct H3K9-active demethylases-the lysine-specific demethylase-1 (LSD1) and several Jumonji C domain-containing moieties (such as JMJD2C)-disable senescence and permit Ras/Braf-evoked transformation. In mouse and zebrafish models, enforced LSD1 or JMJD2C expression promoted Braf-V600E-driven melanomagenesis. A large subset of established melanoma cell lines and primary human melanoma samples presented with a collective upregulation of related and unrelated H3K9 demethylase activities, whose targeted inhibition restored senescence, even in Braf inhibitor-resistant melanomas, evoked secondary immune effects and controlled tumor growth in vivo.Copyright © 2018 Elsevier Inc. All rights reserved. -
Mansour MR, He S, Li Z, Lobbardi R, Abraham BJ, Hug C, Rahman S, Leon TE, Kuang YY, Zimmerman MW, Blonquist T, Gjini E, Gutierrez A, Tang Q, Garcia-Perez L, Pike-Overzet K, Anders L, Berezovskaya A, Zhou Y, Zon LI, Neuberg D, Fielding AK, Staal FJT, Langenau DM, Sanda T, Young RA, Look AT. 2018. JDP2: An oncogenic bZIP transcription factor in T cell acute lymphoblastic leukemia. The Journal of experimental medicine. 215(7):1929-1945. Pubmed: 29941549 DOI:10.1084/jem.20170484 Mansour MR, He S, Li Z, Lobbardi R, Abraham BJ, Hug C, Rahman S, Leon TE, Kuang YY, Zimmerman MW, Blonquist T, Gjini E, Gutierrez A, Tang Q, Garcia-Perez L, Pike-Overzet K, Anders L, Berezovskaya A, Zhou Y, Zon LI, Neuberg D, Fielding AK, Staal FJT, Langenau DM, Sanda T, Young RA, Look AT. 2018. JDP2: An oncogenic bZIP transcription factor in T cell acute lymphoblastic leukemia. The Journal of experimental medicine. 215(7):1929-1945. Pubmed: 29941549 DOI:10.1084/jem.20170484 A substantial subset of patients with T cell acute lymphoblastic leukemia (T-ALL) develops resistance to steroids and succumbs to their disease. encodes a bZIP protein that has been implicated as a T-ALL oncogene from insertional mutagenesis studies in mice, but its role in human T-ALL pathogenesis has remained obscure. Here we show that is aberrantly expressed in a subset of T-ALL patients and is associated with poor survival. JDP2 is required for T-ALL cell survival, as its depletion by short hairpin RNA knockdown leads to apoptosis. Mechanistically, JDP2 regulates prosurvival signaling through direct transcriptional regulation of Furthermore, is one of few oncogenes capable of initiating T-ALL in transgenic zebrafish. Notably, thymocytes from transgenic zebrafish express high levels of and demonstrate resistance to steroids in vivo. These studies establish as a novel oncogene in high-risk T-ALL and implicate overexpression of as a mechanism of steroid resistance in -overexpressing cells.© 2018 Mansour et al. -
Blaser BW, Zon LI. 2018. Making HSCs in vitro: don't forget the hemogenic endothelium. Blood. 132(13):1372-1378. Pubmed: 30089629 DOI:10.1182/blood-2018-04-784140 Blaser BW, Zon LI. 2018. Making HSCs in vitro: don't forget the hemogenic endothelium. Blood. 132(13):1372-1378. Pubmed: 30089629 DOI:10.1182/blood-2018-04-784140 Generating a hematopoietic stem cell (HSC) in vitro from nonhematopoietic tissue has been a goal of experimental hematologists for decades. Until recently, no in vitro-derived cell has closely demonstrated the full lineage potential and self-renewal capacity of a true HSC. Studies revealing stem cell ontogeny from embryonic mesoderm to hemogenic endothelium to HSC provided the key to inducing HSC-like cells in vitro from a variety of cell types. Here we review the path to this discovery and discuss the future of autologous transplantation with in vitro-derived HSCs as a therapeutic modality.© 2018 by The American Society of Hematology. -
Kapp FG, Perlin JR, Hagedorn EJ, Gansner JM, Schwarz DE, O'Connell LA, Johnson NS, Amemiya C, Fisher DE, Wölfle U, Trompouki E, Niemeyer CM, Driever W, Zon LI. 2018. Protection from UV light is an evolutionarily conserved feature of the haematopoietic niche. Nature. 558(7710):445-448. Pubmed: 29899448 DOI:10.1038/s41586-018-0213-0 Kapp FG, Perlin JR, Hagedorn EJ, Gansner JM, Schwarz DE, O'Connell LA, Johnson NS, Amemiya C, Fisher DE, Wölfle U, Trompouki E, Niemeyer CM, Driever W, Zon LI. 2018. Protection from UV light is an evolutionarily conserved feature of the haematopoietic niche. Nature. 558(7710):445-448. Pubmed: 29899448 DOI:10.1038/s41586-018-0213-0 Haematopoietic stem and progenitor cells (HSPCs) require a specific microenvironment, the haematopoietic niche, which regulates HSPC behaviour. The location of this niche varies across species, but the evolutionary pressures that drive HSPCs to different microenvironments remain unknown. The niche is located in the bone marrow in adult mammals, whereas it is found in other locations in non-mammalian vertebrates, for example, in the kidney marrow in teleost fish. Here we show that a melanocyte umbrella above the kidney marrow protects HSPCs against ultraviolet light in zebrafish. Because mutants that lack melanocytes have normal steady-state haematopoiesis under standard laboratory conditions, we hypothesized that melanocytes above the stem cell niche protect HSPCs against ultraviolet-light-induced DNA damage. Indeed, after ultraviolet-light irradiation, unpigmented larvae show higher levels of DNA damage in HSPCs, as indicated by staining of cyclobutane pyrimidine dimers and have reduced numbers of HSPCs, as shown by cmyb (also known as myb) expression. The umbrella of melanocytes associated with the haematopoietic niche is highly evolutionarily conserved in aquatic animals, including the sea lamprey, a basal vertebrate. During the transition from an aquatic to a terrestrial environment, HSPCs relocated into the bone marrow, which is protected from ultraviolet light by the cortical bone around the marrow. Our studies reveal that melanocytes above the haematopoietic niche protect HSPCs from ultraviolet-light-induced DNA damage in aquatic vertebrates and suggest that during the transition to terrestrial life, ultraviolet light was an evolutionary pressure affecting the location of the haematopoietic niche. -
Wattrus SJ, Zon LI. 2018. Stem cell safe harbor: the hematopoietic stem cell niche in zebrafish. Blood advances. 2(21):3063-3069. Pubmed: 30425071 DOI:10.1182/bloodadvances.2018021725 Wattrus SJ, Zon LI. 2018. Stem cell safe harbor: the hematopoietic stem cell niche in zebrafish. Blood advances. 2(21):3063-3069. Pubmed: 30425071 DOI:10.1182/bloodadvances.2018021725 Each stem cell resides in a highly specialized anatomic location known as the niche that protects and regulates stem cell function. The importance of the niche in hematopoiesis has long been appreciated in transplantation, but without methods to observe activity in vivo, the components and mechanisms of the hematopoietic niche have remained incompletely understood. Zebrafish have emerged over the past few decades as an answer to this. Use of zebrafish to study the hematopoietic niche has enabled discovery of novel cell-cell interactions, as well as chemical and genetic regulators of hematopoietic stem cells. Mastery of niche components may improve therapeutic efforts to direct differentiation of hematopoietic stem cells from pluripotent cells, sustain stem cells in culture, or improve stem cell transplant.© 2018 by The American Society of Hematology. -
Zon LI. 2018. Stu Orkin is a superhero. The Journal of clinical investigation. 128(10):4213-4217. Pubmed: 30272583 DOI:124493 Zon LI. 2018. Stu Orkin is a superhero. The Journal of clinical investigation. 128(10):4213-4217. Pubmed: 30272583 DOI:124493 -
Ablain J, Xu M, Rothschild H, Jordan RC, Mito JK, Daniels BH, Bell CF, Joseph NM, Wu H, Bastian BC, Zon LI, Yeh I. 2018. Human tumor genomics and zebrafish modeling identify loss as a driver of mucosal melanoma. Science (New York, N.Y.). 362(6418):1055-1060. Pubmed: 30385465 DOI:10.1126/science.aau6509 Ablain J, Xu M, Rothschild H, Jordan RC, Mito JK, Daniels BH, Bell CF, Joseph NM, Wu H, Bastian BC, Zon LI, Yeh I. 2018. Human tumor genomics and zebrafish modeling identify loss as a driver of mucosal melanoma. Science (New York, N.Y.). 362(6418):1055-1060. Pubmed: 30385465 DOI:10.1126/science.aau6509 Melanomas originating from mucosal surfaces have low mutation burden, genomic instability, and poor prognosis. To identify potential driver genes, we sequenced hundreds of cancer-related genes in 43 human mucosal melanomas, cataloging point mutations, amplifications, and deletions. The gene, which encodes a negative regulator of mitogen-activated protein kinase (MAPK) signaling, was inactivated in 37% of the tumors. Four distinct genotypes were associated with loss. Using a rapid, tissue-specific CRISPR technique to model these genotypes in zebrafish, we found that functions as a tumor suppressor, particularly in the context of mutations. knockdown caused MAPK activation, increased cell proliferation, and conferred resistance to drugs inhibiting KIT tyrosine kinase activity. These findings provide a rationale for MAPK inhibition in SPRED1-deficient melanomas and introduce a zebrafish modeling approach that can be used more generally to dissect genetic interactions in cancer.Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. -
Yien YY, Shi J, Chen C, Cheung JTM, Grillo AS, Shrestha R, Li L, Zhang X, Kafina MD, Kingsley PD, King MJ, Ablain J, Li H, Zon LI, Palis J, Burke MD, Bauer DE, Orkin SH, Koehler CM, Phillips JD, Kaplan J, Ward DM, Lodish HF, Paw BH. 2018. FAM210B is an erythropoietin target and regulates erythroid heme synthesis by controlling mitochondrial iron import and ferrochelatase activity. The Journal of biological chemistry. 293(51):19797-19811. Pubmed: 30366982 DOI:10.1074/jbc.RA118.002742 Yien YY, Shi J, Chen C, Cheung JTM, Grillo AS, Shrestha R, Li L, Zhang X, Kafina MD, Kingsley PD, King MJ, Ablain J, Li H, Zon LI, Palis J, Burke MD, Bauer DE, Orkin SH, Koehler CM, Phillips JD, Kaplan J, Ward DM, Lodish HF, Paw BH. 2018. FAM210B is an erythropoietin target and regulates erythroid heme synthesis by controlling mitochondrial iron import and ferrochelatase activity. The Journal of biological chemistry. 293(51):19797-19811. Pubmed: 30366982 DOI:10.1074/jbc.RA118.002742 Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well-understood. To this end, here we profiled gene expression in EPO-treated 32D pro-B cells and developing fetal liver erythroid cells to identify additional iron regulatory genes. We determined that FAM210B, a mitochondrial inner-membrane protein, is essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation. deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur cluster formation. These defects were corrected with a lipid-soluble, small-molecule iron transporter, hinokitiol, in -deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that FAM210B is not a mitochondrial iron transporter but is required for adequate mitochondrial iron import to sustain heme synthesis and iron-sulfur cluster formation during erythroid differentiation. FAM210B was also required for maximal ferrochelatase activity in differentiating erythroid cells. We propose that FAM210B functions as an adaptor protein that facilitates the formation of an oligomeric mitochondrial iron transport complex, required for the increase in iron acquisition for heme synthesis during terminal erythropoiesis. Collectively, our results reveal a critical mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism.© 2018 Yien et al. -
Mandelbaum J, Shestopalov IA, Henderson RE, Chau NG, Knoechel B, Wick MJ, Zon LI. 2018. Zebrafish blastomere screen identifies retinoic acid suppression of in adenoid cystic carcinoma. The Journal of experimental medicine. 215(10):2673-2685. Pubmed: 30209067 DOI:10.1084/jem.20180939 Mandelbaum J, Shestopalov IA, Henderson RE, Chau NG, Knoechel B, Wick MJ, Zon LI. 2018. Zebrafish blastomere screen identifies retinoic acid suppression of in adenoid cystic carcinoma. The Journal of experimental medicine. 215(10):2673-2685. Pubmed: 30209067 DOI:10.1084/jem.20180939 Pluripotent cells have been used to probe developmental pathways that are involved in genetic diseases and oncogenic events. To find new therapies that would target -driven tumors, we developed a pluripotent zebrafish blastomere culture system. We performed a chemical genetic screen and identified retinoic acid agonists as suppressors of expression. Retinoic acid treatment also decreased gene expression in human leukemia cells. Translocations that drive overexpression of the oncogenic transcription factor are molecular hallmarks of adenoid cystic carcinoma (ACC), a malignant salivary gland tumor with no effective therapy. Retinoic acid agonists inhibited tumor growth in vivo in ACC patient-derived xenograft models and decreased MYB binding at translocated enhancers, thereby potentially diminishing the MYB positive feedback loop driving ACC. Our findings establish the zebrafish pluripotent cell culture system as a method to identify modulators of tumor formation, particularly establishing retinoic acid as a potential new effective therapy for ACC.© 2018 Mandelbaum et al. -
Lahvic JL, Ammerman M, Li P, Blair MC, Stillman ER, Fast EM, Robertson AL, Christodoulou C, Perlin JR, Yang S, Chiang N, Norris PC, Daily ML, Redfield SE, Chan IT, Chatrizeh M, Chase ME, Weis O, Zhou Y, Serhan CN, Zon LI. 2018. Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132. Proceedings of the National Academy of Sciences of the United States of America. 115(37):9252-9257. Pubmed: 30139917 DOI:10.1073/pnas.1806077115 Lahvic JL, Ammerman M, Li P, Blair MC, Stillman ER, Fast EM, Robertson AL, Christodoulou C, Perlin JR, Yang S, Chiang N, Norris PC, Daily ML, Redfield SE, Chan IT, Chatrizeh M, Chase ME, Weis O, Zhou Y, Serhan CN, Zon LI. 2018. Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132. Proceedings of the National Academy of Sciences of the United States of America. 115(37):9252-9257. Pubmed: 30139917 DOI:10.1073/pnas.1806077115 Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based β-arrestin recruitment assay. Knockdown of zebrafish prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure-activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis. -
Kulkeaw K, Inoue T, Ishitani T, Nakanishi Y, Zon LI, Sugiyama D. 2018. Purification of zebrafish erythrocytes as a means of identifying a novel regulator of haematopoiesis. British journal of haematology. 180(3):420-431. Pubmed: 29265183 DOI:10.1111/bjh.15048 Kulkeaw K, Inoue T, Ishitani T, Nakanishi Y, Zon LI, Sugiyama D. 2018. Purification of zebrafish erythrocytes as a means of identifying a novel regulator of haematopoiesis. British journal of haematology. 180(3):420-431. Pubmed: 29265183 DOI:10.1111/bjh.15048 Zebrafish embryos are useful to study haematopoietic gene function in vertebrates, although lack of antibodies to zebrafish proteins has limited the purification of specific cell populations. Here, we purified primitive zebrafish erythrocytes using 1, 5-bis{[2-(di-methylamino)ethyl]amino}-4, 8-dihydroxyanthracene-9, 10-dione (DRAQ5 ), a DNA-staining fluorescent dye. At 48-h post-fertilization, we sorted small-sized cells from embryos using forward scatter and found that they consisted of DRAQ5 and DRAQ5 populations. DRAQ5 cells contained haemoglobin, lacked myeloperoxidase activity and expressed high levels of embryonic globin (hbae3 and hbbe1.1) mRNA, all characteristics of primitive erythrocytes. Following DRAQ5 analysis of gata1:dsRed transgenic embryos, we purified primitive DRAQ5 dsRed+ erythrocytes from haematopoietic progenitor cells. Using this method, we identified docking protein 2 (Dok2) as functioning in differentiation of primitive erythrocytes. We conclude that DRAQ5 -based flow cytometry enables purification of primitive zebrafish erythrocytes.© 2017 John Wiley & Sons Ltd. -
Yu SH, Zhu KY, Zhang F, Wang J, Yuan H, Chen Y, Jin Y, Dong M, Wang L, Jia XE, Gao L, Dong ZW, Ren CG, Chen LT, Huang QH, Deng M, Zon LI, Zhou Y, Zhu J, Xu PF, Liu TX. 2018. The histone demethylase Jmjd3 regulates zebrafish myeloid development by promoting spi1 expression. Biochimica et biophysica acta. Gene regulatory mechanisms. 1861(2):106-116. Pubmed: 29378332 DOI:S1874-9399(17)30309-7 Yu SH, Zhu KY, Zhang F, Wang J, Yuan H, Chen Y, Jin Y, Dong M, Wang L, Jia XE, Gao L, Dong ZW, Ren CG, Chen LT, Huang QH, Deng M, Zon LI, Zhou Y, Zhu J, Xu PF, Liu TX. 2018. The histone demethylase Jmjd3 regulates zebrafish myeloid development by promoting spi1 expression. Biochimica et biophysica acta. Gene regulatory mechanisms. 1861(2):106-116. Pubmed: 29378332 DOI:S1874-9399(17)30309-7 The histone demethylase Jmjd3 plays a critical role in cell lineage specification and differentiation at various stages of development. However, its function during normal myeloid development remains poorly understood. Here, we carried out a systematic in vivo screen of epigenetic factors for their function in hematopoiesis and identified Jmjd3 as a new epigenetic factor that regulates myelopoiesis in zebrafish. We demonstrated that jmjd3 was essential for zebrafish primitive and definitive myelopoiesis, knockdown of jmjd3 suppressed the myeloid commitment and enhanced the erythroid commitment. Only overexpression of spi1 but not the other myeloid regulators rescued the myeloid development in jmjd3 morphants. Furthermore, preliminary mechanistic studies demonstrated that Jmjd3 could directly bind to the spi1 regulatory region to alleviate the repressive H3K27me3 modification and activate spi1 expression. Thus, our studies highlight that Jmjd3 is indispensable for early zebrafish myeloid development by promoting spi1 expression.Copyright © 2018 Elsevier B.V. All rights reserved. -
Parada-Kusz M, Penaranda C, Hagedorn EJ, Clatworthy A, Nair AV, Henninger JE, Ernst C, Li B, Riquelme R, Jijon H, Villablanca EJ, Zon LI, Hung D, Allende ML. 2018. Generation of mouse-zebrafish hematopoietic tissue chimeric embryos for hematopoiesis and host-pathogen interaction studies. Disease models & mechanisms. 11(11). Pubmed: 30266803 DOI:10.1242/dmm.034876 Parada-Kusz M, Penaranda C, Hagedorn EJ, Clatworthy A, Nair AV, Henninger JE, Ernst C, Li B, Riquelme R, Jijon H, Villablanca EJ, Zon LI, Hung D, Allende ML. 2018. Generation of mouse-zebrafish hematopoietic tissue chimeric embryos for hematopoiesis and host-pathogen interaction studies. Disease models & mechanisms. 11(11). Pubmed: 30266803 DOI:10.1242/dmm.034876 Xenografts of the hematopoietic system are extremely useful as disease models and for translational research. Zebrafish xenografts have been widely used to monitor blood cancer cell dissemination and homing due to the optical clarity of embryos and larvae, which allow unrestricted visualization of migratory events. Here, we have developed a xenotransplantation technique that transiently generates hundreds of hematopoietic tissue chimeric embryos by transplanting murine bone marrow cells into zebrafish blastulae. In contrast to previous methods, this procedure allows mammalian cell integration into the fish developmental hematopoietic program, which results in chimeric animals containing distinct phenotypes of murine blood cells in both circulation and the hematopoietic niche. Murine cells in chimeric animals express antigens related to (i) hematopoietic stem and progenitor cells, (ii) active cell proliferation and (iii) myeloid cell lineages. We verified the utility of this method by monitoring zebrafish chimeras during development using non-invasive imaging to show novel murine cell behaviors, such as homing to primitive and definitive hematopoietic tissues, dynamic hematopoietic cell and hematopoietic niche interactions, and response to bacterial infection. Overall, transplantation into the zebrafish blastula provides a useful method that simplifies the generation of numerous chimeric animals and expands the range of murine cell behaviors that can be studied in zebrafish chimeras. In addition, integration of murine cells into the host hematopoietic system during development suggests highly conserved molecular mechanisms of hematopoiesis between zebrafish and mammals.This article has an associated First Person interview with the first author of the paper.© 2018. Published by The Company of Biologists Ltd. 2017
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Neiswender JV, Kortum RL, Bourque C, Kasheta M, Zon LI, Morrison DK, Ceol CJ. 2017. KIT Suppresses BRAF-Mutant Melanoma by Attenuating Oncogenic RAS/MAPK Signaling. Cancer research. 77(21):5820-5830. Pubmed: 28947418 DOI:10.1158/0008-5472.CAN-17-0473 Neiswender JV, Kortum RL, Bourque C, Kasheta M, Zon LI, Morrison DK, Ceol CJ. 2017. KIT Suppresses BRAF-Mutant Melanoma by Attenuating Oncogenic RAS/MAPK Signaling. Cancer research. 77(21):5820-5830. Pubmed: 28947418 DOI:10.1158/0008-5472.CAN-17-0473 The receptor tyrosine kinase KIT promotes survival and migration of melanocytes during development, and excessive KIT activity hyperactivates the RAS/MAPK pathway and can drive formation of melanomas, most notably of rare melanomas that occur on volar and mucosal surfaces of the skin. The much larger fraction of melanomas that occur on sun-exposed skin is driven primarily by BRAF- or NRAS-activating mutations, but these melanomas exhibit a surprising loss of KIT expression, which raises the question of whether loss of KIT in these tumors facilitates tumorigenesis. To address this question, we introduced a mutation into a strain of melanoma-prone zebrafish. Melanoma onset was accelerated in fish. Tumors from animals were more invasive and had higher RAS/MAPK pathway activation. KIT knockdown also increased RAS/MAPK pathway activation in a BRAF-mutant human melanoma cell line. We found that pathway stimulation upstream of BRAF could paradoxically reduce signaling downstream of BRAF, and wild-type BRAF was necessary for this effect, suggesting that its activation can dampen oncogenic BRAF signaling. , expression of wild-type BRAF delayed melanoma onset, but only in a -dependent manner. Together, these results suggest that KIT can activate signaling through wild-type RAF proteins, thus interfering with oncogenic BRAF-driven melanoma formation. .©2017 American Association for Cancer Research. -
Gansner JM, Dang M, Ammerman M, Zon LI. 2017. Transplantation in zebrafish. Methods in cell biology. 138:629-647. Pubmed: 28129861 DOI:S0091-679X(16)30142-X Gansner JM, Dang M, Ammerman M, Zon LI. 2017. Transplantation in zebrafish. Methods in cell biology. 138:629-647. Pubmed: 28129861 DOI:S0091-679X(16)30142-X Tissue or cell transplantation is an invaluable technique with a multitude of applications including studying the developmental potential of certain cell populations, dissecting cell-environment interactions, and identifying stem cells. One key technical requirement for performing transplantation assays is the capability of distinguishing the transplanted donor cells from the endogenous host cells and tracing the donor cells over time. The zebrafish has emerged as an excellent model organism for performing transplantation assays, thanks in part to the transparency of embryos and even adults when pigment mutants are employed. Using transgenic techniques and fast-evolving imaging technology, fluorescence-labeled donor cells can be readily identified and studied during development in vivo. In this chapter, we will discuss the rationale of different types of zebrafish transplantation in both embryos and adults and then focus on four detailed methods of transplantation: blastula/gastrula transplantation for mosaic analysis, hematopoietic stem cell transplantation, chemical screening using a transplantation model, and tumor transplantation.Copyright © 2017 Elsevier Inc. All rights reserved. -
Crawford NG, Kelly DE, Hansen MEB, Beltrame MH, Fan S, Bowman SL, Jewett E, Ranciaro A, Thompson S, Lo Y, Pfeifer SP, Jensen JD, Campbell MC, Beggs W, Hormozdiari F, Mpoloka SW, Mokone GG, Nyambo T, Meskel DW, Belay G, Haut J, Rothschild H, Zon L, Zhou Y, Kovacs MA, Xu M, Zhang T, Bishop K, Sinclair J, Rivas C, Elliot E, Choi J, Li SA, Hicks B, Burgess S, Abnet C, Watkins-Chow DE, Oceana E, Song YS, Eskin E, Brown KM, Marks MS, Loftus SK, Pavan WJ, Yeager M, Chanock S, Tishkoff SA. 2017. Loci associated with skin pigmentation identified in African populations. Science (New York, N.Y.). 358(6365). Pubmed: 29025994 DOI:10.1126/science.aan8433 Crawford NG, Kelly DE, Hansen MEB, Beltrame MH, Fan S, Bowman SL, Jewett E, Ranciaro A, Thompson S, Lo Y, Pfeifer SP, Jensen JD, Campbell MC, Beggs W, Hormozdiari F, Mpoloka SW, Mokone GG, Nyambo T, Meskel DW, Belay G, Haut J, Rothschild H, Zon L, Zhou Y, Kovacs MA, Xu M, Zhang T, Bishop K, Sinclair J, Rivas C, Elliot E, Choi J, Li SA, Hicks B, Burgess S, Abnet C, Watkins-Chow DE, Oceana E, Song YS, Eskin E, Brown KM, Marks MS, Loftus SK, Pavan WJ, Yeager M, Chanock S, Tishkoff SA. 2017. Loci associated with skin pigmentation identified in African populations. Science (New York, N.Y.). 358(6365). Pubmed: 29025994 DOI:10.1126/science.aan8433 Despite the wide range of skin pigmentation in humans, little is known about its genetic basis in global populations. Examining ethnically diverse African genomes, we identify variants in or near , , , , , and that are significantly associated with skin pigmentation. Genetic evidence indicates that the light pigmentation variant at was introduced into East Africa by gene flow from non-Africans. At all other loci, variants associated with dark pigmentation in Africans are identical by descent in South Asian and Australo-Melanesian populations. Functional analyses indicate that encodes a lysosomal protein that affects melanogenesis in zebrafish and mice, and that mutations in melanocyte-specific regulatory regions near correlate with expression of ultraviolet response genes under selection in Eurasians.Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. -
Doulatov S, Vo LT, Macari ER, Wahlster L, Kinney MA, Taylor AM, Barragan J, Gupta M, McGrath K, Lee HY, Humphries JM, DeVine A, Narla A, Alter BP, Beggs AH, Agarwal S, Ebert BL, Gazda HT, Lodish HF, Sieff CA, Schlaeger TM, Zon LI, Daley GQ. 2017. Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors. Science translational medicine. 9(376). Pubmed: 28179501 DOI:10.1126/scitranslmed.aah5645 Doulatov S, Vo LT, Macari ER, Wahlster L, Kinney MA, Taylor AM, Barragan J, Gupta M, McGrath K, Lee HY, Humphries JM, DeVine A, Narla A, Alter BP, Beggs AH, Agarwal S, Ebert BL, Gazda HT, Lodish HF, Sieff CA, Schlaeger TM, Zon LI, Daley GQ. 2017. Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors. Science translational medicine. 9(376). Pubmed: 28179501 DOI:10.1126/scitranslmed.aah5645 Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA.Copyright © 2017, American Association for the Advancement of Science. -
Hockman D, Burns AJ, Schlosser G, Gates KP, Jevans B, Mongera A, Fisher S, Unlu G, Knapik EW, Kaufman CK, Mosimann C, Zon LI, Lancman JJ, Dong PDS, Lickert H, Tucker AS, Baker CV. 2017. Evolution of the hypoxia-sensitive cells involved in amniote respiratory reflexes. eLife. 6. Pubmed: 28387645 DOI:10.7554/eLife.21231 Hockman D, Burns AJ, Schlosser G, Gates KP, Jevans B, Mongera A, Fisher S, Unlu G, Knapik EW, Kaufman CK, Mosimann C, Zon LI, Lancman JJ, Dong PDS, Lickert H, Tucker AS, Baker CV. 2017. Evolution of the hypoxia-sensitive cells involved in amniote respiratory reflexes. eLife. 6. Pubmed: 28387645 DOI:10.7554/eLife.21231 The evolutionary origins of the hypoxia-sensitive cells that trigger amniote respiratory reflexes - carotid body glomus cells, and 'pulmonary neuroendocrine cells' (PNECs) - are obscure. Homology has been proposed between glomus cells, which are neural crest-derived, and the hypoxia-sensitive 'neuroepithelial cells' (NECs) of fish gills, whose embryonic origin is unknown. NECs have also been likened to PNECs, which differentiate in situ within lung airway epithelia. Using genetic lineage-tracing and neural crest-deficient mutants in zebrafish, and physical fate-mapping in frog and lamprey, we find that NECs are not neural crest-derived, but endoderm-derived, like PNECs, whose endodermal origin we confirm. We discover neural crest-derived catecholaminergic cells associated with zebrafish pharyngeal arch blood vessels, and propose a new model for amniote hypoxia-sensitive cell evolution: endoderm-derived NECs were retained as PNECs, while the carotid body evolved via the aggregation of neural crest-derived catecholaminergic (chromaffin) cells already associated with blood vessels in anamniote pharyngeal arches. -
Ludin A, Zon LI. 2017. Cancer immunotherapy: The dark side of PD-1 receptor inhibition. Nature. 552(7683):41-42. Pubmed: 29143822 DOI:10.1038/nature24759 Ludin A, Zon LI. 2017. Cancer immunotherapy: The dark side of PD-1 receptor inhibition. Nature. 552(7683):41-42. Pubmed: 29143822 DOI:10.1038/nature24759 Inhibiting the protein PD-1 can activate T cells that trigger immune responses against tumour cells. But it emerges that, in mice, this immunotherapy exacerbates a cancer that involves the T cells themselves. -
Choudhuri A, Fast EM, Zon LI. 2017. Using Zebrafish to Study Pathways that Regulate Hematopoietic Stem Cell Self-Renewal and Migration. Stem cell reports. 8(6):1465-1471. Pubmed: 28591648 DOI:S2213-6711(17)30225-4 Choudhuri A, Fast EM, Zon LI. 2017. Using Zebrafish to Study Pathways that Regulate Hematopoietic Stem Cell Self-Renewal and Migration. Stem cell reports. 8(6):1465-1471. Pubmed: 28591648 DOI:S2213-6711(17)30225-4 This perspective describes the usefulness of zebrafish as a model to study interaction of hematopoietic stem cells with the associated niche in vivo, explains how such interactions influence regeneration, migration, and clonality of HSCs, and defines their fate during differentiation.Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved. -
van Rooijen E, Fazio M, Zon LI. 2017. From fish bowl to bedside: The power of zebrafish to unravel melanoma pathogenesis and discover new therapeutics. Pigment cell & melanoma research. 30(4):402-412. Pubmed: 28379616 DOI:10.1111/pcmr.12592 van Rooijen E, Fazio M, Zon LI. 2017. From fish bowl to bedside: The power of zebrafish to unravel melanoma pathogenesis and discover new therapeutics. Pigment cell & melanoma research. 30(4):402-412. Pubmed: 28379616 DOI:10.1111/pcmr.12592 Melanoma is the most aggressive and deadliest form of skin cancer. A detailed knowledge of the cellular, molecular, and genetic events underlying melanoma progression is highly relevant to diagnosis, prognosis and risk stratification, and the development of new therapies. In the last decade, zebrafish have emerged as a valuable model system for the study of melanoma. Pathway conservation, coupled with the availability of robust genetic, transgenic, and chemical tools, has made the zebrafish a powerful model for identifying novel disease genes, visualizing cancer initiation, interrogating tumor-microenvironment interactions, and discovering new therapeutics that regulate melanocyte and melanoma development. In this review, we will give an overview of these studies, and highlight recent advancements that will help unravel melanoma pathogenesis and impact human disease.© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. -
Khajavi M, Zhou Y, Birsner AE, Bazinet L, Rosa Di Sant A, Schiffer AJ, Rogers MS, Krishnaji ST, Hu B, Nguyen V, Zon L, D'Amato RJ. 2017. Identification of Padi2 as a novel angiogenesis-regulating gene by genome association studies in mice. PLoS genetics. 13(6):e1006848. Pubmed: 28617813 DOI:10.1371/journal.pgen.1006848 Khajavi M, Zhou Y, Birsner AE, Bazinet L, Rosa Di Sant A, Schiffer AJ, Rogers MS, Krishnaji ST, Hu B, Nguyen V, Zon L, D'Amato RJ. 2017. Identification of Padi2 as a novel angiogenesis-regulating gene by genome association studies in mice. PLoS genetics. 13(6):e1006848. Pubmed: 28617813 DOI:10.1371/journal.pgen.1006848 Recent findings indicate that growth factor-driven angiogenesis is markedly influenced by genetic variation. This variation in angiogenic responsiveness may alter the susceptibility to a number of angiogenesis-dependent diseases. Here, we utilized the genetic diversity available in common inbred mouse strains to identify the loci and candidate genes responsible for differences in angiogenic response. The corneal micropocket neovascularization assay was performed on 42 different inbred mouse strains using basic fibroblast growth factor (bFGF) pellets. We performed a genome-wide association study utilizing efficient mixed-model association (EMMA) mapping using the induced vessel area from all strains. Our analysis yielded five loci with genome-wide significance on chromosomes 4, 8, 11, 15 and 16. We further refined the mapping on chromosome 4 within a haplotype block containing multiple candidate genes. These genes were evaluated by expression analysis in corneas of various inbred strains and in vitro functional assays in human microvascular endothelial cells (HMVECs). Of these, we found the expression of peptidyl arginine deiminase type II (Padi2), known to be involved in metabolic pathways, to have a strong correlation with a haplotype shared by multiple high angiogenic strains. In addition, inhibition of Padi2 demonstrated a dosage-dependent effect in HMVECs. To investigate its role in vivo, we knocked down Padi2 in transgenic kdrl:zsGreen zebrafish embryos using morpholinos. These embryos had disrupted vessel formation compared to control siblings. The impaired vascular pattern was partially rescued by human PADI2 mRNA, providing evidence for the specificity of the morphant phenotype. Taken together, our study is the first to indicate the potential role of Padi2 as an angiogenesis-regulating gene. The characterization of Padi2 and other genes in associated pathways may provide new understanding of angiogenesis regulation and novel targets for diagnosis and treatment of a wide variety of angiogenesis-dependent diseases. -
Perlin JR, Sporrij A, Zon LI. 2017. Blood on the tracks: hematopoietic stem cell-endothelial cell interactions in homing and engraftment. Journal of molecular medicine (Berlin, Germany). 95(8):809-819. Pubmed: 28702683 DOI:10.1007/s00109-017-1559-8 Perlin JR, Sporrij A, Zon LI. 2017. Blood on the tracks: hematopoietic stem cell-endothelial cell interactions in homing and engraftment. Journal of molecular medicine (Berlin, Germany). 95(8):809-819. Pubmed: 28702683 DOI:10.1007/s00109-017-1559-8 Cells of the hematopoietic system undergo rapid turnover. Each day, humans require the production of about one hundred billion new blood cells for proper function. Hematopoietic stem cells (HSCs) are rare cells that reside in specialized niches and are required throughout life to produce specific progenitor cells that will replenish all blood lineages. There is, however, an incomplete understanding of the molecular and physical properties that regulate HSC migration, homing, engraftment, and maintenance in the niche. Endothelial cells (ECs) are intimately associated with HSCs throughout the life of the stem cell, from the specialized endothelial cells that give rise to HSCs, to the perivascular niche endothelial cells that regulate HSC homeostasis. Recent studies have dissected the unique molecular and physical properties of the endothelial cells in the HSC vascular niche and their role in HSC biology, which may be manipulated to enhance hematopoietic stem cell transplantation therapies. -
Liu W, Wu M, Huang Z, Lian J, Chen J, Wang T, Leung AY, Liao Y, Zhang Z, Liu Q, Yen K, Lin S, Zon LI, Wen Z, Zhang Y, Zhang W. 2017. c-myb hyperactivity leads to myeloid and lymphoid malignancies in zebrafish. Leukemia. 31(1):222-233. Pubmed: 27457538 DOI:10.1038/leu.2016.170 Liu W, Wu M, Huang Z, Lian J, Chen J, Wang T, Leung AY, Liao Y, Zhang Z, Liu Q, Yen K, Lin S, Zon LI, Wen Z, Zhang Y, Zhang W. 2017. c-myb hyperactivity leads to myeloid and lymphoid malignancies in zebrafish. Leukemia. 31(1):222-233. Pubmed: 27457538 DOI:10.1038/leu.2016.170 The c-MYB transcription factor is a key regulator of hematopoietic cell proliferation and differentiation, and dysregulation of c-MYB activity often associates with various hematological disorders. Yet, its pathogenic role remains largely unknown due to lack of suitable animal models. Here, we report a detail characterization of a c-myb-gfp transgenic zebrafish harboring c-Myb hyperactivity (named c-myb). This line exhibits abnormal granulocyte expansion that resembles human myelodysplastic syndrome (MDS) from embryonic stage to adulthood. Strikingly, a small portion of c-myb adult fish develops acute myeloid leukemia-like or acute lymphoid leukemia-like disorders with age. The myeloid and lymphoid malignancies in c-myb adult fish are likely caused by the hyperactivity of c-myb, resulting in the dysregulation of a number of cell-cycle-related genes and hyperproliferation of hematopoietic precursor cells. Finally, treatment with c-myb target drug flavopiridol can relieve the MDS-like symptoms in both c-myb embryos and adult fish. Our study establishes a zebrafish model for studying the cellular and molecular mechanisms underlying c-Myb-associated leukemogenesis as well as for anti-leukemic drug screening. -
McGonigle TA, Dwyer AR, Greenland EL, Scott NM, Keane KN, Newsholme P, Goodridge HS, Zon LI, Pixley FJ, Hart PH. 2017. PGE pulsing of murine bone marrow cells reduces migration of daughter monocytes/macrophages in vitro and in vivo. Experimental hematology. 56:64-68. Pubmed: 28822771 DOI:S0301-472X(17)30717-8 McGonigle TA, Dwyer AR, Greenland EL, Scott NM, Keane KN, Newsholme P, Goodridge HS, Zon LI, Pixley FJ, Hart PH. 2017. PGE pulsing of murine bone marrow cells reduces migration of daughter monocytes/macrophages in vitro and in vivo. Experimental hematology. 56:64-68. Pubmed: 28822771 DOI:S0301-472X(17)30717-8 Monocytes/macrophages differentiating from bone marrow (BM) cells pulsed for 2 hours at 37°C with a stabilized derivative of prostaglandin E, 16,16-dimethyl PGE (dmPGE), migrated less efficiently toward a chemoattractant than monocytes/macrophages differentiated from BM cells pulsed with vehicle. To confirm that the effect on BM cells was long lasting and to replicate human BM transplantation, chimeric mice were established with donor BM cells pulsed for 2 hours with dmPGE before injection into marrow-ablated congenic recipient mice. After 12 weeks, when high levels (90%) of engraftment were obtained, regenerated BM-derived monocytes/macrophages differentiating in vitro or in vivo migrated inefficiently toward the chemokines colony-stimulating factor-1 (CSF-1) and chemokine (C-C motif) ligand 2 (CCL2) or thioglycollate, respectively. Our results reveal long-lasting changes to progenitor cells of monocytes/macrophages by a 2-hour dmPGE pulse that, in turn, limits the migration of their daughter cells to chemoattractants and inflammatory mediators.Copyright © 2017 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved. -
Perlin JR, Robertson AL, Zon LI. 2017. Efforts to enhance blood stem cell engraftment: Recent insights from zebrafish hematopoiesis. The Journal of experimental medicine. 214(10):2817-2827. Pubmed: 28830909 DOI:10.1084/jem.20171069 Perlin JR, Robertson AL, Zon LI. 2017. Efforts to enhance blood stem cell engraftment: Recent insights from zebrafish hematopoiesis. The Journal of experimental medicine. 214(10):2817-2827. Pubmed: 28830909 DOI:10.1084/jem.20171069 Hematopoietic stem cell transplantation (HSCT) is an important therapy for patients with a variety of hematological malignancies. HSCT would be greatly improved if patient-specific hematopoietic stem cells (HSCs) could be generated from induced pluripotent stem cells in vitro There is an incomplete understanding of the genes and signals involved in HSC induction, migration, maintenance, and niche engraftment. Recent studies in zebrafish have revealed novel genes that are required for HSC induction and niche regulation of HSC homeostasis. Manipulation of these signaling pathways and cell types may improve HSC bioengineering, which could significantly advance critical, lifesaving HSCT therapies.© 2017 Perlin et al. -
Ciarlo C, Kaufman CK, Kinikoglu B, Michael J, Yang S, D Amato C, Blokzijl-Franke S, den Hertog J, Schlaeger TM, Zhou Y, Liao E, Zon LI. 2017. A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development. eLife. 6. Pubmed: 28832322 DOI:10.7554/eLife.29145 Ciarlo C, Kaufman CK, Kinikoglu B, Michael J, Yang S, D Amato C, Blokzijl-Franke S, den Hertog J, Schlaeger TM, Zhou Y, Liao E, Zon LI. 2017. A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development. eLife. 6. Pubmed: 28832322 DOI:10.7554/eLife.29145 The neural crest is a dynamic progenitor cell population that arises at the border of neural and non-neural ectoderm. The inductive roles of FGF, Wnt, and BMP at the neural plate border are well established, but the signals required for subsequent neural crest development remain poorly characterized. Here, we conducted a screen in primary zebrafish embryo cultures for chemicals that disrupt neural crest development, as read out by expression. We found that the natural product caffeic acid phenethyl ester (CAPE) disrupts neural crest gene expression, migration, and melanocytic differentiation by reducing Sox10 activity. CAPE inhibits FGF-stimulated PI3K/Akt signaling, and neural crest defects in CAPE-treated embryos are suppressed by constitutively active Akt1. Inhibition of Akt activity by constitutively active PTEN similarly decreases expression and Sox10 activity. Our study has identified Akt as a novel intracellular pathway required for neural crest differentiation. -
Norris ML, Pauli A, Gagnon JA, Lord ND, Rogers KW, Mosimann C, Zon LI, Schier AF. 2017. Toddler signaling regulates mesodermal cell migration downstream of Nodal signaling. eLife. 6. Pubmed: 29117894 DOI:10.7554/eLife.22626 Norris ML, Pauli A, Gagnon JA, Lord ND, Rogers KW, Mosimann C, Zon LI, Schier AF. 2017. Toddler signaling regulates mesodermal cell migration downstream of Nodal signaling. eLife. 6. Pubmed: 29117894 DOI:10.7554/eLife.22626 Toddler/Apela/Elabela is a conserved secreted peptide that regulates mesendoderm development during zebrafish gastrulation. Two non-exclusive models have been proposed to explain Toddler function. The 'specification model' postulates that Toddler signaling enhances Nodal signaling to properly specify endoderm, whereas the 'migration model' posits that Toddler signaling regulates mesendodermal cell migration downstream of Nodal signaling. Here, we test key predictions of both models. We find that in mutants Nodal signaling is initially normal and increasing endoderm specification does not rescue mesendodermal cell migration. Mesodermal cell migration defects in mutants result from a decrease in animal pole-directed migration and are independent of endoderm. Conversely, endodermal cell migration defects are dependent on a Cxcr4a-regulated tether of the endoderm to mesoderm. These results suggest that Toddler signaling regulates mesodermal cell migration downstream of Nodal signaling and indirectly affects endodermal cell migration via Cxcr4a-signaling. -
Fazio M, Zon LI. 2017. Fishing for answers in precision cancer medicine. Proceedings of the National Academy of Sciences of the United States of America. 114(39):10306-10308. Pubmed: 28916734 DOI:10.1073/pnas.1713769114 Fazio M, Zon LI. 2017. Fishing for answers in precision cancer medicine. Proceedings of the National Academy of Sciences of the United States of America. 114(39):10306-10308. Pubmed: 28916734 DOI:10.1073/pnas.1713769114 -
Gansner JM, Leung AD, Superdock M, Blair MC, Ammerman MB, Durand EM, Barut B, Handin RI, Stachura DL, Lu C, Springer TA, Zon LI. 2017. Sorting zebrafish thrombocyte lineage cells with a Cd41 monoclonal antibody enriches hematopoietic stem cell activity. Blood. 129(10):1394-1397. Pubmed: 28126924 DOI:10.1182/blood-2016-12-759993 Gansner JM, Leung AD, Superdock M, Blair MC, Ammerman MB, Durand EM, Barut B, Handin RI, Stachura DL, Lu C, Springer TA, Zon LI. 2017. Sorting zebrafish thrombocyte lineage cells with a Cd41 monoclonal antibody enriches hematopoietic stem cell activity. Blood. 129(10):1394-1397. Pubmed: 28126924 DOI:10.1182/blood-2016-12-759993 -
Henninger J, Santoso B, Hans S, Durand E, Moore J, Mosimann C, Brand M, Traver D, Zon L. 2017. Corrigendum: Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development. Nature cell biology. 19(2):142. Pubmed: 28139650 DOI:10.1038/ncb3462 Henninger J, Santoso B, Hans S, Durand E, Moore J, Mosimann C, Brand M, Traver D, Zon L. 2017. Corrigendum: Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development. Nature cell biology. 19(2):142. Pubmed: 28139650 DOI:10.1038/ncb3462 -
van Rooij FJA, Qayyum R, Smith AV, Zhou Y, Trompet S, Tanaka T, Keller MF, Chang LC, Schmidt H, Yang ML, Chen MH, Hayes J, Johnson AD, Yanek LR, Mueller C, Lange L, Floyd JS, Ghanbari M, Zonderman AB, Jukema JW, Hofman A, van Duijn CM, Desch KC, Saba Y, Ozel AB, Snively BM, Wu JY, Schmidt R, Fornage M, Klein RJ, Fox CS, Matsuda K, Kamatani N, Wild PS, Stott DJ, Ford I, Slagboom PE, Yang J, Chu AY, Lambert AJ, Uitterlinden AG, Franco OH, Hofer E, Ginsburg D, Hu B, Keating B, Schick UM, Brody JA, Li JZ, Chen Z, Zeller T, Guralnik JM, Chasman DI, Peters LL, Kubo M, Becker DM, Li J, Eiriksdottir G, Rotter JI, Levy D, Grossmann V, Patel KV, Chen CH, Ridker PM, Tang H, Launer LJ, Rice KM, Li-Gao R, Ferrucci L, Evans MK, Choudhuri A, Trompouki E, Abraham BJ, Yang S, Takahashi A, Kamatani Y, Kooperberg C, Harris TB, Jee SH, Coresh J, Tsai FJ, Longo DL, Chen YT, Felix JF, Yang Q, Psaty BM, Boerwinkle E, Becker LC, Mook-Kanamori DO, Wilson JG, Gudnason V, O'Donnell CJ, Dehghan A, Cupples LA, Nalls MA, Morris AP, Okada Y, Reiner AP, Zon LI, Ganesh SK. 2017. Genome-wide Trans-ethnic Meta-analysis Identifies Seven Genetic Loci Influencing Erythrocyte Traits and a Role for RBPMS in Erythropoiesis. American journal of human genetics. 100(1):51-63. Pubmed: 28017375 DOI:S0002-9297(16)30520-1 van Rooij FJA, Qayyum R, Smith AV, Zhou Y, Trompet S, Tanaka T, Keller MF, Chang LC, Schmidt H, Yang ML, Chen MH, Hayes J, Johnson AD, Yanek LR, Mueller C, Lange L, Floyd JS, Ghanbari M, Zonderman AB, Jukema JW, Hofman A, van Duijn CM, Desch KC, Saba Y, Ozel AB, Snively BM, Wu JY, Schmidt R, Fornage M, Klein RJ, Fox CS, Matsuda K, Kamatani N, Wild PS, Stott DJ, Ford I, Slagboom PE, Yang J, Chu AY, Lambert AJ, Uitterlinden AG, Franco OH, Hofer E, Ginsburg D, Hu B, Keating B, Schick UM, Brody JA, Li JZ, Chen Z, Zeller T, Guralnik JM, Chasman DI, Peters LL, Kubo M, Becker DM, Li J, Eiriksdottir G, Rotter JI, Levy D, Grossmann V, Patel KV, Chen CH, Ridker PM, Tang H, Launer LJ, Rice KM, Li-Gao R, Ferrucci L, Evans MK, Choudhuri A, Trompouki E, Abraham BJ, Yang S, Takahashi A, Kamatani Y, Kooperberg C, Harris TB, Jee SH, Coresh J, Tsai FJ, Longo DL, Chen YT, Felix JF, Yang Q, Psaty BM, Boerwinkle E, Becker LC, Mook-Kanamori DO, Wilson JG, Gudnason V, O'Donnell CJ, Dehghan A, Cupples LA, Nalls MA, Morris AP, Okada Y, Reiner AP, Zon LI, Ganesh SK. 2017. Genome-wide Trans-ethnic Meta-analysis Identifies Seven Genetic Loci Influencing Erythrocyte Traits and a Role for RBPMS in Erythropoiesis. American journal of human genetics. 100(1):51-63. Pubmed: 28017375 DOI:S0002-9297(16)30520-1 Genome-wide association studies (GWASs) have identified loci for erythrocyte traits in primarily European ancestry populations. We conducted GWAS meta-analyses of six erythrocyte traits in 71,638 individuals from European, East Asian, and African ancestries using a Bayesian approach to account for heterogeneity in allelic effects and variation in the structure of linkage disequilibrium between ethnicities. We identified seven loci for erythrocyte traits including a locus (RBPMS/GTF2E2) associated with mean corpuscular hemoglobin and mean corpuscular volume. Statistical fine-mapping at this locus pointed to RBPMS at this locus and excluded nearby GTF2E2. Using zebrafish morpholino to evaluate loss of function, we observed a strong in vivo erythropoietic effect for RBPMS but not for GTF2E2, supporting the statistical fine-mapping at this locus and demonstrating that RBPMS is a regulator of erythropoiesis. Our findings show the utility of trans-ethnic GWASs for discovery and characterization of genetic loci influencing hematologic traits.Copyright © 2017 American Society of Human Genetics. All rights reserved. -
Henninger J, Santoso B, Hans S, Durand E, Moore J, Mosimann C, Brand M, Traver D, Zon L. 2017. Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development. Nature cell biology. 19(1):17-27. Pubmed: 27870830 DOI:10.1038/ncb3444 Henninger J, Santoso B, Hans S, Durand E, Moore J, Mosimann C, Brand M, Traver D, Zon L. 2017. Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development. Nature cell biology. 19(1):17-27. Pubmed: 27870830 DOI:10.1038/ncb3444 Haematopoietic stem cells (HSCs) arise in the developing aorta during embryogenesis. The number of HSC clones born has been estimated through transplantation, but experimental approaches to assess the absolute number of forming HSCs in a native setting have remained challenging. Here, we applied single-cell and clonal analysis of HSCs in zebrafish to quantify developing HSCs. Targeting creER in developing cd41:eGFP HSCs enabled long-term assessment of their blood contribution. We also applied the Brainbow-based multicolour Zebrabow system with drl:creER that is active in early haematopoiesis to induce heritable colour barcoding unique to each HSC and its progeny. Our findings reveal that approximately 21 HSC clones exist prior to HSC emergence and 30 clones are present during peak production from aortic endothelium. Our methods further reveal that stress haematopoiesis, including sublethal irradiation and transplantation, reduces clonal diversity. Our findings provide quantitative insights into the early clonal events that regulate haematopoietic development. -
Ben Nasr M, Tezza S, D'Addio F, Mameli C, Usuelli V, Maestroni A, Corradi D, Belletti S, Albarello L, Becchi G, Fadini GP, Schuetz C, Markmann J, Wasserfall C, Zon L, Zuccotti GV, Fiorina P. 2017. PD-L1 genetic overexpression or pharmacological restoration in hematopoietic stem and progenitor cells reverses autoimmune diabetes. Science translational medicine. 9(416). Pubmed: 29141886 DOI:10.1126/scitranslmed.aam7543 Ben Nasr M, Tezza S, D'Addio F, Mameli C, Usuelli V, Maestroni A, Corradi D, Belletti S, Albarello L, Becchi G, Fadini GP, Schuetz C, Markmann J, Wasserfall C, Zon L, Zuccotti GV, Fiorina P. 2017. PD-L1 genetic overexpression or pharmacological restoration in hematopoietic stem and progenitor cells reverses autoimmune diabetes. Science translational medicine. 9(416). Pubmed: 29141886 DOI:10.1126/scitranslmed.aam7543 Immunologically based clinical trials performed thus far have failed to cure type 1 diabetes (T1D), in part because these approaches were nonspecific. Because the disease is driven by autoreactive CD4 T cells, which destroy β cells, transplantation of hematopoietic stem and progenitor cells (HSPCs) has been recently offered as a therapy for T1D. Our transcriptomic profiling of HSPCs revealed that these cells are deficient in programmed death ligand 1 (PD-L1), an important immune checkpoint, in the T1D nonobese diabetic (NOD) mouse model. Notably, the immunoregulatory molecule PD-L1 plays a determinant role in controlling/inhibiting activated T cells and thus maintains immune tolerance. Furthermore, our genome-wide and bioinformatic analysis revealed the existence of a network of microRNAs (miRNAs) controlling PD-L1 expression, and silencing one of key altered miRNAs restored PD-L1 expression in HSPCs. We therefore sought to determine whether restoration of this defect would cure T1D as an alternative to immunosuppression. Genetically engineered or pharmacologically modulated HSPCs overexpressing PD-L1 inhibited the autoimmune response in vitro, reverted diabetes in newly hyperglycemic NOD mice in vivo, and homed to the pancreas of hyperglycemic NOD mice. The PD-L1 expression defect was confirmed in human HSPCs in T1D patients as well, and pharmacologically modulated human HSPCs also inhibited the autoimmune response in vitro. Targeting a specific immune checkpoint defect in HSPCs thus may contribute to establishing a cure for T1D.Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. -
Fazio M, Avagyan S, van Rooijen E, Mannherz W, Kaufman CK, Lobbardi R, Langenau DM, Zon LI. 2017. Efficient Transduction of Zebrafish Melanoma Cell Lines and Embryos Using Lentiviral Vectors. Zebrafish. 14(4):379-382. Pubmed: 28557653 DOI:10.1089/zeb.2017.1434 Fazio M, Avagyan S, van Rooijen E, Mannherz W, Kaufman CK, Lobbardi R, Langenau DM, Zon LI. 2017. Efficient Transduction of Zebrafish Melanoma Cell Lines and Embryos Using Lentiviral Vectors. Zebrafish. 14(4):379-382. Pubmed: 28557653 DOI:10.1089/zeb.2017.1434 The establishment of in vitro cultures of zebrafish cancer cells has expanded the potential of zebrafish as a disease model. However, the lack of effective methods for gene delivery and genetic manipulation has limited the experimental applications of these cultures. To overcome this barrier, we tested and optimized vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral and retroviral vector transduction protocols. We show that lentivirus successfully and efficiently transduced zebrafish melanoma cell lines in vitro, allowing antibiotic selection, fluorescence-based sorting, and in vivo allotransplantation. In addition, injection of concentrated lentiviral particles into embryos and tumors established the feasibility of in vivo gene delivery. -
Blaser BW, Moore JL, Hagedorn EJ, Li B, Riquelme R, Lichtig A, Yang S, Zhou Y, Tamplin OJ, Binder V, Zon LI. 2017. CXCR1 remodels the vascular niche to promote hematopoietic stem and progenitor cell engraftment. The Journal of experimental medicine. 214(4):1011-1027. Pubmed: 28351983 DOI:10.1084/jem.20161616 Blaser BW, Moore JL, Hagedorn EJ, Li B, Riquelme R, Lichtig A, Yang S, Zhou Y, Tamplin OJ, Binder V, Zon LI. 2017. CXCR1 remodels the vascular niche to promote hematopoietic stem and progenitor cell engraftment. The Journal of experimental medicine. 214(4):1011-1027. Pubmed: 28351983 DOI:10.1084/jem.20161616 The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSPC) biology. Recent advances marking fluorescent HSPCs have allowed exquisite visualization of HSPCs in the caudal hematopoietic tissue (CHT) of the developing zebrafish. Here, we show that the chemokine and its receptor, , are expressed by zebrafish endothelial cells, and we identify signaling as a positive regulator of HSPC colonization. Single-cell tracking experiments demonstrated that this is a result of increases in HSPC-endothelial cell "cuddling," HSPC residency time within the CHT, and HSPC mitotic rate. Enhanced signaling was associated with an increase in the volume of the CHT and induction of expression. Finally, using parabiotic zebrafish, we show that acts HSPC nonautonomously to improve the efficiency of donor HSPC engraftment. This work identifies a mechanism by which the hematopoietic niche remodels to promote HSPC engraftment and suggests that signaling is a potential therapeutic target in patients undergoing hematopoietic stem cell transplantation.© 2017 Blaser et al. -
Wiley DS, Redfield SE, Zon LI. 2017. Chemical screening in zebrafish for novel biological and therapeutic discovery. Methods in cell biology. 138:651-679. Pubmed: 28129862 DOI:S0091-679X(16)30147-9 Wiley DS, Redfield SE, Zon LI. 2017. Chemical screening in zebrafish for novel biological and therapeutic discovery. Methods in cell biology. 138:651-679. Pubmed: 28129862 DOI:S0091-679X(16)30147-9 Zebrafish chemical screening allows for an in vivo assessment of small molecule modulation of biological processes. Compound toxicities, chemical alterations by metabolism, pharmacokinetic and pharmacodynamic properties, and modulation of cell niches can be studied with this method. Furthermore, zebrafish screening is straightforward and cost effective. Zebrafish provide an invaluable platform for novel therapeutic discovery through chemical screening.Copyright © 2017 Elsevier Inc. All rights reserved. -
Theodore LN, Hagedorn EJ, Cortes M, Natsuhara K, Liu SY, Perlin JR, Yang S, Daily ML, Zon LI, North TE. 2017. Distinct Roles for Matrix Metalloproteinases 2 and 9 in Embryonic Hematopoietic Stem Cell Emergence, Migration, and Niche Colonization. Stem cell reports. 8(5):1226-1241. Pubmed: 28416284 DOI:S2213-6711(17)30122-4 Theodore LN, Hagedorn EJ, Cortes M, Natsuhara K, Liu SY, Perlin JR, Yang S, Daily ML, Zon LI, North TE. 2017. Distinct Roles for Matrix Metalloproteinases 2 and 9 in Embryonic Hematopoietic Stem Cell Emergence, Migration, and Niche Colonization. Stem cell reports. 8(5):1226-1241. Pubmed: 28416284 DOI:S2213-6711(17)30122-4 Hematopoietic stem/progenitor cells (HSPCs) are formed during ontogeny from hemogenic endothelium in the ventral wall of the dorsal aorta (VDA). Critically, the cellular mechanism(s) allowing HSPC egress and migration to secondary niches are incompletely understood. Matrix metalloproteinases (MMPs) are inflammation-responsive proteins that regulate extracellular matrix (ECM) remodeling, cellular interactions, and signaling. Here, inhibition of vascular-associated Mmp2 function caused accumulation of fibronectin-rich ECM, retention of runx1/cmyb HSPCs in the VDA, and delayed caudal hematopoietic tissue (CHT) colonization; these defects were absent in fibronectin mutants, indicating that Mmp2 facilitates endothelial-to-hematopoietic transition via ECM remodeling. In contrast, Mmp9 was dispensable for HSPC budding, being instead required for proper colonization of secondary niches. Significantly, these migration defects were mimicked by overexpression and blocked by knockdown of C-X-C motif chemokine-12 (cxcl12), suggesting that Mmp9 controls CHT homeostasis through chemokine regulation. Our findings indicate Mmp2 and Mmp9 play distinct but complementary roles in developmental HSPC production and migration.Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved. 2016
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Srivastava A, Kay MA, Athanasopoulos T, Angastiniotis M, Anagnostopoulos A, Karponi G, Yannaki E, Zon LI, Lederer CW, Phylactides MS, Kleanthous M. 2016. A Tribute to George Stamatoyannopoulos. Human gene therapy. 27(4):280-6. Pubmed: 27074151 DOI:10.1089/hum.2016.29025.gst Srivastava A, Kay MA, Athanasopoulos T, Angastiniotis M, Anagnostopoulos A, Karponi G, Yannaki E, Zon LI, Lederer CW, Phylactides MS, Kleanthous M. 2016. A Tribute to George Stamatoyannopoulos. Human gene therapy. 27(4):280-6. Pubmed: 27074151 DOI:10.1089/hum.2016.29025.gst -
Ciarlo CA, Zon LI. 2016. Embryonic cell culture in zebrafish. Methods in cell biology. 133:1-10. Pubmed: 27263406 DOI:S0091-679X(16)00045-5 Ciarlo CA, Zon LI. 2016. Embryonic cell culture in zebrafish. Methods in cell biology. 133:1-10. Pubmed: 27263406 DOI:S0091-679X(16)00045-5 Zebrafish embryonic cell cultures have many useful properties that make them complementary to intact embryos for a wide range of studies. Embryonic cell cultures allow for maintenance of transient cell populations, control of chemical and mechanical cues received by cells, and facile chemical screening. Zebrafish cells can be cultured in either heterogeneous or homogeneous cultures from a wide range of developmental time points. Here we describe two methods with particular applicability to chemical screening: a method for the culture of blastomeres for directed differentiation toward the myogenic lineage and a method for the culture of neural crest cells in heterogeneous cultures from early somitogenesis embryos.Copyright © 2016 Elsevier Inc. All rights reserved. -
Burberry A, Suzuki N, Wang JY, Moccia R, Mordes DA, Stewart MH, Suzuki-Uematsu S, Ghosh S, Singh A, Merkle FT, Koszka K, Li QZ, Zon L, Rossi DJ, Trowbridge JJ, Notarangelo LD, Eggan K. 2016. Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease. Science translational medicine. 8(347):347ra93. Pubmed: 27412785 DOI:10.1126/scitranslmed.aaf6038 Burberry A, Suzuki N, Wang JY, Moccia R, Mordes DA, Stewart MH, Suzuki-Uematsu S, Ghosh S, Singh A, Merkle FT, Koszka K, Li QZ, Zon L, Rossi DJ, Trowbridge JJ, Notarangelo LD, Eggan K. 2016. Loss-of-function mutations in the C9ORF72 mouse ortholog cause fatal autoimmune disease. Science translational medicine. 8(347):347ra93. Pubmed: 27412785 DOI:10.1126/scitranslmed.aaf6038 C9ORF72 mutations are found in a significant fraction of patients suffering from amyotrophic lateral sclerosis and frontotemporal dementia, yet the function of the C9ORF72 gene product remains poorly understood. We show that mice harboring loss-of-function mutations in the ortholog of C9ORF72 develop splenomegaly, neutrophilia, thrombocytopenia, increased expression of inflammatory cytokines, and severe autoimmunity, ultimately leading to a high mortality rate. Transplantation of mutant mouse bone marrow into wild-type recipients was sufficient to recapitulate the phenotypes observed in the mutant animals, including autoimmunity and premature mortality. Reciprocally, transplantation of wild-type mouse bone marrow into mutant mice improved their phenotype. We conclude that C9ORF72 serves an important function within the hematopoietic system to restrict inflammation and the development of autoimmunity.Copyright © 2016, American Association for the Advancement of Science. -
Yang S, Ott CJ, Rossmann MP, Superdock M, Zon LI, Zhou Y. 2016. Chromatin immunoprecipitation and an open chromatin assay in zebrafish erythrocytes. Methods in cell biology. 135:387-412. Pubmed: 27443937 DOI:S0091-679X(16)30053-X Yang S, Ott CJ, Rossmann MP, Superdock M, Zon LI, Zhou Y. 2016. Chromatin immunoprecipitation and an open chromatin assay in zebrafish erythrocytes. Methods in cell biology. 135:387-412. Pubmed: 27443937 DOI:S0091-679X(16)30053-X Zebrafish is an excellent genetic and developmental model for the study of vertebrate development and disease. Its ability to produce an abundance of transparent, externally developed embryos has facilitated large-scale genetic and chemical screens for the identification of critical genes and chemical factors that modulate developmental pathways. These studies can have profound implications for the diagnosis and treatment of a variety of human diseases. Recent advancements in molecular and genomic studies have provided valuable tools and resources for comprehensive and high-resolution analysis of epigenomes during cell specification and lineage differentiation throughout development. In this chapter, we describe two simple methods to evaluate protein-DNA interaction and chromatin architecture in erythrocytes from adult zebrafish. These are chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) and an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). These techniques, together with gene expression profiling, are useful for analyzing epigenomic regulation of cell specification, differentiation, and function during zebrafish development in both normal and disease models.Copyright © 2016 Elsevier Inc. All rights reserved. -
Nasrallah R, Fast EM, Solaimani P, Knezevic K, Eliades A, Patel R, Thambyrajah R, Unnikrishnan A, Thoms J, Beck D, Vink CS, Smith A, Wong J, Shepherd M, Kent D, Roychoudhuri R, Paul F, Klippert J, Hammes A, Willnow T, Göttgens B, Dzierzak E, Zon LI, Lacaud G, Kouskoff V, Pimanda JE. 2016. Identification of novel regulators of developmental hematopoiesis using Endoglin regulatory elements as molecular probes. Blood. 128(15):1928-1939. Pubmed: 27554085 Nasrallah R, Fast EM, Solaimani P, Knezevic K, Eliades A, Patel R, Thambyrajah R, Unnikrishnan A, Thoms J, Beck D, Vink CS, Smith A, Wong J, Shepherd M, Kent D, Roychoudhuri R, Paul F, Klippert J, Hammes A, Willnow T, Göttgens B, Dzierzak E, Zon LI, Lacaud G, Kouskoff V, Pimanda JE. 2016. Identification of novel regulators of developmental hematopoiesis using Endoglin regulatory elements as molecular probes. Blood. 128(15):1928-1939. Pubmed: 27554085 Enhancers are the primary determinants of cell identity, and specific promoter/enhancer combinations of Endoglin (ENG) have been shown to target blood and endothelium in the embryo. Here, we generated a series of embryonic stem cell lines, each targeted with reporter constructs driven by specific promoter/enhancer combinations of ENG, to evaluate their discriminative potential and value as molecular probes of the corresponding transcriptome. The Eng promoter (P) in combination with the -8/+7/+9-kb enhancers, targeted cells in FLK1 mesoderm that were enriched for blast colony forming potential, whereas the P/-8-kb enhancer targeted TIE2+/c-KIT+/CD41- endothelial cells that were enriched for hematopoietic potential. These fractions were isolated using reporter expression and their transcriptomes profiled by RNA-seq. There was high concordance between our signatures and those from embryos with defects at corresponding stages of hematopoiesis. Of the 6 genes that were upregulated in both hemogenic mesoderm and hemogenic endothelial fractions targeted by the reporters, LRP2, a multiligand receptor, was the only gene that had not previously been associated with hematopoiesis. We show that LRP2 is indeed involved in definitive hematopoiesis and by doing so validate the use of reporter gene-coupled enhancers as probes to gain insights into transcriptional changes that facilitate cell fate transitions.© 2016 by The American Society of Hematology. -
Aranda-Orgilles B, Saldaña-Meyer R, Wang E, Trompouki E, Fassl A, Lau S, Mullenders J, Rocha PP, Raviram R, Guillamot M, Sánchez-Díaz M, Wang K, Kayembe C, Zhang N, Amoasii L, Choudhuri A, Skok JA, Schober M, Reinberg D, Sicinski P, Schrewe H, Tsirigos A, Zon LI, Aifantis I. 2016. MED12 Regulates HSC-Specific Enhancers Independently of Mediator Kinase Activity to Control Hematopoiesis. Cell stem cell. 19(6):784-799. Pubmed: 27570068 DOI:S1934-5909(16)30251-X Aranda-Orgilles B, Saldaña-Meyer R, Wang E, Trompouki E, Fassl A, Lau S, Mullenders J, Rocha PP, Raviram R, Guillamot M, Sánchez-Díaz M, Wang K, Kayembe C, Zhang N, Amoasii L, Choudhuri A, Skok JA, Schober M, Reinberg D, Sicinski P, Schrewe H, Tsirigos A, Zon LI, Aifantis I. 2016. MED12 Regulates HSC-Specific Enhancers Independently of Mediator Kinase Activity to Control Hematopoiesis. Cell stem cell. 19(6):784-799. Pubmed: 27570068 DOI:S1934-5909(16)30251-X Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis.Copyright © 2016 Elsevier Inc. All rights reserved. -
Robertson AL, Avagyan S, Gansner JM, Zon LI. 2016. Understanding the regulation of vertebrate hematopoiesis and blood disorders - big lessons from a small fish. FEBS letters. 590(22):4016-4033. Pubmed: 27616157 DOI:10.1002/1873-3468.12415 Robertson AL, Avagyan S, Gansner JM, Zon LI. 2016. Understanding the regulation of vertebrate hematopoiesis and blood disorders - big lessons from a small fish. FEBS letters. 590(22):4016-4033. Pubmed: 27616157 DOI:10.1002/1873-3468.12415 Hematopoietic stem cells (HSCs) give rise to all differentiated blood cells. Understanding the mechanisms that regulate self-renewal and lineage specification of HSCs is key for developing treatments for many human diseases. Zebrafish have emerged as an excellent model for studying vertebrate hematopoiesis. This review will highlight the unique strengths of zebrafish and important findings that have emerged from studies of blood development and disorders using this system. We discuss recent advances in our understanding of hematopoiesis, including the origin of HSCs, molecular control of their development, and key signaling pathways involved in their regulation. We highlight significant findings from zebrafish models of blood disorders and discuss their application for investigating stem cell dysfunction in disease and for the development of new therapeutics.© 2016 Federation of European Biochemical Societies. -
Rossmann MP, Zhou Y, Zon LI. 2016. Development: For cloche the Bell Tolls. Current biology : CB. 26(19):R890-R892. Pubmed: 27728792 DOI:S0960-9822(16)30921-6 Rossmann MP, Zhou Y, Zon LI. 2016. Development: For cloche the Bell Tolls. Current biology : CB. 26(19):R890-R892. Pubmed: 27728792 DOI:S0960-9822(16)30921-6 A recent publication identifies npas4l as the gene defective in the well-known cloche mutant that lacks most endothelial as well as hematopoietic cells. This work poses intriguing questions as to the genetic and molecular nature of the origin of hemato-vascular lineages during early embryogenesis.Copyright © 2016 Elsevier Ltd. All rights reserved. -
Fast EM, Zon LI. 2016. Aging Hematopoietic Stem Cells Make Their History. Developmental cell. 39(4):390-391. Pubmed: 27875682 DOI:S1534-5807(16)30789-4 Fast EM, Zon LI. 2016. Aging Hematopoietic Stem Cells Make Their History. Developmental cell. 39(4):390-391. Pubmed: 27875682 DOI:S1534-5807(16)30789-4 A major hallmark of aging is a decline in tissue regeneration. In a recent issue of Cell, Bernitz and colleagues (2016) determine the divisional history of hematopoietic stem cells (HSCs) to be a key player of regenerative potential in the aging mouse.Copyright © 2016 Elsevier Inc. All rights reserved. -
Wakabayashi A, Ulirsch JC, Ludwig LS, Fiorini C, Yasuda M, Choudhuri A, McDonel P, Zon LI, Sankaran VG. 2016. Insight into GATA1 transcriptional activity through interrogation of cis elements disrupted in human erythroid disorders. Proceedings of the National Academy of Sciences of the United States of America. 113(16):4434-9. Pubmed: 27044088 DOI:10.1073/pnas.1521754113 Wakabayashi A, Ulirsch JC, Ludwig LS, Fiorini C, Yasuda M, Choudhuri A, McDonel P, Zon LI, Sankaran VG. 2016. Insight into GATA1 transcriptional activity through interrogation of cis elements disrupted in human erythroid disorders. Proceedings of the National Academy of Sciences of the United States of America. 113(16):4434-9. Pubmed: 27044088 DOI:10.1073/pnas.1521754113 Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptionalcis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders. -
Tan JL, Fogley RD, Flynn RA, Ablain J, Yang S, Saint-André V, Fan ZP, Do BT, Laga AC, Fujinaga K, Santoriello C, Greer CB, Kim YJ, Clohessy JG, Bothmer A, Pandell N, Avagyan S, Brogie JE, van Rooijen E, Hagedorn EJ, Shyh-Chang N, White RM, Price DH, Pandolfi PP, Peterlin BM, Zhou Y, Kim TH, Asara JM, Chang HY, Young RA, Zon LI. 2016. Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma. Molecular cell. 62(1):34-46. Pubmed: 27058786 DOI:S1097-2765(16)00223-9 Tan JL, Fogley RD, Flynn RA, Ablain J, Yang S, Saint-André V, Fan ZP, Do BT, Laga AC, Fujinaga K, Santoriello C, Greer CB, Kim YJ, Clohessy JG, Bothmer A, Pandell N, Avagyan S, Brogie JE, van Rooijen E, Hagedorn EJ, Shyh-Chang N, White RM, Price DH, Pandolfi PP, Peterlin BM, Zhou Y, Kim TH, Asara JM, Chang HY, Young RA, Zon LI. 2016. Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma. Molecular cell. 62(1):34-46. Pubmed: 27058786 DOI:S1097-2765(16)00223-9 Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.Copyright © 2016 Elsevier Inc. All rights reserved. -
Ablain J, Zon LI. 2016. Tissue-specific gene targeting using CRISPR/Cas9. Methods in cell biology. 135:189-202. Pubmed: 27443926 DOI:S0091-679X(16)00049-2 Ablain J, Zon LI. 2016. Tissue-specific gene targeting using CRISPR/Cas9. Methods in cell biology. 135:189-202. Pubmed: 27443926 DOI:S0091-679X(16)00049-2 The zebrafish has been a powerful model in forward genetic screens to identify genes essential for organogenesis and embryonic development. Conversely, using reverse genetics to investigate specific gene function requires phenotypic analysis of complete gene inactivation. Despite the availability and efficacy of morpholinos, the lack of tractable and efficient knockout technologies has impeded reverse genetic studies in the zebrafish, particularly in adult animals. The recent development of genome-editing technologies such as CRISPR/Cas9 greatly widened the scope of loss-of-function studies in the zebrafish, allowing for the rapid phenotypic assessment of gene silencing in embryos, the generation of knockout lines, and large-scale reverse genetic screens. Tissue-specific gene inactivation would be ideal for these studies given the caveats of whole-embryo gene silencing, yet spatial control of gene targeting remains a challenge. In this chapter, we focus on tissue-specific gene inactivation using the CRISPR/Cas9 technology. We first explain the rationale for this technique, including some of its potential applications to tackle important biological issues and the inability of current technologies to address these issues. We then present a method to target genes in a tissue-specific manner in the zebrafish. Finally, we discuss technical difficulties and limitations of this method as well as possible future developments.Copyright © 2016 Elsevier Inc. All rights reserved. -
Avagyan S, Zon LI. 2016. Fish to Learn: Insights into Blood Development and Blood Disorders from Zebrafish Hematopoiesis. Human gene therapy. 27(4):287-94. Pubmed: 27018965 DOI:10.1089/hum.2016.024 Avagyan S, Zon LI. 2016. Fish to Learn: Insights into Blood Development and Blood Disorders from Zebrafish Hematopoiesis. Human gene therapy. 27(4):287-94. Pubmed: 27018965 DOI:10.1089/hum.2016.024 Since its introduction in early 1980s, the zebrafish (Danio rerio) has become an invaluable vertebrate animal model system to study many human disorders in almost all systems, from hepatic and brain pathology, to autoimmune and psychiatric disorders. Hematopoiesis between zebrafish and mammals is highly conserved, making the zebrafish an attractive model to study hematopoietic development and blood disorders. Unique attributes of the zebrafish include the ability to perform large-scale genetic and chemical screens in vivo, study development at the cellular level, and use transgenic fish to dissect mechanisms of disease or drug effects. This review summarizes major discoveries that helped define molecular control of hematopoiesis in vertebrates and specific contributions from studies in zebrafish. -
Svoboda O, Stachura DL, Machonova O, Zon LI, Traver D, Bartunek P. 2016. Ex vivo tools for the clonal analysis of zebrafish hematopoiesis. Nature protocols. 11(5):1007-20. Pubmed: 27123951 DOI:10.1038/nprot.2016.053 Svoboda O, Stachura DL, Machonova O, Zon LI, Traver D, Bartunek P. 2016. Ex vivo tools for the clonal analysis of zebrafish hematopoiesis. Nature protocols. 11(5):1007-20. Pubmed: 27123951 DOI:10.1038/nprot.2016.053 This protocol describes the ex vivo characterization of zebrafish hematopoietic progenitors. We show how to isolate zebrafish hematopoietic cells for cultivation and differentiation in colony assays in semi-solid media. We also describe procedures for the generation of recombinant zebrafish cytokines and for the isolation of carp serum, which are essential components of the medium required to grow zebrafish hematopoietic cells ex vivo. The outcome of these clonal assays can easily be evaluated using standard microscopy techniques after 3-10 d in culture. In addition, we describe how to isolate individual colonies for further imaging and gene expression profiling. In other vertebrate model organisms, ex vivo assays have been crucial for elucidating the relationships among hematopoietic stem cells (HSCs), progenitor cells and their mature progeny. The present protocol should facilitate such studies on cells derived from zebrafish. -
Zon L. 2016. Modeling human diseases: an education in interactions and interdisciplinary approaches. Disease models & mechanisms. 9(6):597-600. Pubmed: 27483497 DOI:10.1242/dmm.025882 Zon L. 2016. Modeling human diseases: an education in interactions and interdisciplinary approaches. Disease models & mechanisms. 9(6):597-600. Pubmed: 27483497 DOI:10.1242/dmm.025882 Traditionally, most investigators in the biomedical arena exploit one model system in the course of their careers. Occasionally, an investigator will switch models. The selection of a suitable model system is a crucial step in research design. Factors to consider include the accuracy of the model as a reflection of the human disease under investigation, the numbers of animals needed and ease of husbandry, its physiology and developmental biology, and the ability to apply genetics and harness the model for drug discovery. In my lab, we have primarily used the zebrafish but combined it with other animal models and provided a framework for others to consider the application of developmental biology for therapeutic discovery. Our interdisciplinary approach has led to many insights into human diseases and to the advancement of candidate drugs to clinical trials. Here, I draw on my experiences to highlight the importance of combining multiple models, establishing infrastructure and genetic tools, forming collaborations, and interfacing with the medical community for successful translation of basic findings to the clinic.© 2016. Published by The Company of Biologists Ltd. -
Rowe RG, Mandelbaum J, Zon LI, Daley GQ. 2016. Engineering Hematopoietic Stem Cells: Lessons from Development. Cell stem cell. 18(6):707-720. Pubmed: 27257760 DOI:S1934-5909(16)30100-X Rowe RG, Mandelbaum J, Zon LI, Daley GQ. 2016. Engineering Hematopoietic Stem Cells: Lessons from Development. Cell stem cell. 18(6):707-720. Pubmed: 27257760 DOI:S1934-5909(16)30100-X Cell engineering has brought us tantalizingly close to the goal of deriving patient-specific hematopoietic stem cells (HSCs). While directed differentiation and transcription factor-mediated conversion strategies have generated progenitor cells with multilineage potential, to date, therapy-grade engineered HSCs remain elusive due to insufficient long-term self-renewal and inadequate differentiated progeny functionality. A cross-species approach involving zebrafish and mammalian systems offers complementary methodologies to improve understanding of native HSCs. Here, we discuss the role of conserved developmental timing processes in vertebrate hematopoiesis, highlighting how identification and manipulation of stage-specific factors that specify HSC developmental state must be harnessed to engineer HSCs for therapy.Copyright © 2016 Elsevier Inc. All rights reserved. -
Hagedorn EJ, Cillis JL, Curley CR, Patch TC, Li B, Blaser BW, Riquelme R, Zon LI, Shah DI. 2016. Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae. Journal of visualized experiments : JoVE. Pubmed: 27341538 DOI:10.3791/54168 Hagedorn EJ, Cillis JL, Curley CR, Patch TC, Li B, Blaser BW, Riquelme R, Zon LI, Shah DI. 2016. Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae. Journal of visualized experiments : JoVE. Pubmed: 27341538 DOI:10.3791/54168 Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development. -
Giani FC, Fiorini C, Wakabayashi A, Ludwig LS, Salem RM, Jobaliya CD, Regan SN, Ulirsch JC, Liang G, Steinberg-Shemer O, Guo MH, Esko T, Tong W, Brugnara C, Hirschhorn JN, Weiss MJ, Zon LI, Chou ST, French DL, Musunuru K, Sankaran VG. 2016. Targeted Application of Human Genetic Variation Can Improve Red Blood Cell Production from Stem Cells. Cell stem cell. 18(1):73-78. Pubmed: 26607381 DOI:10.1016/j.stem.2015.09.015 Giani FC, Fiorini C, Wakabayashi A, Ludwig LS, Salem RM, Jobaliya CD, Regan SN, Ulirsch JC, Liang G, Steinberg-Shemer O, Guo MH, Esko T, Tong W, Brugnara C, Hirschhorn JN, Weiss MJ, Zon LI, Chou ST, French DL, Musunuru K, Sankaran VG. 2016. Targeted Application of Human Genetic Variation Can Improve Red Blood Cell Production from Stem Cells. Cell stem cell. 18(1):73-78. Pubmed: 26607381 DOI:10.1016/j.stem.2015.09.015 Multipotent and pluripotent stem cells are potential sources for cell and tissue replacement therapies. For example, stem cell-derived red blood cells (RBCs) are a potential alternative to donated blood, but yield and quality remain a challenge. Here, we show that application of insight from human population genetic studies can enhance RBC production from stem cells. The SH2B3 gene encodes a negative regulator of cytokine signaling and naturally occurring loss-of-function variants in this gene increase RBC counts in vivo. Targeted suppression of SH2B3 in primary human hematopoietic stem and progenitor cells enhanced the maturation and overall yield of in-vitro-derived RBCs. Moreover, inactivation of SH2B3 by CRISPR/Cas9 genome editing in human pluripotent stem cells allowed enhanced erythroid cell expansion with preserved differentiation. Our findings therefore highlight the potential for combining human genome variation studies with genome editing approaches to improve cell and tissue production for regenerative medicine.Copyright © 2016 Elsevier Inc. All rights reserved. -
Dang M, Fogley R, Zon LI. 2016. Identifying Novel Cancer Therapies Using Chemical Genetics and Zebrafish. Advances in experimental medicine and biology. 916:103-24. Pubmed: 27165351 DOI:10.1007/978-3-319-30654-4_5 Dang M, Fogley R, Zon LI. 2016. Identifying Novel Cancer Therapies Using Chemical Genetics and Zebrafish. Advances in experimental medicine and biology. 916:103-24. Pubmed: 27165351 DOI:10.1007/978-3-319-30654-4_5 Chemical genetics is the use of small molecules to perturb biological pathways. This technique is a powerful tool for implicating genes and pathways in developmental programs and disease, and simultaneously provides a platform for the discovery of novel therapeutics. The zebrafish is an advantageous model for in vivo high-throughput small molecule screening due to translational appeal, high fecundity, and a unique set of developmental characteristics that support genetic manipulation, chemical treatment, and phenotype detection. Chemical genetic screens in zebrafish can identify hit compounds that target oncogenic processes-including cancer initiation and maintenance, metastasis, and angiogenesis-and may serve as cancer therapies. Notably, by combining drug discovery and animal testing, in vivo screening of small molecules in zebrafish has enabled rapid translation of hit anti-cancer compounds to the clinic, especially through the repurposing of FDA-approved drugs. Future technological advancements in automation and high-powered imaging, as well as the development and characterization of new mutant and transgenic lines, will expand the scope of chemical genetics in zebrafish. -
Huang J, Liu X, Li D, Shao Z, Cao H, Zhang Y, Trompouki E, Bowman TV, Zon LI, Yuan GC, Orkin SH, Xu J. 2016. Dynamic Control of Enhancer Repertoires Drives Lineage and Stage-Specific Transcription during Hematopoiesis. Developmental cell. 36(1):9-23. Pubmed: 26766440 DOI:10.1016/j.devcel.2015.12.014 Huang J, Liu X, Li D, Shao Z, Cao H, Zhang Y, Trompouki E, Bowman TV, Zon LI, Yuan GC, Orkin SH, Xu J. 2016. Dynamic Control of Enhancer Repertoires Drives Lineage and Stage-Specific Transcription during Hematopoiesis. Developmental cell. 36(1):9-23. Pubmed: 26766440 DOI:10.1016/j.devcel.2015.12.014 Enhancers are the primary determinants of cell identity, but the regulatory components controlling enhancer turnover during lineage commitment remain largely unknown. Here we compare the enhancer landscape, transcriptional factor occupancy, and transcriptomic changes in human fetal and adult hematopoietic stem/progenitor cells and committed erythroid progenitors. We find that enhancers are modulated pervasively and direct lineage- and stage-specific transcription. GATA2-to-GATA1 switch is prevalent at dynamic enhancers and drives erythroid enhancer commissioning. Examination of lineage-specific enhancers identifies transcription factors and their combinatorial patterns in enhancer turnover. Importantly, by CRISPR/Cas9-mediated genomic editing, we uncover functional hierarchy of constituent enhancers within the SLC25A37 super-enhancer. Despite indistinguishable chromatin features, we reveal through genomic editing the functional diversity of several GATA switch enhancers in which enhancers with opposing functions cooperate to coordinate transcription. Thus, genome-wide enhancer profiling coupled with in situ enhancer editing provide critical insights into the functional complexity of enhancers during development.Copyright © 2016 Elsevier Inc. All rights reserved. -
Kaufman CK, Mosimann C, Fan ZP, Yang S, Thomas AJ, Ablain J, Tan JL, Fogley RD, van Rooijen E, Hagedorn EJ, Ciarlo C, White RM, Matos DA, Puller AC, Santoriello C, Liao EC, Young RA, Zon LI. 2016. A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science (New York, N.Y.). 351(6272):aad2197. Pubmed: 26823433 DOI:10.1126/science.aad2197 Kaufman CK, Mosimann C, Fan ZP, Yang S, Thomas AJ, Ablain J, Tan JL, Fogley RD, van Rooijen E, Hagedorn EJ, Ciarlo C, White RM, Matos DA, Puller AC, Santoriello C, Liao EC, Young RA, Zon LI. 2016. A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science (New York, N.Y.). 351(6272):aad2197. Pubmed: 26823433 DOI:10.1126/science.aad2197 The "cancerized field" concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAF(V600E) mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAF(V600E)-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.Copyright © 2016, American Association for the Advancement of Science. -
Gao X, Wu T, Johnson KD, Lahvic JL, Ranheim EA, Zon LI, Bresnick EH. 2016. GATA Factor-G-Protein-Coupled Receptor Circuit Suppresses Hematopoiesis. Stem cell reports. 6(3):368-82. Pubmed: 26905203 DOI:S2213-6711(16)00025-4 Gao X, Wu T, Johnson KD, Lahvic JL, Ranheim EA, Zon LI, Bresnick EH. 2016. GATA Factor-G-Protein-Coupled Receptor Circuit Suppresses Hematopoiesis. Stem cell reports. 6(3):368-82. Pubmed: 26905203 DOI:S2213-6711(16)00025-4 Hematopoietic stem cells (HSCs) originate from hemogenic endothelium within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo. The relationship between genetic circuits controlling stem cell genesis and multi-potency is not understood. A Gata2 cis element (+9.5) enhances Gata2 expression in the AGM and induces the endothelial to HSC transition. We demonstrated that GATA-2 rescued hematopoiesis in +9.5(-/-) AGMs. As G-protein-coupled receptors (GPCRs) are the most common targets for FDA-approved drugs, we analyzed the GPCR gene ensemble to identify GATA-2-regulated GPCRs. Of the 20 GATA-2-activated GPCR genes, four were GATA-1-activated, and only Gpr65 expression resembled Gata2. Contrasting with the paradigm in which GATA-2-activated genes promote hematopoietic stem and progenitor cell genesis/function, our mouse and zebrafish studies indicated that GPR65 suppressed hematopoiesis. GPR65 established repressive chromatin at the +9.5 site, restricted occupancy by the activator Scl/TAL1, and repressed Gata2 transcription. Thus, a Gata2 cis element creates a GATA-2-GPCR circuit that limits positive regulators that promote hematopoiesis.Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved. -
Adatto I, Krug L, Zon LI. 2016. The Red Light District and Its Effects on Zebrafish Reproduction. Zebrafish. 13(3):226-9. Pubmed: 26978703 DOI:10.1089/zeb.2015.1228 Adatto I, Krug L, Zon LI. 2016. The Red Light District and Its Effects on Zebrafish Reproduction. Zebrafish. 13(3):226-9. Pubmed: 26978703 DOI:10.1089/zeb.2015.1228 Light-dark cycles mimicking natural settings in a zebrafish facility are crucial for maintaining fish with an entrained circadian clock making them an ideal vertebrate model to study such rhythms. However, failure to provide optimal conditions to include complete darkness can lead to a disturbed circadian pacemaker affecting physiology and behavior in zebrafish. To meet building code requirements, the aquatics facility in use was outfitted with EXIT signs emitting a constant light. To determine if light radiating from the EXIT sign has an effect on zebrafish embryo production, 100 fish (1:1 m/f ratio) were split and housed at 10 fish/L. Half were housed directly in front of the EXIT sign, whereas the other half (control) were housed under a true 14-h light-10-h dark cycle. Reproductive success was evaluated by recording fecundity and viability from 10 weekly matings under two light colors: red (640 nm) and green (560 nm). On average the control group spawned twice as many embryos compared to those housed in front of a red EXIT sign, whereas green EXIT sign showed no difference. This suggests the importance of providing a complete dark environment within the night cycle and a recommendation toward dim green EXIT signs to avoid a decline in reproductive performance. -
Dang M, Henderson RE, Garraway LA, Zon LI. 2016. Long-term drug administration in the adult zebrafish using oral gavage for cancer preclinical studies. Disease models & mechanisms. 9(7):811-20. Pubmed: 27482819 DOI:10.1242/dmm.024166 Dang M, Henderson RE, Garraway LA, Zon LI. 2016. Long-term drug administration in the adult zebrafish using oral gavage for cancer preclinical studies. Disease models & mechanisms. 9(7):811-20. Pubmed: 27482819 DOI:10.1242/dmm.024166 Zebrafish are a major model for chemical genetics, and most studies use embryos when investigating small molecules that cause interesting phenotypes or that can rescue disease models. Limited studies have dosed adults with small molecules by means of water-borne exposure or injection techniques. Challenges in the form of drug delivery-related trauma and anesthesia-related toxicity have excluded the adult zebrafish from long-term drug efficacy studies. Here, we introduce a novel anesthetic combination of MS-222 and isoflurane to an oral gavage technique for a non-toxic, non-invasive and long-term drug administration platform. As a proof of principle, we established drug efficacy of the FDA-approved BRAF(V600E) inhibitor, Vemurafenib, in adult zebrafish harboring BRAF(V600E) melanoma tumors. In the model, adult casper zebrafish intraperitoneally transplanted with a zebrafish melanoma cell line (ZMEL1) and exposed to daily sub-lethal dosing at 100 mg/kg of Vemurafenib for 2 weeks via oral gavage resulted in an average 65% decrease in tumor burden and a 15% mortality rate. In contrast, Vemurafenib-resistant ZMEL1 cell lines, generated in culture from low-dose drug exposure for 4 months, did not respond to the oral gavage treatment regimen. Similarly, this drug treatment regimen can be applied for treatment of primary melanoma tumors in the zebrafish. Taken together, we developed an effective long-term drug treatment system that will allow the adult zebrafish to be used to identify more effective anti-melanoma combination therapies and opens up possibilities for treating adult models of other diseases.© 2016. Published by The Company of Biologists Ltd. -
Ray MK, Wiskow O, King MJ, Ismail N, Ergun A, Wang Y, Plys AJ, Davis CP, Kathrein K, Sadreyev R, Borowsky ML, Eggan K, Zon L, Galloway JL, Kingston RE. 2016. CAT7 and cat7l Long Non-coding RNAs Tune Polycomb Repressive Complex 1 Function during Human and Zebrafish Development. The Journal of biological chemistry. 291(37):19558-72. Pubmed: 27405765 DOI:10.1074/jbc.M116.730853 Ray MK, Wiskow O, King MJ, Ismail N, Ergun A, Wang Y, Plys AJ, Davis CP, Kathrein K, Sadreyev R, Borowsky ML, Eggan K, Zon L, Galloway JL, Kingston RE. 2016. CAT7 and cat7l Long Non-coding RNAs Tune Polycomb Repressive Complex 1 Function during Human and Zebrafish Development. The Journal of biological chemistry. 291(37):19558-72. Pubmed: 27405765 DOI:10.1074/jbc.M116.730853 The essential functions of polycomb repressive complex 1 (PRC1) in development and gene silencing are thought to involve long non-coding RNAs (lncRNAs), but few specific lncRNAs that guide PRC1 activity are known. We screened for lncRNAs, which co-precipitate with PRC1 from chromatin and found candidates that impact polycomb group protein (PcG)-regulated gene expression in vivo A novel lncRNA from this screen, CAT7, regulates expression and polycomb group binding at the MNX1 locus during early neuronal differentiation. CAT7 contains a unique tandem repeat domain that shares high sequence similarity to a non-syntenic zebrafish analog, cat7l Defects caused by interference of cat7l RNA during zebrafish embryogenesis were rescued by human CAT7 RNA, enhanced by interference of a PRC1 component, and suppressed by interference of a known PRC1 target gene, demonstrating cat7l genetically interacts with a PRC1. We propose a model whereby PRC1 acts in concert with specific lncRNAs and that CAT7/cat7l represents convergent lncRNAs that independently evolved to tune PRC1 repression at individual loci.© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. 2015
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Dang M, Zon LI. 2015. Screening for melanoma resistance genes in vivo. Pigment cell & melanoma research. 28(4):375-6. Pubmed: 25818368 DOI:10.1111/pcmr.12368 Dang M, Zon LI. 2015. Screening for melanoma resistance genes in vivo. Pigment cell & melanoma research. 28(4):375-6. Pubmed: 25818368 DOI:10.1111/pcmr.12368 -
Lin MI, Price EN, Boatman S, Hagedorn EJ, Trompouki E, Satishchandran S, Carspecken CW, Uong A, DiBiase A, Yang S, Canver MC, Dahlberg A, Lu Z, Zhang CC, Orkin SH, Bernstein ID, Aster JC, White RM, Zon LI. 2015. Angiopoietin-like proteins stimulate HSPC development through interaction with notch receptor signaling. eLife. 4. Pubmed: 25714926 DOI:10.7554/eLife.05544 Lin MI, Price EN, Boatman S, Hagedorn EJ, Trompouki E, Satishchandran S, Carspecken CW, Uong A, DiBiase A, Yang S, Canver MC, Dahlberg A, Lu Z, Zhang CC, Orkin SH, Bernstein ID, Aster JC, White RM, Zon LI. 2015. Angiopoietin-like proteins stimulate HSPC development through interaction with notch receptor signaling. eLife. 4. Pubmed: 25714926 DOI:10.7554/eLife.05544 Angiopoietin-like proteins (angptls) are capable of ex vivo expansion of mouse and human hematopoietic stem and progenitor cells (HSPCs). Despite this intriguing ability, their mechanism is unknown. In this study, we show that angptl2 overexpression is sufficient to expand definitive HSPCs in zebrafish embryos. Angptl1/2 are required for definitive hematopoiesis and vascular specification of the hemogenic endothelium. The loss-of-function phenotype is reminiscent of the notch mutant mindbomb (mib), and a strong genetic interaction occurs between angptls and notch. Overexpressing angptl2 rescues mib while overexpressing notch rescues angptl1/2 morphants. Gene expression studies in ANGPTL2-stimulated CD34(+) cells showed a strong MYC activation signature and myc overexpression in angptl1/2 morphants or mib restored HSPCs formation. ANGPTL2 can increase NOTCH activation in cultured cells and ANGPTL receptor interacted with NOTCH to regulate NOTCH cleavage. Together our data provide insight to the angptl-mediated notch activation through receptor interaction and subsequent activation of myc targets. -
Peng X, Dong M, Ma L, Jia XE, Mao J, Jin C, Chen Y, Gao L, Liu X, Ma K, Wang L, Du T, Jin Y, Huang Q, Li K, Zon LI, Liu T, Deng M, Zhou Y, Xi X, Zhou Y, Chen S. 2015. A point mutation of zebrafish c-cbl gene in the ring finger domain produces a phenotype mimicking human myeloproliferative disease. Leukemia. 29(12):2355-65. Pubmed: 26104663 DOI:10.1038/leu.2015.154 Peng X, Dong M, Ma L, Jia XE, Mao J, Jin C, Chen Y, Gao L, Liu X, Ma K, Wang L, Du T, Jin Y, Huang Q, Li K, Zon LI, Liu T, Deng M, Zhou Y, Xi X, Zhou Y, Chen S. 2015. A point mutation of zebrafish c-cbl gene in the ring finger domain produces a phenotype mimicking human myeloproliferative disease. Leukemia. 29(12):2355-65. Pubmed: 26104663 DOI:10.1038/leu.2015.154 Controlled self-renewal and differentiation of hematopoietic stem/progenitor cells (HSPCs) are critical for vertebrate development and survival. These processes are tightly regulated by the transcription factors, signaling molecules and epigenetic factors. Impaired regulations of their function could result in hematological malignancies. Using a large-scale zebrafish N-ethyl-N-nitrosourea mutagenesis screening, we identified a line named LDD731, which presented significantly increased HSPCs in hematopoietic organs. Further analysis revealed that the cells of erythroid/myeloid lineages in definitive hematopoiesis were increased while the primitive hematopoiesis was not affected. The homozygous mutation was lethal with a median survival time around 14-15 days post fertilization. The causal mutation was located by positional cloning in the c-cbl gene, the human ortholog of which, c-CBL, is found frequently mutated in myeloproliferative neoplasms (MPN) or acute leukemia. Sequence analysis showed the mutation in LDD731 caused a histidine-to-tyrosine substitution of the amino acid codon 382 within the RING finger domain of c-Cbl. Moreover, the myeloproliferative phenotype in zebrafish seemed dependent on the Flt3 (fms-like tyrosine kinase 3) signaling, consistent with that observed in both mice and humans. Our study may shed new light on the pathogenesis of MPN and provide a useful in vivo vertebrate model of this syndrome for screening drugs. -
Gao L, Li D, Ma K, Zhang W, Xu T, Fu C, Jing C, Jia X, Wu S, Sun X, Dong M, Deng M, Chen Y, Zhu W, Peng J, Wan F, Zhou Y, Zon LI, Pan W. 2015. TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis. PLoS genetics. 11(7):e1005346. Pubmed: 26131719 DOI:10.1371/journal.pgen.1005346 Gao L, Li D, Ma K, Zhang W, Xu T, Fu C, Jing C, Jia X, Wu S, Sun X, Dong M, Deng M, Chen Y, Zhu W, Peng J, Wan F, Zhou Y, Zon LI, Pan W. 2015. TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis. PLoS genetics. 11(7):e1005346. Pubmed: 26131719 DOI:10.1371/journal.pgen.1005346 In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II β binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion. -
Jia XE, Ma K, Xu T, Gao L, Wu S, Fu C, Zhang W, Wang Z, Liu K, Dong M, Jing C, Ren C, Dong Z, Chen Y, Jin Y, Huang Q, Chang X, Deng M, Li L, Luo L, Zhu J, Dang Y, Chang HC, Zon LI, Zhou Y, Chen S, Pan W. 2015. Mutation of kri1l causes definitive hematopoiesis failure via PERK-dependent excessive autophagy induction. Cell research. 25(8):946-62. Pubmed: 26138676 DOI:10.1038/cr.2015.81 Jia XE, Ma K, Xu T, Gao L, Wu S, Fu C, Zhang W, Wang Z, Liu K, Dong M, Jing C, Ren C, Dong Z, Chen Y, Jin Y, Huang Q, Chang X, Deng M, Li L, Luo L, Zhu J, Dang Y, Chang HC, Zon LI, Zhou Y, Chen S, Pan W. 2015. Mutation of kri1l causes definitive hematopoiesis failure via PERK-dependent excessive autophagy induction. Cell research. 25(8):946-62. Pubmed: 26138676 DOI:10.1038/cr.2015.81 Dysregulation of ribosome biogenesis causes human diseases, such as Diamond-Blackfan anemia, del (5q-) syndrome and bone marrow failure. However, the mechanisms of blood disorders in these diseases remain elusive. Through genetic mapping, molecular cloning and mechanism characterization of the zebrafish mutant cas002, we reveal a novel connection between ribosomal dysfunction and excessive autophagy in the regulation of hematopoietic stem/progenitor cells (HSPCs). cas002 carries a recessive lethal mutation in kri1l gene that encodes an essential component of rRNA small subunit processome. We show that Kri1l is required for normal ribosome biogenesis, expansion of definitive HSPCs and subsequent lineage differentiation. Through live imaging and biochemical studies, we find that loss of Kri1l causes the accumulation of misfolded proteins and excessive PERK activation-dependent autophagy in HSPCs. Blocking autophagy but not inhibiting apoptosis by Bcl2 overexpression can fully rescue hematopoietic defects, but not the lethality of kri1l(cas002) embryos. Treatment with autophagy inhibitors (3-MA and Baf A1) or PERK inhibitor (GSK2656157), or knockdown of beclin1 or perk can markedly restore HSPC proliferation and definitive hematopoietic cell differentiation. These results may provide leads for effective therapeutics that benefit patients with anemia or bone marrow failure caused by ribosome disorders. -
Li P, Lahvic JL, Binder V, Pugach EK, Riley EB, Tamplin OJ, Panigrahy D, Bowman TV, Barrett FG, Heffner GC, McKinney-Freeman S, Schlaeger TM, Daley GQ, Zeldin DC, Zon LI. 2015. Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment. Nature. 523(7561):468-71. Pubmed: 26201599 DOI:10.1038/nature14569 Li P, Lahvic JL, Binder V, Pugach EK, Riley EB, Tamplin OJ, Panigrahy D, Bowman TV, Barrett FG, Heffner GC, McKinney-Freeman S, Schlaeger TM, Daley GQ, Zeldin DC, Zon LI. 2015. Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment. Nature. 523(7561):468-71. Pubmed: 26201599 DOI:10.1038/nature14569 Haematopoietic stem and progenitor cell (HSPC) transplant is a widely used treatment for life-threatening conditions such as leukaemia; however, the molecular mechanisms regulating HSPC engraftment of the recipient niche remain incompletely understood. Here we develop a competitive HSPC transplant method in adult zebrafish, using in vivo imaging as a non-invasive readout. We use this system to conduct a chemical screen, and identify epoxyeicosatrienoic acids (EETs) as a family of lipids that enhance HSPC engraftment. The pro-haematopoietic effects of EETs were conserved in the developing zebrafish embryo, where 11,12-EET promoted HSPC specification by activating a unique activator protein 1 (AP-1) and runx1 transcription program autonomous to the haemogenic endothelium. This effect required the activation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, specifically PI(3)Kγ. In adult HSPCs, 11,12-EET induced transcriptional programs, including AP-1 activation, which modulate several cellular processes, such as migration, to promote engraftment. Furthermore, we demonstrate that the EET effects on enhancing HSPC homing and engraftment are conserved in mammals. Our study establishes a new method to explore the molecular mechanisms of HSPC engraftment, and discovers a previously unrecognized, evolutionarily conserved pathway regulating multiple haematopoietic generation and regeneration processes. EETs may have clinical application in marrow or cord blood transplantation. -
Patsch C, Challet-Meylan L, Thoma EC, Urich E, Heckel T, O'Sullivan JF, Grainger SJ, Kapp FG, Sun L, Christensen K, Xia Y, Florido MH, He W, Pan W, Prummer M, Warren CR, Jakob-Roetne R, Certa U, Jagasia R, Freskgård PO, Adatto I, Kling D, Huang P, Zon LI, Chaikof EL, Gerszten RE, Graf M, Iacone R, Cowan CA. 2015. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. Nature cell biology. 17(8):994-1003. Pubmed: 26214132 DOI:10.1038/ncb3205 Patsch C, Challet-Meylan L, Thoma EC, Urich E, Heckel T, O'Sullivan JF, Grainger SJ, Kapp FG, Sun L, Christensen K, Xia Y, Florido MH, He W, Pan W, Prummer M, Warren CR, Jakob-Roetne R, Certa U, Jagasia R, Freskgård PO, Adatto I, Kling D, Huang P, Zon LI, Chaikof EL, Gerszten RE, Graf M, Iacone R, Cowan CA. 2015. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. Nature cell biology. 17(8):994-1003. Pubmed: 26214132 DOI:10.1038/ncb3205 The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease. -
Heilmann S, Ratnakumar K, Langdon E, Kansler E, Kim I, Campbell NR, Perry E, McMahon A, Kaufman C, van Rooijen E, Lee W, Iacobuzio-Donahue C, Hynes R, Zon L, Xavier J, White RM. 2015. A Quantitative System for Studying Metastasis Using Transparent Zebrafish. Cancer research. 75(20):4272-4282. Pubmed: 26282170 DOI:10.1158/0008-5472.CAN-14-3319 Heilmann S, Ratnakumar K, Langdon E, Kansler E, Kim I, Campbell NR, Perry E, McMahon A, Kaufman C, van Rooijen E, Lee W, Iacobuzio-Donahue C, Hynes R, Zon L, Xavier J, White RM. 2015. A Quantitative System for Studying Metastasis Using Transparent Zebrafish. Cancer research. 75(20):4272-4282. Pubmed: 26282170 DOI:10.1158/0008-5472.CAN-14-3319 Metastasis is the defining feature of advanced malignancy, yet remains challenging to study in laboratory environments. Here, we describe a high-throughput zebrafish system for comprehensive, in vivo assessment of metastatic biology. First, we generated several stable cell lines from melanomas of transgenic mitfa-BRAF(V600E);p53(-/-) fish. We then transplanted the melanoma cells into the transparent casper strain to enable highly quantitative measurement of the metastatic process at single-cell resolution. Using computational image analysis of the resulting metastases, we generated a metastasis score, μ, that can be applied to quantitative comparison of metastatic capacity between experimental conditions. Furthermore, image analysis also provided estimates of the frequency of metastasis-initiating cells (∼1/120,000 cells). Finally, we determined that the degree of pigmentation is a key feature defining cells with metastatic capability. The small size and rapid generation of progeny combined with superior imaging tools make zebrafish ideal for unbiased high-throughput investigations of cell-intrinsic or microenvironmental modifiers of metastasis. The approaches described here are readily applicable to other tumor types and thus serve to complement studies also employing murine and human cell culture systems.©2015 American Association for Cancer Research. -
Mosimann C, Panáková D, Werdich AA, Musso G, Burger A, Lawson KL, Carr LA, Nevis KR, Sabeh MK, Zhou Y, Davidson AJ, DiBiase A, Burns CE, Burns CG, MacRae CA, Zon LI. 2015. Chamber identity programs drive early functional partitioning of the heart. Nature communications. 6:8146. Pubmed: 26306682 DOI:10.1038/ncomms9146 Mosimann C, Panáková D, Werdich AA, Musso G, Burger A, Lawson KL, Carr LA, Nevis KR, Sabeh MK, Zhou Y, Davidson AJ, DiBiase A, Burns CE, Burns CG, MacRae CA, Zon LI. 2015. Chamber identity programs drive early functional partitioning of the heart. Nature communications. 6:8146. Pubmed: 26306682 DOI:10.1038/ncomms9146 The vertebrate heart muscle (myocardium) develops from the first heart field (FHF) and expands by adding second heart field (SHF) cells. While both lineages exist already in teleosts, the primordial contributions of FHF and SHF to heart structure and function remain incompletely understood. Here we delineate the functional contribution of the FHF and SHF to the zebrafish heart using the cis-regulatory elements of the draculin (drl) gene. The drl reporters initially delineate the lateral plate mesoderm, including heart progenitors. Subsequent myocardial drl reporter expression restricts to FHF descendants. We harnessed this unique feature to uncover that loss of tbx5a and pitx2 affect relative FHF versus SHF contributions to the heart. High-resolution physiology reveals distinctive electrical properties of each heart field territory that define a functional boundary within the single zebrafish ventricle. Our data establish that the transcriptional program driving cardiac septation regulates physiologic ventricle partitioning, which successively provides mechanical advantages of sequential contraction. -
Musso G, Mosimann C, Panáková D, Burger A, Zhou Y, Zon LI, MacRae CA. 2015. Generating and evaluating a ranked candidate gene list for potential vertebrate heart field regulators. Genomics data. 6:199-201. Pubmed: 26697374 DOI:10.1016/j.gdata.2015.09.015 Musso G, Mosimann C, Panáková D, Burger A, Zhou Y, Zon LI, MacRae CA. 2015. Generating and evaluating a ranked candidate gene list for potential vertebrate heart field regulators. Genomics data. 6:199-201. Pubmed: 26697374 DOI:10.1016/j.gdata.2015.09.015 The vertebrate heart develops from two distinct lineages of cardiomyocytes that arise from the first and second heart fields (FHF and SHF, respectively). The FHF forms the primitive heart tube, while adding cells from the SHF allows elongation at both poles of the tube. Initially seen as an exclusive characteristic of higher vertebrates, recent work has demonstrated the presence of a distinct FHF and SHF in lower vertebrates, including zebrafish. We found that key transcription factors that regulate septation and chamber formation in higher vertebrates, including Tbx5 and Pitx2, influence relative FHF and SHF contributions to the zebrafish heart tube. To identify molecular modulators of heart field migration, we used microarray-based expression profiling following inhibition of tbx5a and pitx2ab in embryonic zebrafish (Mosimann & Panakova, et al, 2015; GSE70750). Here, we describe in more detail the procedure used to process, prioritize, and analyze the expression data for functional enrichment. -
Anderson H, Patch TC, Reddy PN, Hagedorn EJ, Kim PG, Soltis KA, Chen MJ, Tamplin OJ, Frye M, MacLean GA, Hübner K, Bauer DE, Kanki JP, Vogin G, Huston NC, Nguyen M, Fujiwara Y, Paw BH, Vestweber D, Zon LI, Orkin SH, Daley GQ, Shah DI. 2015. Hematopoietic stem cells develop in the absence of endothelial cadherin 5 expression. Blood. 126(26):2811-20. Pubmed: 26385351 DOI:10.1182/blood-2015-07-659276 Anderson H, Patch TC, Reddy PN, Hagedorn EJ, Kim PG, Soltis KA, Chen MJ, Tamplin OJ, Frye M, MacLean GA, Hübner K, Bauer DE, Kanki JP, Vogin G, Huston NC, Nguyen M, Fujiwara Y, Paw BH, Vestweber D, Zon LI, Orkin SH, Daley GQ, Shah DI. 2015. Hematopoietic stem cells develop in the absence of endothelial cadherin 5 expression. Blood. 126(26):2811-20. Pubmed: 26385351 DOI:10.1182/blood-2015-07-659276 Rare endothelial cells in the aorta-gonad-mesonephros (AGM) transition into hematopoietic stem cells (HSCs) during embryonic development. Lineage tracing experiments indicate that HSCs emerge from cadherin 5 (Cdh5; vascular endothelial-cadherin)(+) endothelial precursors, and isolated populations of Cdh5(+) cells from mouse embryos and embryonic stem cells can be differentiated into hematopoietic cells. Cdh5 has also been widely implicated as a marker of AGM-derived hemogenic endothelial cells. Because Cdh5(-/-) mice embryos die before the first HSCs emerge, it is unknown whether Cdh5 has a direct role in HSC emergence. Our previous genetic screen yielded malbec (mlb(bw306)), a zebrafish mutant for cdh5, with normal embryonic and definitive blood. Using time-lapse confocal imaging, parabiotic surgical pairing of zebrafish embryos, and blastula transplantation assays, we show that HSCs emerge, migrate, engraft, and differentiate in the absence of cdh5 expression. By tracing Cdh5(-/-)green fluorescent protein (GFP)(+/+) cells in chimeric mice, we demonstrated that Cdh5(-/-)GFP(+/+) HSCs emerging from embryonic day 10.5 and 11.5 (E10.5 and E11.5) AGM or derived from E13.5 fetal liver not only differentiate into hematopoietic colonies but also engraft and reconstitute multilineage adult blood. We also developed a conditional mouse Cdh5 knockout (Cdh5(flox/flox):Scl-Cre-ER(T)) and demonstrated that multipotent hematopoietic colonies form despite the absence of Cdh5. These data establish that Cdh5, a marker of hemogenic endothelium in the AGM, is dispensable for the transition of hemogenic endothelium to HSCs.© 2015 by The American Society of Hematology. -
Gjini E, Mansour MR, Sander JD, Moritz N, Nguyen AT, Kesarsing M, Gans E, He S, Chen S, Ko M, Kuang YY, Yang S, Zhou Y, Rodig S, Zon LI, Joung JK, Rao A, Look AT. 2015. A zebrafish model of myelodysplastic syndrome produced through tet2 genomic editing. Molecular and cellular biology. 35(5):789-804. Pubmed: 25512612 DOI:10.1128/MCB.00971-14 Gjini E, Mansour MR, Sander JD, Moritz N, Nguyen AT, Kesarsing M, Gans E, He S, Chen S, Ko M, Kuang YY, Yang S, Zhou Y, Rodig S, Zon LI, Joung JK, Rao A, Look AT. 2015. A zebrafish model of myelodysplastic syndrome produced through tet2 genomic editing. Molecular and cellular biology. 35(5):789-804. Pubmed: 25512612 DOI:10.1128/MCB.00971-14 The ten-eleven translocation 2 gene (TET2) encodes a member of the TET family of DNA methylcytosine oxidases that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) to initiate the demethylation of DNA within genomic CpG islands. Somatic loss-of-function mutations of TET2 are frequently observed in human myelodysplastic syndrome (MDS), which is a clonal malignancy characterized by dysplastic changes of developing blood cell progenitors, leading to ineffective hematopoiesis. We used genome-editing technology to disrupt the zebrafish Tet2 catalytic domain. tet2(m/m) (homozygous for the mutation) zebrafish exhibited normal embryonic and larval hematopoiesis but developed progressive clonal myelodysplasia as they aged, culminating in myelodysplastic syndromes (MDS) at 24 months of age, with dysplasia of myeloid progenitor cells and anemia with abnormal circulating erythrocytes. The resultant tet2(m/m) mutant zebrafish lines show decreased levels of 5hmC in hematopoietic cells of the kidney marrow but not in other cell types, most likely reflecting the ability of other Tet family members to provide this enzymatic activity in nonhematopoietic tissues but not in hematopoietic cells. tet2(m/m) zebrafish are viable and fertile, providing an ideal model to dissect altered pathways in hematopoietic cells and, for small-molecule screens in embryos, to identify compounds with specific activity against tet2 mutant cells.Copyright © 2015, American Society for Microbiology. All Rights Reserved. -
Jang IH, Lu YF, Zhao L, Wenzel PL, Kume T, Datta SM, Arora N, Guiu J, Lagha M, Kim PG, Do EK, Kim JH, Schlaeger TM, Zon LI, Bigas A, Burns CE, Daley GQ. 2015. Notch1 acts via Foxc2 to promote definitive hematopoiesis via effects on hemogenic endothelium. Blood. 125(9):1418-26. Pubmed: 25587036 DOI:10.1182/blood-2014-04-568170 Jang IH, Lu YF, Zhao L, Wenzel PL, Kume T, Datta SM, Arora N, Guiu J, Lagha M, Kim PG, Do EK, Kim JH, Schlaeger TM, Zon LI, Bigas A, Burns CE, Daley GQ. 2015. Notch1 acts via Foxc2 to promote definitive hematopoiesis via effects on hemogenic endothelium. Blood. 125(9):1418-26. Pubmed: 25587036 DOI:10.1182/blood-2014-04-568170 Hematopoietic and vascular development share many common features, including cell surface markers and sites of origin. Recent lineage-tracing studies have established that definitive hematopoietic stem and progenitor cells arise from vascular endothelial-cadherin(+) hemogenic endothelial cells of the aorta-gonad-mesonephros region, but the genetic programs underlying the specification of hemogenic endothelial cells remain poorly defined. Here, we discovered that Notch induction enhances hematopoietic potential and promotes the specification of hemogenic endothelium in differentiating cultures of mouse embryonic stem cells, and we identified Foxc2 as a highly upregulated transcript in the hemogenic endothelial population. Studies in zebrafish and mouse embryos revealed that Foxc2 and its orthologs are required for the proper development of definitive hematopoiesis and function downstream of Notch signaling in the hemogenic endothelium. These data establish a pathway linking Notch signaling to Foxc2 in hemogenic endothelial cells to promote definitive hematopoiesis.© 2015 by The American Society of Hematology. -
Tamplin OJ, Durand EM, Carr LA, Childs SJ, Hagedorn EJ, Li P, Yzaguirre AD, Speck NA, Zon LI. 2015. Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche. Cell. 160(1-2):241-52. Pubmed: 25594182 DOI:S0092-8674(14)01638-9 Tamplin OJ, Durand EM, Carr LA, Childs SJ, Hagedorn EJ, Li P, Yzaguirre AD, Speck NA, Zon LI. 2015. Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche. Cell. 160(1-2):241-52. Pubmed: 25594182 DOI:S0092-8674(14)01638-9 Hematopoietic stem and progenitor cells (HSPCs) can reconstitute and sustain the entire blood system. We generated a highly specific transgenic reporter of HSPCs in zebrafish. This allowed us to perform high-resolution live imaging on endogenous HSPCs not currently possible in mammalian bone marrow. Using this system, we have uncovered distinct interactions between single HSPCs and their niche. When an HSPC arrives in the perivascular niche, a group of endothelial cells remodel to form a surrounding pocket. This structure appears conserved in mouse fetal liver. Correlative light and electron microscopy revealed that endothelial cells surround a single HSPC attached to a single mesenchymal stromal cell. Live imaging showed that mesenchymal stromal cells anchor HSPCs and orient their divisions. A chemical genetic screen found that the compound lycorine promotes HSPC-niche interactions during development and ultimately expands the stem cell pool into adulthood. Our studies provide evidence for dynamic niche interactions upon stem cell colonization. PAPERFLICK:Copyright © 2015 Elsevier Inc. All rights reserved. -
Liu X, Jia X, Yuan H, Ma K, Chen Y, Jin Y, Deng M, Pan W, Chen S, Chen Z, de The H, Zon LI, Zhou Y, Zhou J, Zhu J. 2015. DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish. Journal of hematology & oncology. 8:15. Pubmed: 25886310 DOI:10.1186/s13045-015-0115-7 Liu X, Jia X, Yuan H, Ma K, Chen Y, Jin Y, Deng M, Pan W, Chen S, Chen Z, de The H, Zon LI, Zhou Y, Zhou J, Zhu J. 2015. DNA methyltransferase 1 functions through C/ebpa to maintain hematopoietic stem and progenitor cells in zebrafish. Journal of hematology & oncology. 8:15. Pubmed: 25886310 DOI:10.1186/s13045-015-0115-7 Array -
Schlaeger TM, Daheron L, Brickler TR, Entwisle S, Chan K, Cianci A, DeVine A, Ettenger A, Fitzgerald K, Godfrey M, Gupta D, McPherson J, Malwadkar P, Gupta M, Bell B, Doi A, Jung N, Li X, Lynes MS, Brookes E, Cherry AB, Demirbas D, Tsankov AM, Zon LI, Rubin LL, Feinberg AP, Meissner A, Cowan CA, Daley GQ. 2015. A comparison of non-integrating reprogramming methods. Nature biotechnology. 33(1):58-63. Pubmed: 25437882 DOI:10.1038/nbt.3070 Schlaeger TM, Daheron L, Brickler TR, Entwisle S, Chan K, Cianci A, DeVine A, Ettenger A, Fitzgerald K, Godfrey M, Gupta D, McPherson J, Malwadkar P, Gupta M, Bell B, Doi A, Jung N, Li X, Lynes MS, Brookes E, Cherry AB, Demirbas D, Tsankov AM, Zon LI, Rubin LL, Feinberg AP, Meissner A, Cowan CA, Daley GQ. 2015. A comparison of non-integrating reprogramming methods. Nature biotechnology. 33(1):58-63. Pubmed: 25437882 DOI:10.1038/nbt.3070 Human induced pluripotent stem cells (hiPSCs) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV), episomal (Epi) and mRNA transfection mRNA methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation. -
Ablain J, Durand EM, Yang S, Zhou Y, Zon LI. 2015. A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish. Developmental cell. 32(6):756-64. Pubmed: 25752963 DOI:S1534-5807(15)00075-1 Ablain J, Durand EM, Yang S, Zhou Y, Zon LI. 2015. A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish. Developmental cell. 32(6):756-64. Pubmed: 25752963 DOI:S1534-5807(15)00075-1 CRISPR/Cas9 technology of genome editing has greatly facilitated the targeted inactivation of genes in vitro and in vivo in a wide range of organisms. In zebrafish, it allows the rapid generation of knockout lines by simply injecting a guide RNA (gRNA) and Cas9 mRNA into one-cell stage embryos. Here, we report a simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, specifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish embryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very penetrant in stable F1 fish. This vector system constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies in zebrafish.Copyright © 2015 Elsevier Inc. All rights reserved. -
Jing L, Tamplin OJ, Chen MJ, Deng Q, Patterson S, Kim PG, Durand EM, McNeil A, Green JM, Matsuura S, Ablain J, Brandt MK, Schlaeger TM, Huttenlocher A, Daley GQ, Ravid K, Zon LI. 2015. Adenosine signaling promotes hematopoietic stem and progenitor cell emergence. The Journal of experimental medicine. 212(5):649-63. Pubmed: 25870200 DOI:10.1084/jem.20141528 Jing L, Tamplin OJ, Chen MJ, Deng Q, Patterson S, Kim PG, Durand EM, McNeil A, Green JM, Matsuura S, Ablain J, Brandt MK, Schlaeger TM, Huttenlocher A, Daley GQ, Ravid K, Zon LI. 2015. Adenosine signaling promotes hematopoietic stem and progenitor cell emergence. The Journal of experimental medicine. 212(5):649-63. Pubmed: 25870200 DOI:10.1084/jem.20141528 Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1(+)/cmyb(+) HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl(+) hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP-protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates.© 2015 Jing et al. -
Kim PG, Nakano H, Das PP, Chen MJ, Rowe RG, Chou SS, Ross SJ, Sakamoto KM, Zon LI, Schlaeger TM, Orkin SH, Nakano A, Daley GQ. 2015. Flow-induced protein kinase A-CREB pathway acts via BMP signaling to promote HSC emergence. The Journal of experimental medicine. 212(5):633-48. Pubmed: 25870201 DOI:10.1084/jem.20141514 Kim PG, Nakano H, Das PP, Chen MJ, Rowe RG, Chou SS, Ross SJ, Sakamoto KM, Zon LI, Schlaeger TM, Orkin SH, Nakano A, Daley GQ. 2015. Flow-induced protein kinase A-CREB pathway acts via BMP signaling to promote HSC emergence. The Journal of experimental medicine. 212(5):633-48. Pubmed: 25870201 DOI:10.1084/jem.20141514 Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta-gonad-mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)-cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA-CREB and BMP pathways in isolated AGM VE-cadherin(+) cells from mid-gestation embryos, we demonstrate that PKA-CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA-CREB-BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.© 2015 Kim et al. 2014
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Zhang X, Ling J, Barcia G, Jing L, Wu J, Barry BJ, Mochida GH, Hill RS, Weimer JM, Stein Q, Poduri A, Partlow JN, Ville D, Dulac O, Yu TW, Lam AT, Servattalab S, Rodriguez J, Boddaert N, Munnich A, Colleaux L, Zon LI, Söll D, Walsh CA, Nabbout R. 2014. Mutations in QARS, encoding glutaminyl-tRNA synthetase, cause progressive microcephaly, cerebral-cerebellar atrophy, and intractable seizures. American journal of human genetics. 94(4):547-58. Pubmed: 24656866 DOI:S0002-9297(14)00105-0 Zhang X, Ling J, Barcia G, Jing L, Wu J, Barry BJ, Mochida GH, Hill RS, Weimer JM, Stein Q, Poduri A, Partlow JN, Ville D, Dulac O, Yu TW, Lam AT, Servattalab S, Rodriguez J, Boddaert N, Munnich A, Colleaux L, Zon LI, Söll D, Walsh CA, Nabbout R. 2014. Mutations in QARS, encoding glutaminyl-tRNA synthetase, cause progressive microcephaly, cerebral-cerebellar atrophy, and intractable seizures. American journal of human genetics. 94(4):547-58. Pubmed: 24656866 DOI:S0002-9297(14)00105-0 Progressive microcephaly is a heterogeneous condition with causes including mutations in genes encoding regulators of neuronal survival. Here, we report the identification of mutations in QARS (encoding glutaminyl-tRNA synthetase [QARS]) as the causative variants in two unrelated families affected by progressive microcephaly, severe seizures in infancy, atrophy of the cerebral cortex and cerebellar vermis, and mild atrophy of the cerebellar hemispheres. Whole-exome sequencing of individuals from each family independently identified compound-heterozygous mutations in QARS as the only candidate causative variants. QARS was highly expressed in the developing fetal human cerebral cortex in many cell types. The four QARS mutations altered highly conserved amino acids, and the aminoacylation activity of QARS was significantly impaired in mutant cell lines. Variants p.Gly45Val and p.Tyr57His were located in the N-terminal domain required for QARS interaction with proteins in the multisynthetase complex and potentially with glutamine tRNA, and recombinant QARS proteins bearing either substitution showed an over 10-fold reduction in aminoacylation activity. Conversely, variants p.Arg403Trp and p.Arg515Trp, each occurring in a different family, were located in the catalytic core and completely disrupted QARS aminoacylation activity in vitro. Furthermore, p.Arg403Trp and p.Arg515Trp rendered QARS less soluble, and p.Arg403Trp disrupted QARS-RARS (arginyl-tRNA synthetase 1) interaction. In zebrafish, homozygous qars loss of function caused decreased brain and eye size and extensive cell death in the brain. Our results highlight the importance of QARS during brain development and that epilepsy due to impairment of QARS activity is unusually severe in comparison to other aminoacyl-tRNA synthetase disorders.Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved. -
Kong Y, Grimaldi M, Curtin E, Dougherty M, Kaufman C, White RM, Zon LI, Liao EC. 2014. Neural crest development and craniofacial morphogenesis is coordinated by nitric oxide and histone acetylation. Chemistry & biology. 21(4):488-501. Pubmed: 24684905 DOI:S1074-5521(14)00072-6 Kong Y, Grimaldi M, Curtin E, Dougherty M, Kaufman C, White RM, Zon LI, Liao EC. 2014. Neural crest development and craniofacial morphogenesis is coordinated by nitric oxide and histone acetylation. Chemistry & biology. 21(4):488-501. Pubmed: 24684905 DOI:S1074-5521(14)00072-6 Cranial neural crest (CNC) cells are patterned and coalesce to facial prominences that undergo convergence and extension to generate the craniofacial form. We applied a chemical genetics approach to identify pathways that regulate craniofacial development during embryogenesis. Treatment with the nitric oxide synthase inhibitor 1-(2-[trifluoromethyl] phenyl) imidazole (TRIM) abrogated first pharyngeal arch structures and induced ectopic ceratobranchial formation. TRIM promoted a progenitor CNC fate and inhibited chondrogenic differentiation, which were mediated through impaired nitric oxide (NO) production without appreciable effect on global protein S-nitrosylation. Instead, TRIM perturbed hox gene patterning and caused histone hypoacetylation. Rescue of TRIM phenotype was achieved with overexpression of histone acetyltransferase kat6a, inhibition of histone deacetylase, and complementary NO. These studies demonstrate that NO signaling and histone acetylation are coordinated mechanisms that regulate CNC patterning, differentiation, and convergence during craniofacial morphogenesis.Copyright © 2014 Elsevier Ltd. All rights reserved. -
Mirabello L, Macari ER, Jessop L, Ellis SR, Myers T, Giri N, Taylor AM, McGrath KE, Humphries JM, Ballew BJ, Yeager M, Boland JF, He J, Hicks BD, Burdett L, Alter BP, Zon L, Savage SA. 2014. Whole-exome sequencing and functional studies identify RPS29 as a novel gene mutated in multicase Diamond-Blackfan anemia families. Blood. 124(1):24-32. Pubmed: 24829207 DOI:10.1182/blood-2013-11-540278 Mirabello L, Macari ER, Jessop L, Ellis SR, Myers T, Giri N, Taylor AM, McGrath KE, Humphries JM, Ballew BJ, Yeager M, Boland JF, He J, Hicks BD, Burdett L, Alter BP, Zon L, Savage SA. 2014. Whole-exome sequencing and functional studies identify RPS29 as a novel gene mutated in multicase Diamond-Blackfan anemia families. Blood. 124(1):24-32. Pubmed: 24829207 DOI:10.1182/blood-2013-11-540278 Diamond-Blackfan anemia (DBA) is a cancer-prone inherited bone marrow failure syndrome. Approximately half of DBA patients have a germ-line mutation in a ribosomal protein gene. We used whole-exome sequencing to identify disease-causing genes in 2 large DBA families. After filtering, 1 nonsynonymous mutation (p.I31F) in the ribosomal protein S29 (RPS29[AUQ1]) gene was present in all 5 DBA-affected individuals and the obligate carrier, and absent from the unaffected noncarrier parent in 1 DBA family. A second DBA family was found to have a different nonsynonymous mutation (p.I50T) in RPS29. Both mutations are amino acid substitutions in exon 2 predicted to be deleterious and resulted in haploinsufficiency of RPS29 expression compared with wild-type RPS29 expression from an unaffected control. The DBA proband with the p.I31F RPS29 mutation had a pre-ribosomal RNA (rRNA) processing defect compared with the healthy control. We demonstrated that both RPS29 mutations failed to rescue the defective erythropoiesis in the rps29(-/-) mutant zebra fish DBA model. RPS29 is a component of the small 40S ribosomal subunit and essential for rRNA processing and ribosome biogenesis. We uncovered a novel DBA causative gene, RPS29, and showed that germ-line mutations in RPS29 can cause a defective erythropoiesis phenotype using a zebra fish model. -
Svoboda O, Stachura DL, Machoňová O, Pajer P, Brynda J, Zon LI, Traver D, Bartůněk P. 2014. Dissection of vertebrate hematopoiesis using zebrafish thrombopoietin. Blood. 124(2):220-8. Pubmed: 24869937 DOI:10.1182/blood-2014-03-564682 Svoboda O, Stachura DL, Machoňová O, Pajer P, Brynda J, Zon LI, Traver D, Bartůněk P. 2014. Dissection of vertebrate hematopoiesis using zebrafish thrombopoietin. Blood. 124(2):220-8. Pubmed: 24869937 DOI:10.1182/blood-2014-03-564682 In nonmammalian vertebrates, the functional units of hemostasis are thrombocytes. Thrombocytes are thought to arise from bipotent thrombocytic/erythroid progenitors (TEPs). TEPs have been experimentally demonstrated in avian models of hematopoiesis, and mammals possess functional equivalents known as megakaryocyte/erythroid progenitors (MEPs). However, the presence of TEPs in teleosts has only been speculated. To identify and prospectively isolate TEPs, we identified, cloned, and generated recombinant zebrafish thrombopoietin (Tpo). Tpo mRNA expanded itga2b:GFP(+) (cd41:GFP(+)) thrombocytes as well as hematopoietic stem and progenitor cells (HSPCs) in the zebrafish embryo. Utilizing Tpo in clonal methylcellulose assays, we describe for the first time the prospective isolation and characterization of TEPs from transgenic zebrafish. Combinatorial use of zebrafish Tpo, erythropoietin, and granulocyte colony stimulating factor (Gcsf) allowed the investigation of HSPCs responsible for erythro-, myelo-, and thrombo-poietic differentiation. Utilizing these assays allowed the visualization and differentiation of hematopoietic progenitors ex vivo in real-time with time-lapse and high-throughput microscopy, allowing analyses of their clonogenic and proliferative capacity. These studies indicate that the functional role of Tpo in the differentiation of thrombocytes from HSPCs is well conserved among vertebrate organisms, positing the zebrafish as an excellent model to investigate diseases caused by dysregulated erythro- and thrombo-poietic differentiation.© 2014 by The American Society of Hematology. -
Zon L, Cagan R. 2014. From fish tank to bedside in cancer therapy: an interview with Leonard Zon. Disease models & mechanisms. 7(7):735-8. Pubmed: 24973742 DOI:10.1242/dmm.016642 Zon L, Cagan R. 2014. From fish tank to bedside in cancer therapy: an interview with Leonard Zon. Disease models & mechanisms. 7(7):735-8. Pubmed: 24973742 DOI:10.1242/dmm.016642 Leonard Zon, who is based at Harvard Medical School, is internationally recognized for his pioneering work in hematology and stem cell biology. His lab uses zebrafish as a model to understand blood cell development and, in recent years, has made inspiring breakthroughs in the treatment of blood diseases and cancer, helping to establish zebrafish as a powerful model for translational research. In this interview, Leonard speaks to Disease Models & Mechanisms Editor-in-Chief, Ross Cagan, about the evolution of his career from developmental biologist to physician-scientist and the stories behind some of his major research accomplishments. He also discusses challenges and opportunities in zebrafish research and provides advice on translating basic research findings to the clinic. -
Guo C, Chen X, Song H, Maynard MA, Zhou Y, Lobanov AV, Gladyshev VN, Ganis JJ, Wiley D, Jugo RH, Lee NY, Castroneves LA, Zon LI, Scanlan TS, Feldman HA, Huang SA. 2014. Intrinsic expression of a multiexon type 3 deiodinase gene controls zebrafish embryo size. Endocrinology. 155(10):4069-80. Pubmed: 25004091 DOI:10.1210/en.2013-2029 Guo C, Chen X, Song H, Maynard MA, Zhou Y, Lobanov AV, Gladyshev VN, Ganis JJ, Wiley D, Jugo RH, Lee NY, Castroneves LA, Zon LI, Scanlan TS, Feldman HA, Huang SA. 2014. Intrinsic expression of a multiexon type 3 deiodinase gene controls zebrafish embryo size. Endocrinology. 155(10):4069-80. Pubmed: 25004091 DOI:10.1210/en.2013-2029 Thyroid hormone is a master regulator of differentiation and growth, and its action is terminated by the enzymatic removal of an inner-ring iodine catalyzed by the selenoenzyme type 3 deiodinase (dio3). Our studies of the zebrafish reveal that the dio3 gene is duplicated in this species and that embryonic deiodination is an important determinant of embryo size. Although both dio3 paralogs encode enzymatically active proteins with high affinity for thyroid hormones, their anatomic patterns of expression are markedly divergent and only embryos with knockdown of dio3b, a biallelically expressed selenoenzyme expressed in the developing central nervous system, manifest severe thyroid hormone-dependent growth restriction at 72 hours post fertilization. This indicates that the embryonic deficiency of dio3, once considered only a placental enzyme, causes microsomia independently of placental physiology and raises the intriguing possibility that fetal abnormalities in human deiodination may present as intrauterine growth retardation. By mapping the gene structures and enzymatic properties of all four zebrafish deiodinases, we also identify dio3b as the first multiexon dio3 gene, containing a large intron separating its open reading frame from its selenocysteine insertion sequence (SECIS) element. -
Hagedorn EJ, Durand EM, Fast EM, Zon LI. 2014. Getting more for your marrow: boosting hematopoietic stem cell numbers with PGE2. Experimental cell research. 329(2):220-6. Pubmed: 25094063 DOI:S0014-4827(14)00321-8 Hagedorn EJ, Durand EM, Fast EM, Zon LI. 2014. Getting more for your marrow: boosting hematopoietic stem cell numbers with PGE2. Experimental cell research. 329(2):220-6. Pubmed: 25094063 DOI:S0014-4827(14)00321-8 Throughout the lifetime of an individual, hematopoietic stem cells (HSCs) self-renew and differentiate into lineages that include erythrocytes, platelets and all immune cells. HSC transplantation offers a potentially curative treatment for a number of hematopoietic and non-hematopoietic malignancies as well as immune and genetic disorders. Limited availability of immune-matched donors reduces the viable options for many patients in need of HSC transplantation, particularly those of diverse racial and ethnic backgrounds. Due to rapid availability and less stringent immune-matching requirements, umbilical cord blood (UCB) has emerged as a valuable source of transplantable HSCs. A single UCB unit contains a suboptimal number of HSCs for treating larger children or adults and there has thus been great clinical interest in expanding UCB HSCs ex vivo for use in transplantation. In this review we discuss the latest research and future avenues for the therapeutic use of small lipid mediator dmPGE2 to expand HSC numbers for transplantation. Originally identified in a chemical screen in zebrafish, dmPGE2 has now advanced to a phase II clinical trial as a therapy for patients with leukemia and lymphoma who are undergoing UCB transplantation.Copyright © 2014 Elsevier Inc. All rights reserved. -
Thornton JE, Du P, Jing L, Sjekloca L, Lin S, Grossi E, Sliz P, Zon LI, Gregory RI. 2014. Selective microRNA uridylation by Zcchc6 (TUT7) and Zcchc11 (TUT4). Nucleic acids research. 42(18):11777-91. Pubmed: 25223788 DOI:10.1093/nar/gku805 Thornton JE, Du P, Jing L, Sjekloca L, Lin S, Grossi E, Sliz P, Zon LI, Gregory RI. 2014. Selective microRNA uridylation by Zcchc6 (TUT7) and Zcchc11 (TUT4). Nucleic acids research. 42(18):11777-91. Pubmed: 25223788 DOI:10.1093/nar/gku805 Recent small RNA sequencing data has uncovered 3' end modification of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3' mono-uridylate a specific subset of miRNAs involved in cell differentiation and Homeobox (Hox) gene control. Zcchc6/11 selectively uridylates these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3' mono-uridylation of many of the same miRNAs. Upon TUTase-dependent loss of uridylation, we observe a concomitant increase in non-templated 3' mono-adenylation. Furthermore, TUTase inhibition in Zebrafish embryos causes developmental defects and aberrant Hox expression. Our results uncover the molecular basis for selective miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3' miRNA modifications in cells and have implications for miRNA and Hox gene regulation during development.© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. -
Alcaraz-Pérez F, García-Castillo J, García-Moreno D, López-Muñoz A, Anchelin M, Angosto D, Zon LI, Mulero V, Cayuela ML. 2014. A non-canonical function of telomerase RNA in the regulation of developmental myelopoiesis in zebrafish. Nature communications. 5:3228. Pubmed: 24496182 DOI:10.1038/ncomms4228 Alcaraz-Pérez F, García-Castillo J, García-Moreno D, López-Muñoz A, Anchelin M, Angosto D, Zon LI, Mulero V, Cayuela ML. 2014. A non-canonical function of telomerase RNA in the regulation of developmental myelopoiesis in zebrafish. Nature communications. 5:3228. Pubmed: 24496182 DOI:10.1038/ncomms4228 Dyskeratosis congenita (DC) is an inherited disorder with mutations affecting telomerase or telomeric proteins. DC patients usually die of bone marrow failure. Here we show that genetic depletion of the telomerase RNA component (TR) in the zebrafish results in impaired myelopoiesis, despite normal development of haematopoietic stem cells (HSCs). The neutropenia caused by TR depletion is independent of telomere length and telomerase activity. Genetic analysis shows that TR modulates the myeloid-erythroid fate decision by controlling the levels of the master myeloid and erythroid transcription factors spi1 and gata1, respectively. The alteration in spi1 and gata1 levels occurs through stimulation of gcsf and mcsf. Our model of TR deficiency in the zebrafish illuminates the non-canonical roles of TR, and could establish therapeutic targets for DC. -
Musso G, Tasan M, Mosimann C, Beaver JE, Plovie E, Carr LA, Chua HN, Dunham J, Zuberi K, Rodriguez H, Morris Q, Zon L, Roth FP, MacRae CA. 2014. Novel cardiovascular gene functions revealed via systematic phenotype prediction in zebrafish. Development (Cambridge, England). 141(1):224-35. Pubmed: 24346703 DOI:10.1242/dev.099796 Musso G, Tasan M, Mosimann C, Beaver JE, Plovie E, Carr LA, Chua HN, Dunham J, Zuberi K, Rodriguez H, Morris Q, Zon L, Roth FP, MacRae CA. 2014. Novel cardiovascular gene functions revealed via systematic phenotype prediction in zebrafish. Development (Cambridge, England). 141(1):224-35. Pubmed: 24346703 DOI:10.1242/dev.099796 Comprehensive functional annotation of vertebrate genomes is fundamental to biological discovery. Reverse genetic screening has been highly useful for determination of gene function, but is untenable as a systematic approach in vertebrate model organisms given the number of surveyable genes and observable phenotypes. Unbiased prediction of gene-phenotype relationships offers a strategy to direct finite experimental resources towards likely phenotypes, thus maximizing de novo discovery of gene functions. Here we prioritized genes for phenotypic assay in zebrafish through machine learning, predicting the effect of loss of function of each of 15,106 zebrafish genes on 338 distinct embryonic anatomical processes. Focusing on cardiovascular phenotypes, the learning procedure predicted known knockdown and mutant phenotypes with high precision. In proof-of-concept studies we validated 16 high-confidence cardiac predictions using targeted morpholino knockdown and initial blinded phenotyping in embryonic zebrafish, confirming a significant enrichment for cardiac phenotypes as compared with morpholino controls. Subsequent detailed analyses of cardiac function confirmed these results, identifying novel physiological defects for 11 tested genes. Among these we identified tmem88a, a recently described attenuator of Wnt signaling, as a discrete regulator of the patterning of intercellular coupling in the zebrafish cardiac epithelium. Thus, we show that systematic prioritization in zebrafish can accelerate the pace of developmental gene function discovery. -
Zhao C, Andreeva V, Gibert Y, LaBonty M, Lattanzi V, Prabhudesai S, Zhou Y, Zon L, McCann KL, Baserga S, Yelick PC. 2014. Tissue specific roles for the ribosome biogenesis factor Wdr43 in zebrafish development. PLoS genetics. 10(1):e1004074. Pubmed: 24497835 DOI:10.1371/journal.pgen.1004074 Zhao C, Andreeva V, Gibert Y, LaBonty M, Lattanzi V, Prabhudesai S, Zhou Y, Zon L, McCann KL, Baserga S, Yelick PC. 2014. Tissue specific roles for the ribosome biogenesis factor Wdr43 in zebrafish development. PLoS genetics. 10(1):e1004074. Pubmed: 24497835 DOI:10.1371/journal.pgen.1004074 During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome. -
Hagedorn EJ, Zon LI. 2014. Unexpectedly uneven: posttransplant skeletal distribution of hematopoietic stem cell clones. The Journal of experimental medicine. 211(3):384-5. Pubmed: 24616474 DOI:10.1084/jem.2113insight2 Hagedorn EJ, Zon LI. 2014. Unexpectedly uneven: posttransplant skeletal distribution of hematopoietic stem cell clones. The Journal of experimental medicine. 211(3):384-5. Pubmed: 24616474 DOI:10.1084/jem.2113insight2 -
Zon L. 2014. Translational research: the path for bringing discovery to patients. Cell stem cell. 14(2):146-8. Pubmed: 24506882 DOI:S1934-5909(14)00005-8 Zon L. 2014. Translational research: the path for bringing discovery to patients. Cell stem cell. 14(2):146-8. Pubmed: 24506882 DOI:S1934-5909(14)00005-8 Translating basic research findings into therapeutic settings presents many scientific, logistic, and financial challenges for academic researchers. Here, I highlight some key insights for navigating such challenges based on recent clinical trials initiated by basic research from my lab.Copyright © 2014 Elsevier Inc. All rights reserved. 2013
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Xu C, Tabebordbar M, Iovino S, Ciarlo C, Liu J, Castiglioni A, Price E, Liu M, Barton ER, Kahn CR, Wagers AJ, Zon LI. 2013. A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species. Cell. 155(4):909-921. Pubmed: 24209627 DOI:10.1016/j.cell.2013.10.023 Xu C, Tabebordbar M, Iovino S, Ciarlo C, Liu J, Castiglioni A, Price E, Liu M, Barton ER, Kahn CR, Wagers AJ, Zon LI. 2013. A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species. Cell. 155(4):909-921. Pubmed: 24209627 DOI:10.1016/j.cell.2013.10.023 Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3β inhibitors, two calpain inhibitors, and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin, and the GSK3β inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle.Copyright © 2013 Elsevier Inc. All rights reserved. -
Paik EJ, Mahony S, White RM, Price EN, Dibiase A, Dorjsuren B, Mosimann C, Davidson AJ, Gifford D, Zon LI. 2013. A Cdx4-Sall4 regulatory module controls the transition from mesoderm formation to embryonic hematopoiesis. Stem cell reports. 1(5):425-36. Pubmed: 24286030 DOI:10.1016/j.stemcr.2013.10.001 Paik EJ, Mahony S, White RM, Price EN, Dibiase A, Dorjsuren B, Mosimann C, Davidson AJ, Gifford D, Zon LI. 2013. A Cdx4-Sall4 regulatory module controls the transition from mesoderm formation to embryonic hematopoiesis. Stem cell reports. 1(5):425-36. Pubmed: 24286030 DOI:10.1016/j.stemcr.2013.10.001 Deletion of caudal/cdx genes alters hox gene expression and causes defects in posterior tissues and hematopoiesis. Yet, the defects in hox gene expression only partially explain these phenotypes. To gain deeper insight into Cdx4 function, we performed chromatin immunoprecipitation sequencing (ChIP-seq) combined with gene-expression profiling in zebrafish, and identified the transcription factor spalt-like 4 (sall4) as a Cdx4 target. ChIP-seq revealed that Sall4 bound to its own gene locus and the cdx4 locus. Expression profiling showed that Cdx4 and Sall4 coregulate genes that initiate hematopoiesis, such as hox, scl, and lmo2. Combined cdx4/sall4 gene knockdown impaired erythropoiesis, and overexpression of the Cdx4 and Sall4 target genes scl and lmo2 together rescued the erythroid program. These findings suggest that auto- and cross-regulation of Cdx4 and Sall4 establish a stable molecular circuit in the mesoderm that facilitates the activation of the blood-specific program as development proceeds. -
Yen J, White RM, Wedge DC, Van Loo P, de Ridder J, Capper A, Richardson J, Jones D, Raine K, Watson IR, Wu CJ, Cheng J, Martincorena I, Nik-Zainal S, Mudie L, Moreau Y, Marshall J, Ramakrishna M, Tarpey P, Shlien A, Whitmore I, Gamble S, Latimer C, Langdon E, Kaufman C, Dovey M, Taylor A, Menzies A, McLaren S, O'Meara S, Butler A, Teague J, Lister J, Chin L, Campbell P, Adams DJ, Zon LI, Patton EE, Stemple DL, Futreal PA. 2013. The genetic heterogeneity and mutational burden of engineered melanomas in zebrafish models. Genome biology. 14(10):R113. Pubmed: 24148783 Yen J, White RM, Wedge DC, Van Loo P, de Ridder J, Capper A, Richardson J, Jones D, Raine K, Watson IR, Wu CJ, Cheng J, Martincorena I, Nik-Zainal S, Mudie L, Moreau Y, Marshall J, Ramakrishna M, Tarpey P, Shlien A, Whitmore I, Gamble S, Latimer C, Langdon E, Kaufman C, Dovey M, Taylor A, Menzies A, McLaren S, O'Meara S, Butler A, Teague J, Lister J, Chin L, Campbell P, Adams DJ, Zon LI, Patton EE, Stemple DL, Futreal PA. 2013. The genetic heterogeneity and mutational burden of engineered melanomas in zebrafish models. Genome biology. 14(10):R113. Pubmed: 24148783 Array -
Kim PG, Albacker CE, Lu YF, Jang IH, Lim Y, Heffner GC, Arora N, Bowman TV, Lin MI, Lensch MW, De Los Angeles A, Zon LI, Loewer S, Daley GQ. 2013. Signaling axis involving Hedgehog, Notch, and Scl promotes the embryonic endothelial-to-hematopoietic transition. Proceedings of the National Academy of Sciences of the United States of America. 110(2):E141-50. Pubmed: 23236128 DOI:10.1073/pnas.1214361110 Kim PG, Albacker CE, Lu YF, Jang IH, Lim Y, Heffner GC, Arora N, Bowman TV, Lin MI, Lensch MW, De Los Angeles A, Zon LI, Loewer S, Daley GQ. 2013. Signaling axis involving Hedgehog, Notch, and Scl promotes the embryonic endothelial-to-hematopoietic transition. Proceedings of the National Academy of Sciences of the United States of America. 110(2):E141-50. Pubmed: 23236128 DOI:10.1073/pnas.1214361110 During development, the hematopoietic lineage transits through hemogenic endothelium, but the signaling pathways effecting this transition are incompletely characterized. Although the Hedgehog (Hh) pathway is hypothesized to play a role in patterning blood formation, early embryonic lethality of mice lacking Hh signaling precludes such analysis. To determine a role for Hh signaling in patterning of hemogenic endothelium, we assessed the effect of altered Hh signaling in differentiating mouse ES cells, cultured mouse embryos, and developing zebrafish embryos. In differentiating mouse ES cells and mouse yolk sac cultures, addition of Indian Hh ligand increased hematopoietic progenitors, whereas chemical inhibition of Hh signaling reduced hematopoietic progenitors without affecting primitive streak mesoderm formation. In the setting of Hh inhibition, induction of either Notch signaling or overexpression of Stem cell leukemia (Scl)/T-cell acute lymphocytic leukemia protein 1 rescued hemogenic vascular-endothelial cadherin(+) cells and hematopoietic progenitor formation. Together, our results reveal that Scl overexpression is sufficient to rescue the developmental defects caused by blocking the Hh and Notch pathways, and inform our understanding of the embryonic endothelial-to-hematopoietic transition. -
Jing CB, Chen Y, Dong M, Peng XL, Jia XE, Gao L, Ma K, Deng M, Liu TX, Zon LI, Zhu J, Zhou Y, Zhou Y. 2013. Phospholipase C gamma-1 is required for granulocyte maturation in zebrafish. Developmental biology. 374(1):24-31. Pubmed: 23220656 DOI:S0012-1606(12)00651-3 Jing CB, Chen Y, Dong M, Peng XL, Jia XE, Gao L, Ma K, Deng M, Liu TX, Zon LI, Zhu J, Zhou Y, Zhou Y. 2013. Phospholipase C gamma-1 is required for granulocyte maturation in zebrafish. Developmental biology. 374(1):24-31. Pubmed: 23220656 DOI:S0012-1606(12)00651-3 The regulation of hematopoiesis is generally evolutionarily conserved from zebrafish to mammals, including hematopoietic stem cell formation and blood cell lineage differentiation. In zebrafish, primitive granulocytes originate at two distinct regions, the anterior lateral plate mesoderm (A-LPM) and the intermediate cell mass (ICM). Few studies in the zebrafish have examined genes specifically required for the granulocytic lineage. In this study, we identified the responsible gene for a zebrafish mutant that has relatively normal hematopoiesis, except decreased expression of the granulocyte-specific gene mpx. Positional cloning revealed that phospholipase C gamma-1 (plcg1) was mutated. Deficiency of plcg1 function specifically affected development of granulocytes, especially the maturation process. These results suggested that plcg1 functioned specifically in zebrafish ICM granulopoiesis for the first time. Our studies suggest that specific pathways regulate the differentiation of the hematopoietic lineages.Copyright © 2012 Elsevier Inc. All rights reserved. -
Bai X, Trowbridge JJ, Riley E, Lee JA, DiBiase A, Kaartinen VM, Orkin SH, Zon LI. 2013. TiF1-gamma plays an essential role in murine hematopoiesis and regulates transcriptional elongation of erythroid genes. Developmental biology. 373(2):422-30. Pubmed: 23159334 DOI:S0012-1606(12)00549-0 Bai X, Trowbridge JJ, Riley E, Lee JA, DiBiase A, Kaartinen VM, Orkin SH, Zon LI. 2013. TiF1-gamma plays an essential role in murine hematopoiesis and regulates transcriptional elongation of erythroid genes. Developmental biology. 373(2):422-30. Pubmed: 23159334 DOI:S0012-1606(12)00549-0 Transcriptional regulators play critical roles in the regulation of cell fate during hematopoiesis. Previous studies in zebrafish have identified an essential role for the transcriptional intermediary factor TIF1γ in erythropoiesis by regulating the transcription elongation of erythroid genes. To study if TIF1γ plays a similar role in murine erythropoiesis and to assess its function in other blood lineages, we generated mouse models with hematopoietic deletion of TIF1γ. Our results showed a block in erythroid maturation in the bone marrow following tif1γ deletion that was compensated with enhanced spleen erythropoiesis. Further analyses revealed a defect in transcription elongation of erythroid genes in the bone marrow. In addition, loss of TIF1γ resulted in defects in other blood compartments, including a profound loss of B cells, a dramatic expansion of granulocytes and decreased HSC function. TIF1γ exerts its functions in a cell-autonomous manner as revealed by competitive transplantation experiments. Our study therefore demonstrates that TIF1γ plays essential roles in multiple murine blood lineages and that its function in transcription elongation is evolutionally conserved.Published by Elsevier Inc. -
Storer NY, White RM, Uong A, Price E, Nielsen GP, Langenau DM, Zon LI. 2013. Zebrafish rhabdomyosarcoma reflects the developmental stage of oncogene expression during myogenesis. Development (Cambridge, England). 140(14):3040-50. Pubmed: 23821038 DOI:10.1242/dev.087858 Storer NY, White RM, Uong A, Price E, Nielsen GP, Langenau DM, Zon LI. 2013. Zebrafish rhabdomyosarcoma reflects the developmental stage of oncogene expression during myogenesis. Development (Cambridge, England). 140(14):3040-50. Pubmed: 23821038 DOI:10.1242/dev.087858 Rhabdomyosarcoma is a pediatric malignancy thought to arise from the uncontrolled proliferation of myogenic cells. Here, we have generated models of rhabdomyosarcoma in the zebrafish by inducing oncogenic KRAS(G12D) expression at different stages during muscle development. Several zebrafish promoters were used, including the cdh15 and rag2 promoters, which drive gene expression in early muscle progenitors, and the mylz2 promoter, which is expressed in differentiating myoblasts. The tumors that developed differed in their ability to recapitulate normal myogenesis. cdh15:KRAS(G12D) and rag2:KRAS(G12D) fish developed tumors that displayed an inability to complete muscle differentiation as determined by histological appearance and gene expression analyses. By contrast, mylz2:KRAS(G12D) tumors more closely resembled mature skeletal muscle and were most similar to well-differentiated human rhabdomyosarcoma in terms of gene expression. mylz2:KRAS(G12D) fish showed significantly improved survival compared with cdh15:KRAS(G12D) and rag2:KRAS(G12D) fish. Tumor-propagating activity was enriched in myf5-expressing cell populations within all of the tumor types. Our results demonstrate that oncogenic KRAS(G12D) expression at different stages during muscle development has profound effects on the ability of tumor cells to recapitulate normal myogenesis, altering the tumorigenic capability of these cells. -
Jagannathan-Bogdan M, Zon LI. 2013. Hematopoiesis. Development (Cambridge, England). 140(12):2463-7. Pubmed: 23715539 DOI:10.1242/dev.083147 Jagannathan-Bogdan M, Zon LI. 2013. Hematopoiesis. Development (Cambridge, England). 140(12):2463-7. Pubmed: 23715539 DOI:10.1242/dev.083147 Hematopoiesis - the process by which blood cells are formed - has been studied intensely for over a century using a variety of model systems. There is conservation of the overall hematopoietic process between vertebrates, although some differences do exist. Over the last decade, the zebrafish has come to the forefront as a new model in hematopoiesis research, as it allows the use of large-scale genetics, chemical screens and transgenics. This comparative approach to understanding hematopoiesis has led to fundamental knowledge about the process and to the development of new therapies for disease. Here, we provide a broad overview of vertebrate hematopoiesis. We also highlight the benefits of using zebrafish as a model. -
Albacker CE, Storer NY, Langdon EM, Dibiase A, Zhou Y, Langenau DM, Zon LI. 2013. The histone methyltransferase SUV39H1 suppresses embryonal rhabdomyosarcoma formation in zebrafish. PloS one. 8(5):e64969. Pubmed: 23705022 DOI:10.1371/journal.pone.0064969 Albacker CE, Storer NY, Langdon EM, Dibiase A, Zhou Y, Langenau DM, Zon LI. 2013. The histone methyltransferase SUV39H1 suppresses embryonal rhabdomyosarcoma formation in zebrafish. PloS one. 8(5):e64969. Pubmed: 23705022 DOI:10.1371/journal.pone.0064969 Epigenetics, or the reversible and heritable marks of gene regulation not including DNA sequence, encompasses chromatin modifications on both the DNA and histones and is as important as the DNA sequence itself. Chromatin-modifying factors are playing an increasingly important role in tumorigenesis, particularly among pediatric rhabdomyosarcomas (RMS), revealing potential novel therapeutic targets. We performed an overexpression screen of chromatin-modifying factors in a KRAS(G12D)-driven zebrafish model for RMS. Here, we describe the identification of a histone H3 lysine 9 histone methyltransferase, SUV39H1, as a suppressor of embryonal RMS formation in zebrafish. This suppression is specific to the histone methyltransferase activity of SUV39H1, as point mutations in the SET domain lacked the effect. SUV39H1-overexpressing and control tumors have a similar proliferation rate, muscle differentiation state, and tumor growth rate. Strikingly, SUV39H1-overexpressing fish initiate fewer tumors, which results in the observed suppressive phenotype. We demonstrate that the delayed tumor onset occurs between 5 and 7 days post fertilization. Gene expression profiling at these stages revealed that in the context of KRAS(G12D) overexpression, SUV39H1 may suppress cell cycle progression. Our studies provide evidence for the role of SUV39H1 as a tumor suppressor. -
Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, Collins JE, Humphray S, McLaren K, Matthews L, McLaren S, Sealy I, Caccamo M, Churcher C, Scott C, Barrett JC, Koch R, Rauch GJ, White S, Chow W, Kilian B, Quintais LT, Guerra-Assunção JA, Zhou Y, Gu Y, Yen J, Vogel JH, Eyre T, Redmond S, Banerjee R, Chi J, Fu B, Langley E, Maguire SF, Laird GK, Lloyd D, Kenyon E, Donaldson S, Sehra H, Almeida-King J, Loveland J, Trevanion S, Jones M, Quail M, Willey D, Hunt A, Burton J, Sims S, McLay K, Plumb B, Davis J, Clee C, Oliver K, Clark R, Riddle C, Elliot D, Threadgold G, Harden G, Ware D, Begum S, Mortimore B, Kerry G, Heath P, Phillimore B, Tracey A, Corby N, Dunn M, Johnson C, Wood J, Clark S, Pelan S, Griffiths G, Smith M, Glithero R, Howden P, Barker N, Lloyd C, Stevens C, Harley J, Holt K, Panagiotidis G, Lovell J, Beasley H, Henderson C, Gordon D, Auger K, Wright D, Collins J, Raisen C, Dyer L, Leung K, Robertson L, Ambridge K, Leongamornlert D, McGuire S, Gilderthorp R, Griffiths C, Manthravadi D, Nichol S, Barker G, Whitehead S, Kay M, Brown J, Murnane C, Gray E, Humphries M, Sycamore N, Barker D, Saunders D, Wallis J, Babbage A, Hammond S, Mashreghi-Mohammadi M, Barr L, Martin S, Wray P, Ellington A, Matthews N, Ellwood M, Woodmansey R, Clark G, Cooper J, Tromans A, Grafham D, Skuce C, Pandian R, Andrews R, Harrison E, Kimberley A, Garnett J, Fosker N, Hall R, Garner P, Kelly D, Bird C, Palmer S, Gehring I, Berger A, Dooley CM, Ersan-Ürün Z, Eser C, Geiger H, Geisler M, Karotki L, Kirn A, Konantz J, Konantz M, Oberländer M, Rudolph-Geiger S, Teucke M, Lanz C, Raddatz G, Osoegawa K, Zhu B, Rapp A, Widaa S, Langford C, Yang F, Schuster SC, Carter NP, Harrow J, Ning Z, Herrero J, Searle SM, Enright A, Geisler R, Plasterk RH, Lee C, Westerfield M, de Jong PJ, Zon LI, Postlethwait JH, Nüsslein-Volhard C, Hubbard TJ, Roest Crollius H, Rogers J, Stemple DL. 2013. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 496(7446):498-503. Pubmed: 23594743 DOI:10.1038/nature12111 Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, Collins JE, Humphray S, McLaren K, Matthews L, McLaren S, Sealy I, Caccamo M, Churcher C, Scott C, Barrett JC, Koch R, Rauch GJ, White S, Chow W, Kilian B, Quintais LT, Guerra-Assunção JA, Zhou Y, Gu Y, Yen J, Vogel JH, Eyre T, Redmond S, Banerjee R, Chi J, Fu B, Langley E, Maguire SF, Laird GK, Lloyd D, Kenyon E, Donaldson S, Sehra H, Almeida-King J, Loveland J, Trevanion S, Jones M, Quail M, Willey D, Hunt A, Burton J, Sims S, McLay K, Plumb B, Davis J, Clee C, Oliver K, Clark R, Riddle C, Elliot D, Threadgold G, Harden G, Ware D, Begum S, Mortimore B, Kerry G, Heath P, Phillimore B, Tracey A, Corby N, Dunn M, Johnson C, Wood J, Clark S, Pelan S, Griffiths G, Smith M, Glithero R, Howden P, Barker N, Lloyd C, Stevens C, Harley J, Holt K, Panagiotidis G, Lovell J, Beasley H, Henderson C, Gordon D, Auger K, Wright D, Collins J, Raisen C, Dyer L, Leung K, Robertson L, Ambridge K, Leongamornlert D, McGuire S, Gilderthorp R, Griffiths C, Manthravadi D, Nichol S, Barker G, Whitehead S, Kay M, Brown J, Murnane C, Gray E, Humphries M, Sycamore N, Barker D, Saunders D, Wallis J, Babbage A, Hammond S, Mashreghi-Mohammadi M, Barr L, Martin S, Wray P, Ellington A, Matthews N, Ellwood M, Woodmansey R, Clark G, Cooper J, Tromans A, Grafham D, Skuce C, Pandian R, Andrews R, Harrison E, Kimberley A, Garnett J, Fosker N, Hall R, Garner P, Kelly D, Bird C, Palmer S, Gehring I, Berger A, Dooley CM, Ersan-Ürün Z, Eser C, Geiger H, Geisler M, Karotki L, Kirn A, Konantz J, Konantz M, Oberländer M, Rudolph-Geiger S, Teucke M, Lanz C, Raddatz G, Osoegawa K, Zhu B, Rapp A, Widaa S, Langford C, Yang F, Schuster SC, Carter NP, Harrow J, Ning Z, Herrero J, Searle SM, Enright A, Geisler R, Plasterk RH, Lee C, Westerfield M, de Jong PJ, Zon LI, Postlethwait JH, Nüsslein-Volhard C, Hubbard TJ, Roest Crollius H, Rogers J, Stemple DL. 2013. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 496(7446):498-503. Pubmed: 23594743 DOI:10.1038/nature12111 Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination. -
Bowman TV, Zon LI. 2013. Ageing: Stem cells on a stress-busting diet. Nature. 494(7437):317-8. Pubmed: 23389448 DOI:10.1038/nature11948 Bowman TV, Zon LI. 2013. Ageing: Stem cells on a stress-busting diet. Nature. 494(7437):317-8. Pubmed: 23389448 DOI:10.1038/nature11948 -
Ciarlo CA, Zon LI. 2013. A model for primary melanoma of the CNS implicates NRAS. Cancer discovery. 3(4):382-3. Pubmed: 23580282 DOI:10.1158/2159-8290.CD-13-0076 Ciarlo CA, Zon LI. 2013. A model for primary melanoma of the CNS implicates NRAS. Cancer discovery. 3(4):382-3. Pubmed: 23580282 DOI:10.1158/2159-8290.CD-13-0076 In this issue of Cancer Discovery, Pedersen and colleagues present the first mouse model of primary CNS melanoma, which arises when oncogenic NRAS is expressed from the endogenous Nras promoter in melanocytes during embryogenesis. In support of this model, two pediatric cases of NRAS-mutant primary melanoma of the CNS are identified.©2013 AACR. -
Stachura DL, Svoboda O, Campbell CA, Espín-Palazón R, Lau RP, Zon LI, Bartunek P, Traver D. 2013. The zebrafish granulocyte colony-stimulating factors (Gcsfs): 2 paralogous cytokines and their roles in hematopoietic development and maintenance. Blood. 122(24):3918-28. Pubmed: 24128862 DOI:10.1182/blood-2012-12-475392 Stachura DL, Svoboda O, Campbell CA, Espín-Palazón R, Lau RP, Zon LI, Bartunek P, Traver D. 2013. The zebrafish granulocyte colony-stimulating factors (Gcsfs): 2 paralogous cytokines and their roles in hematopoietic development and maintenance. Blood. 122(24):3918-28. Pubmed: 24128862 DOI:10.1182/blood-2012-12-475392 Granulocyte colony-stimulating factor (Gcsf) drives the proliferation and differentiation of granulocytes, monocytes, and macrophages (mφs) from hematopoietic stem and progenitor cells (HSPCs). Analysis of the zebrafish genome indicates the presence of 2 Gcsf ligands, likely resulting from a duplication event in teleost evolution. Although Gcsfa and Gcsfb share low sequence conservation, they share significant similarity in their predicted ligand/receptor interaction sites and structure. Each ligand displays differential temporal expression patterns during embryogenesis and spatial expression patterns in adult animals. To determine the functions of each ligand, we performed loss- and gain-of-function experiments. Both ligands signal through the Gcsf receptor to expand primitive neutrophils and mφs, as well as definitive granulocytes. To further address their functions, we generated recombinant versions and tested them in clonal progenitor assays. These sensitive in vitro techniques indicated similar functional attributes in supporting HSPC growth and differentiation. Finally, in addition to supporting myeloid differentiation, zebrafish Gcsf is required for the specification and proliferation of hematopoietic stem cells, suggesting that Gcsf represents an ancestral cytokine responsible for the broad support of HSPCs. These findings may inform how hematopoietic cytokines evolved following the diversification of teleosts and mammals from a common ancestor. -
Doulatov S, Vo LT, Chou SS, Kim PG, Arora N, Li H, Hadland BK, Bernstein ID, Collins JJ, Zon LI, Daley GQ. 2013. Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via respecification of lineage-restricted precursors. Cell stem cell. 13(4):459-70. Pubmed: 24094326 DOI:S1934-5909(13)00402-5 Doulatov S, Vo LT, Chou SS, Kim PG, Arora N, Li H, Hadland BK, Bernstein ID, Collins JJ, Zon LI, Daley GQ. 2013. Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via respecification of lineage-restricted precursors. Cell stem cell. 13(4):459-70. Pubmed: 24094326 DOI:S1934-5909(13)00402-5 Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor cells (HSPCs) has limited their characterization to in vitro assays. We report a strategy to respecify lineage-restricted CD34(+)CD45(+) myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engrafted in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34(+)CD38(-) cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, conferred engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription-factor-mediated engraftment of blood progenitors from human pluripotent cells.Copyright © 2013 Elsevier Inc. All rights reserved. -
White R, Rose K, Zon L. 2013. Zebrafish cancer: the state of the art and the path forward. Nature reviews. Cancer. 13(9):624-36. Pubmed: 23969693 DOI:10.1038/nrc3589 White R, Rose K, Zon L. 2013. Zebrafish cancer: the state of the art and the path forward. Nature reviews. Cancer. 13(9):624-36. Pubmed: 23969693 DOI:10.1038/nrc3589 The zebrafish is a recent addition to animal models of human cancer, and studies using this model are rapidly contributing major insights. Zebrafish develop cancer spontaneously, after mutagen exposure and through transgenesis. The tumours resemble human cancers at the histological, gene expression and genomic levels. The ability to carry out in vivo imaging, chemical and genetic screens, and high-throughput transgenesis offers a unique opportunity to functionally characterize the cancer genome. Moreover, increasingly sophisticated modelling of combinations of genetic and epigenetic alterations will allow the zebrafish to complement what can be achieved in other models, such as mouse and human cell culture systems. -
Cutler C, Multani P, Robbins D, Kim HT, Le T, Hoggatt J, Pelus LM, Desponts C, Chen YB, Rezner B, Armand P, Koreth J, Glotzbecker B, Ho VT, Alyea E, Isom M, Kao G, Armant M, Silberstein L, Hu P, Soiffer RJ, Scadden DT, Ritz J, Goessling W, North TE, Mendlein J, Ballen K, Zon LI, Antin JH, Shoemaker DD. 2013. Prostaglandin-modulated umbilical cord blood hematopoietic stem cell transplantation. Blood. 122(17):3074-81. Pubmed: 23996087 DOI:10.1182/blood-2013-05-503177 Cutler C, Multani P, Robbins D, Kim HT, Le T, Hoggatt J, Pelus LM, Desponts C, Chen YB, Rezner B, Armand P, Koreth J, Glotzbecker B, Ho VT, Alyea E, Isom M, Kao G, Armant M, Silberstein L, Hu P, Soiffer RJ, Scadden DT, Ritz J, Goessling W, North TE, Mendlein J, Ballen K, Zon LI, Antin JH, Shoemaker DD. 2013. Prostaglandin-modulated umbilical cord blood hematopoietic stem cell transplantation. Blood. 122(17):3074-81. Pubmed: 23996087 DOI:10.1182/blood-2013-05-503177 Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells (HSCs) for use in allogeneic transplantation. Key advantages of UCB are rapid availability and less stringent requirements for HLA matching. However, UCB contains an inherently limited HSC count, which is associated with delayed time to engraftment, high graft failure rates, and early mortality. 16,16-Dimethyl prostaglandin E2 (dmPGE2) was previously identified to be a critical regulator of HSC homeostasis, and we hypothesized that brief ex vivo modulation with dmPGE2 could improve patient outcomes by increasing the "effective dose" of HSCs. Molecular profiling approaches were used to determine the optimal ex vivo modulation conditions (temperature, time, concentration, and media) for use in the clinical setting. A phase 1 trial was performed to evaluate the safety and therapeutic potential of ex vivo modulation of a single UCB unit using dmPGE2 before reduced-intensity, double UCB transplantation. Results from this study demonstrated clear safety with durable, multilineage engraftment of dmPGE2-treated UCB units. We observed encouraging trends in efficacy, with accelerated neutrophil recovery (17.5 vs 21 days, P = .045), coupled with preferential, long-term engraftment of the dmPGE2-treated UCB unit in 10 of 12 treated participants. -
Boatman S, Barrett F, Satishchandran S, Jing L, Shestopalov I, Zon LI. 2013. Assaying hematopoiesis using zebrafish. Blood cells, molecules & diseases. 51(4):271-6. Pubmed: 23916372 DOI:S1079-9796(13)00160-5 Boatman S, Barrett F, Satishchandran S, Jing L, Shestopalov I, Zon LI. 2013. Assaying hematopoiesis using zebrafish. Blood cells, molecules & diseases. 51(4):271-6. Pubmed: 23916372 DOI:S1079-9796(13)00160-5 The zebrafish has become a commonly used model for studying hematopoiesis as a result of its unique attributes. Zebrafish are highly suitable for large-scale genetic and chemical screens compared to other vertebrate systems. It is now possible to analyze hematopoietic lineages in zebrafish and validate cell function via transplantation assays. Here, we review advancements over the past decade in forward genetic screens, chemical screens, fluorescence-activated cell sorting analysis, and transplantation assays. Integrating these approaches enables new chemical and genetic screens that assay cell function within the hematopoietic system. Studies in zebrafish will continue to contribute and expand our knowledge about hematopoiesis, and develop novel treatments for clinical applications.© 2013. -
Mosimann C, Puller AC, Lawson KL, Tschopp P, Amsterdam A, Zon LI. 2013. Site-directed zebrafish transgenesis into single landing sites with the phiC31 integrase system. Developmental dynamics : an official publication of the American Association of Anatomists. 242(8):949-963. Pubmed: 23723152 DOI:10.1002/dvdy.23989 Mosimann C, Puller AC, Lawson KL, Tschopp P, Amsterdam A, Zon LI. 2013. Site-directed zebrafish transgenesis into single landing sites with the phiC31 integrase system. Developmental dynamics : an official publication of the American Association of Anatomists. 242(8):949-963. Pubmed: 23723152 DOI:10.1002/dvdy.23989 ArrayCopyright © 2013 Wiley Periodicals, Inc. -
Ablain J, Zon LI. 2013. Of fish and men: using zebrafish to fight human diseases. Trends in cell biology. 23(12):584-6. Pubmed: 24275383 DOI:S0962-8924(13)00166-9 Ablain J, Zon LI. 2013. Of fish and men: using zebrafish to fight human diseases. Trends in cell biology. 23(12):584-6. Pubmed: 24275383 DOI:S0962-8924(13)00166-9 Long restricted to the field of developmental biology, the use of the zebrafish (Danio rerio) has extended to the study of human pathogenesis. Fostered by the rapid adaptation of new technologies, the design and analysis of fish models of human diseases have contributed important findings that are now making their way from aquariums to clinics. Here we outline the clinical relevance of the zebrafish as a model organism.Copyright © 2013 Elsevier Ltd. All rights reserved. -
Huang HT, Kathrein KL, Barton A, Gitlin Z, Huang YH, Ward TP, Hofmann O, Dibiase A, Song A, Tyekucheva S, Hide W, Zhou Y, Zon LI. 2013. A network of epigenetic regulators guides developmental haematopoiesis in vivo. Nature cell biology. 15(12):1516-25. Pubmed: 24240475 DOI:10.1038/ncb2870 Huang HT, Kathrein KL, Barton A, Gitlin Z, Huang YH, Ward TP, Hofmann O, Dibiase A, Song A, Tyekucheva S, Hide W, Zhou Y, Zon LI. 2013. A network of epigenetic regulators guides developmental haematopoiesis in vivo. Nature cell biology. 15(12):1516-25. Pubmed: 24240475 DOI:10.1038/ncb2870 The initiation of cellular programs is orchestrated by key transcription factors and chromatin regulators that activate or inhibit target gene expression. To generate a compendium of chromatin factors that establish the epigenetic code during developmental haematopoiesis, a large-scale reverse genetic screen was conducted targeting orthologues of 425 human chromatin factors in zebrafish. A set of chromatin regulators was identified that target different stages of primitive and definitive blood formation, including factors not previously implicated in haematopoiesis. We identified 15 factors that regulate development of primitive erythroid progenitors and 29 factors that regulate development of definitive haematopoietic stem and progenitor cells. These chromatin factors are associated with SWI/SNF and ISWI chromatin remodelling, SET1 methyltransferase, CBP-p300-HBO1-NuA4 acetyltransferase, HDAC-NuRD deacetylase, and Polycomb repressive complexes. Our work provides a comprehensive view of how specific chromatin factors and their associated complexes play a major role in the establishment of haematopoietic cells in vivo. -
Chen CH, Durand E, Wang J, Zon LI, Poss KD. 2013. zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish. Development (Cambridge, England). 140(24):4988-97. Pubmed: 24198277 DOI:10.1242/dev.102053 Chen CH, Durand E, Wang J, Zon LI, Poss KD. 2013. zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish. Development (Cambridge, England). 140(24):4988-97. Pubmed: 24198277 DOI:10.1242/dev.102053 The zebrafish has become a standard model system for stem cell and tissue regeneration research, based on powerful genetics, high tissue regenerative capacity and low maintenance costs. Yet, these studies can be challenged by current limitations of tissue visualization techniques in adult animals. Here we describe new imaging methodology and present several ubiquitous and tissue-specific luciferase-based transgenic lines, which we have termed zebraflash, that facilitate the assessment of regeneration and engraftment in freely moving adult zebrafish. We show that luciferase-based live imaging reliably estimates muscle quantity in an internal organ, the heart, and can longitudinally follow cardiac regeneration in individual animals after major injury. Furthermore, luciferase-based detection enables visualization and quantification of engraftment in live recipients of transplanted hematopoietic stem cell progeny, with advantages in sensitivity and gross spatial resolution over fluorescence detection. Our findings present a versatile resource for monitoring and dissecting vertebrate stem cell and regeneration biology. -
Cooney JD, Hildick-Smith GJ, Shafizadeh E, McBride PF, Carroll KJ, Anderson H, Shaw GC, Tamplin OJ, Branco DS, Dalton AJ, Shah DI, Wong C, Gallagher PG, Zon LI, North TE, Paw BH. 2013. Teleost growth factor independence (gfi) genes differentially regulate successive waves of hematopoiesis. Developmental biology. 373(2):431-41. Pubmed: 22960038 DOI:S0012-1606(12)00455-1 Cooney JD, Hildick-Smith GJ, Shafizadeh E, McBride PF, Carroll KJ, Anderson H, Shaw GC, Tamplin OJ, Branco DS, Dalton AJ, Shah DI, Wong C, Gallagher PG, Zon LI, North TE, Paw BH. 2013. Teleost growth factor independence (gfi) genes differentially regulate successive waves of hematopoiesis. Developmental biology. 373(2):431-41. Pubmed: 22960038 DOI:S0012-1606(12)00455-1 Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.Copyright © 2012 Elsevier Inc. All rights reserved. -
Le X, Pugach EK, Hettmer S, Storer NY, Liu J, Wills AA, DiBiase A, Chen EY, Ignatius MS, Poss KD, Wagers AJ, Langenau DM, Zon LI. 2013. A novel chemical screening strategy in zebrafish identifies common pathways in embryogenesis and rhabdomyosarcoma development. Development (Cambridge, England). 140(11):2354-64. Pubmed: 23615277 DOI:10.1242/dev.088427 Le X, Pugach EK, Hettmer S, Storer NY, Liu J, Wills AA, DiBiase A, Chen EY, Ignatius MS, Poss KD, Wagers AJ, Langenau DM, Zon LI. 2013. A novel chemical screening strategy in zebrafish identifies common pathways in embryogenesis and rhabdomyosarcoma development. Development (Cambridge, England). 140(11):2354-64. Pubmed: 23615277 DOI:10.1242/dev.088427 The zebrafish is a powerful genetic model that has only recently been used to dissect developmental pathways involved in oncogenesis. We hypothesized that operative pathways during embryogenesis would also be used for oncogenesis. In an effort to define RAS target genes during embryogenesis, gene expression was evaluated in Tg(hsp70-HRAS(G12V)) zebrafish embryos subjected to heat shock. dusp6 was activated by RAS, and this was used as the basis for a chemical genetic screen to identify small molecules that interfere with RAS signaling during embryogenesis. A KRAS(G12D)-induced zebrafish embryonal rhabdomyosarcoma was then used to assess the therapeutic effects of the small molecules. Two of these inhibitors, PD98059 and TPCK, had anti-tumor activity as single agents in both zebrafish embryonal rhabdomyosarcoma and a human cell line of rhabdomyosarcoma that harbored activated mutations in NRAS. PD98059 inhibited MEK1 whereas TPCK suppressed S6K1 activity; however, the combined treatment completely suppressed eIF4B phosphorylation and decreased translation initiation. Our work demonstrates that the activated pathways in RAS induction during embryogenesis are also important in oncogenesis and that inhibition of these pathways suppresses tumor growth. 2012
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Gupta VA, Kawahara G, Myers JA, Chen AT, Hall TE, Manzini MC, Currie PD, Zhou Y, Zon LI, Kunkel LM, Beggs AH. 2012. A splice site mutation in laminin-α2 results in a severe muscular dystrophy and growth abnormalities in zebrafish. PloS one. 7(8):e43794. Pubmed: 22952766 DOI:10.1371/journal.pone.0043794 Gupta VA, Kawahara G, Myers JA, Chen AT, Hall TE, Manzini MC, Currie PD, Zhou Y, Zon LI, Kunkel LM, Beggs AH. 2012. A splice site mutation in laminin-α2 results in a severe muscular dystrophy and growth abnormalities in zebrafish. PloS one. 7(8):e43794. Pubmed: 22952766 DOI:10.1371/journal.pone.0043794 Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A) is a form of CMD caused by a defect in the laminin-α2 gene (LAMA2). Laminin-α2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC) complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2(cl501/cl501), exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8-15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-α2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-α2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD. -
Mochida GH, Ganesh VS, de Michelena MI, Dias H, Atabay KD, Kathrein KL, Huang HT, Hill RS, Felie JM, Rakiec D, Gleason D, Hill AD, Malik AN, Barry BJ, Partlow JN, Tan WH, Glader LJ, Barkovich AJ, Dobyns WB, Zon LI, Walsh CA. 2012. CHMP1A encodes an essential regulator of BMI1-INK4A in cerebellar development. Nature genetics. 44(11):1260-4. Pubmed: 23023333 DOI:10.1038/ng.2425 Mochida GH, Ganesh VS, de Michelena MI, Dias H, Atabay KD, Kathrein KL, Huang HT, Hill RS, Felie JM, Rakiec D, Gleason D, Hill AD, Malik AN, Barry BJ, Partlow JN, Tan WH, Glader LJ, Barkovich AJ, Dobyns WB, Zon LI, Walsh CA. 2012. CHMP1A encodes an essential regulator of BMI1-INK4A in cerebellar development. Nature genetics. 44(11):1260-4. Pubmed: 23023333 DOI:10.1038/ng.2425 Charged multivesicular body protein 1A (CHMP1A; also known as chromatin-modifying protein 1A) is a member of the ESCRT-III (endosomal sorting complex required for transport-III) complex but is also suggested to localize to the nuclear matrix and regulate chromatin structure. Here, we show that loss-of-function mutations in human CHMP1A cause reduced cerebellar size (pontocerebellar hypoplasia) and reduced cerebral cortical size (microcephaly). CHMP1A-mutant cells show impaired proliferation, with increased expression of INK4A, a negative regulator of stem cell proliferation. Chromatin immunoprecipitation suggests loss of the normal INK4A repression by BMI in these cells. Morpholino-based knockdown of zebrafish chmp1a resulted in brain defects resembling those seen after bmi1a and bmi1b knockdown, which were partially rescued by INK4A ortholog knockdown, further supporting links between CHMP1A and BMI1-mediated regulation of INK4A. Our results suggest that CHMP1A serves as a critical link between cytoplasmic signals and BMI1-mediated chromatin modifications that regulate proliferation of central nervous system progenitor cells. -
McKinney-Freeman S, Cahan P, Li H, Lacadie SA, Huang HT, Curran M, Loewer S, Naveiras O, Kathrein KL, Konantz M, Langdon EM, Lengerke C, Zon LI, Collins JJ, Daley GQ. 2012. The transcriptional landscape of hematopoietic stem cell ontogeny. Cell stem cell. 11(5):701-14. Pubmed: 23122293 DOI:S1934-5909(12)00476-6 McKinney-Freeman S, Cahan P, Li H, Lacadie SA, Huang HT, Curran M, Loewer S, Naveiras O, Kathrein KL, Konantz M, Langdon EM, Lengerke C, Zon LI, Collins JJ, Daley GQ. 2012. The transcriptional landscape of hematopoietic stem cell ontogeny. Cell stem cell. 11(5):701-14. Pubmed: 23122293 DOI:S1934-5909(12)00476-6 Transcriptome analysis of adult hematopoietic stem cells (HSCs) and their progeny has revealed mechanisms of blood differentiation and leukemogenesis, but a similar analysis of HSC development is lacking. Here, we acquired the transcriptomes of developing HSCs purified from >2,500 murine embryos and adult mice. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs undergoing specification, and definitive HSCs. We applied a network-biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development and functionally validated candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knockdown in zebrafish embryos. Moreover, we found that HSCs from in vitro differentiated embryonic stem cells closely resemble definitive HSCs, yet lack a Notch-signaling signature, likely accounting for their defective lymphopoiesis. Our analysis and web resource will enhance efforts to identify regulators of HSC ontogeny and facilitate the engineering of hematopoietic specification.Copyright © 2012 Elsevier Inc. All rights reserved. -
de Jong JL, Zon LI. 2012. Histocompatibility and hematopoietic transplantation in the zebrafish. Advances in hematology. 2012:282318. Pubmed: 22778744 DOI:10.1155/2012/282318 de Jong JL, Zon LI. 2012. Histocompatibility and hematopoietic transplantation in the zebrafish. Advances in hematology. 2012:282318. Pubmed: 22778744 DOI:10.1155/2012/282318 The zebrafish has proven to be an excellent model for human disease, particularly hematopoietic diseases, since these fish make similar types of blood cells as humans and other mammals. The genetic program that regulates the development and differentiation of hematopoietic cells is highly conserved. Hematopoietic stem cells (HSCs) are the source of all the blood cells needed by an organism during its lifetime. Identifying an HSC requires a functional assay, namely, a transplantation assay consisting of multilineage engraftment of a recipient and subsequent serial transplant recipients. In the past decade, several types of hematopoietic transplant assays have been developed in the zebrafish. An understanding of the major histocompatibility complex (MHC) genes in the zebrafish has lagged behind transplantation experiments, limiting the ability to perform unbiased competitive transplantation assays. This paper summarizes the different hematopoietic transplantation experiments performed in the zebrafish, both with and without immunologic matching, and discusses future directions for this powerful experimental model of human blood diseases. -
Santoriello C, Zon LI. 2012. Hooked! Modeling human disease in zebrafish. The Journal of clinical investigation. 122(7):2337-43. Pubmed: 22751109 DOI:60434 Santoriello C, Zon LI. 2012. Hooked! Modeling human disease in zebrafish. The Journal of clinical investigation. 122(7):2337-43. Pubmed: 22751109 DOI:60434 Zebrafish have been widely used as a model system for studying developmental processes, but in the last decade, they have also emerged as a valuable system for modeling human disease. The development and function of zebrafish organs are strikingly similar to those of humans, and the ease of creating mutant or transgenic fish has facilitated the generation of disease models. Here, we highlight the use of zebrafish for defining disease pathways and for discovering new therapies. -
Mione M, Zon LI. 2012. Cancer and inflammation: an aspirin a day keeps the cancer at bay. Current biology : CB. 22(13):R522-5. Pubmed: 22789999 DOI:10.1016/j.cub.2012.05.037 Mione M, Zon LI. 2012. Cancer and inflammation: an aspirin a day keeps the cancer at bay. Current biology : CB. 22(13):R522-5. Pubmed: 22789999 DOI:10.1016/j.cub.2012.05.037 Live imaging of the interactions between oncogene-transformed cells and leukocytes in zebrafish reveals that PGE(2) promotes the survival and proliferation of cancer cells. Non-steroid anti-inflammatory drugs, like aspirin, are the effective inhibitors of PGE(2) production and could be used with other anti-tumor agents in the treatment of cancer.Copyright © 2012 Elsevier Ltd. All rights reserved. -
Oubaha M, Lin MI, Margaron Y, Filion D, Price EN, Zon LI, Côté JF, Gratton JP. 2012. Formation of a PKCζ/β-catenin complex in endothelial cells promotes angiopoietin-1-induced collective directional migration and angiogenic sprouting. Blood. 120(16):3371-81. Pubmed: 22936663 DOI:10.1182/blood-2012-03-419721 Oubaha M, Lin MI, Margaron Y, Filion D, Price EN, Zon LI, Côté JF, Gratton JP. 2012. Formation of a PKCζ/β-catenin complex in endothelial cells promotes angiopoietin-1-induced collective directional migration and angiogenic sprouting. Blood. 120(16):3371-81. Pubmed: 22936663 DOI:10.1182/blood-2012-03-419721 Angiogenic sprouting requires that cell-cell contacts be maintained during migration of endothelial cells. Angiopoietin-1 (Ang-1) and vascular endothelial growth factor act oppositely on endothelial cell junctions. We found that Ang-1 promotes collective and directional migration and, in contrast to VEGF, induces the formation of a complex formed of atypical protein kinase C (PKC)-ζ and β-catenin at cell-cell junctions and at the leading edge of migrating endothelial cells. This complex brings Par3, Par6, and adherens junction proteins at the front of migrating cells to locally activate Rac1 in response to Ang-1. The colocalization of PKCζ and β-catenin at leading edge along with PKCζ-dependent stabilization of cell-cell contacts promotes directed and collective endothelial cell migration. Consistent with these results, down-regulation of PKCζ in endothelial cells alters Ang-1-induced sprouting in vitro and knockdown in developing zebrafish results in intersegmental vessel defects caused by a perturbed directionality of tip cells and by loss of cell contacts between tip and stalk cells. These results reveal that PKCζ and β-catenin function in a complex at adherens junctions and at the leading edge of migrating endothelial cells to modulate collective and directional migration during angiogenesis. -
Sorrells S, Carbonneau S, Harrington E, Chen AT, Hast B, Milash B, Pyati U, Major MB, Zhou Y, Zon LI, Stewart RA, Look AT, Jette C. 2012. Ccdc94 protects cells from ionizing radiation by inhibiting the expression of p53. PLoS genetics. 8(8):e1002922. Pubmed: 22952453 DOI:10.1371/journal.pgen.1002922 Sorrells S, Carbonneau S, Harrington E, Chen AT, Hast B, Milash B, Pyati U, Major MB, Zhou Y, Zon LI, Stewart RA, Look AT, Jette C. 2012. Ccdc94 protects cells from ionizing radiation by inhibiting the expression of p53. PLoS genetics. 8(8):e1002922. Pubmed: 22952453 DOI:10.1371/journal.pgen.1002922 DNA double-strand breaks (DSBs) represent one of the most deleterious forms of DNA damage to a cell. In cancer therapy, induction of cell death by DNA DSBs by ionizing radiation (IR) and certain chemotherapies is thought to mediate the successful elimination of cancer cells. However, cancer cells often evolve to evade the cytotoxicity induced by DNA DSBs, thereby forming the basis for treatment resistance. As such, a better understanding of the DSB DNA damage response (DSB-DDR) pathway will facilitate the design of more effective strategies to overcome chemo- and radioresistance. To identify novel mechanisms that protect cells from the cytotoxic effects of DNA DSBs, we performed a forward genetic screen in zebrafish for recessive mutations that enhance the IR-induced apoptotic response. Here, we describe radiosensitizing mutation 7 (rs7), which causes a severe sensitivity of zebrafish embryonic neurons to IR-induced apoptosis and is required for the proper development of the central nervous system. The rs7 mutation disrupts the coding sequence of ccdc94, a highly conserved gene that has no previous links to the DSB-DDR pathway. We demonstrate that Ccdc94 is a functional member of the Prp19 complex and that genetic knockdown of core members of this complex causes increased sensitivity to IR-induced apoptosis. We further show that Ccdc94 and the Prp19 complex protect cells from IR-induced apoptosis by repressing the expression of p53 mRNA. In summary, we have identified a new gene regulating a dosage-sensitive response to DNA DSBs during embryonic development. Future studies in human cancer cells will determine whether pharmacological inactivation of CCDC94 reduces the threshold of the cancer cell apoptotic response. -
Lian CG, Xu Y, Ceol C, Wu F, Larson A, Dresser K, Xu W, Tan L, Hu Y, Zhan Q, Lee CW, Hu D, Lian BQ, Kleffel S, Yang Y, Neiswender J, Khorasani AJ, Fang R, Lezcano C, Duncan LM, Scolyer RA, Thompson JF, Kakavand H, Houvras Y, Zon LI, Mihm MC, Kaiser UB, Schatton T, Woda BA, Murphy GF, Shi YG. 2012. Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma. Cell. 150(6):1135-46. Pubmed: 22980977 DOI:S0092-8674(12)01012-4 Lian CG, Xu Y, Ceol C, Wu F, Larson A, Dresser K, Xu W, Tan L, Hu Y, Zhan Q, Lee CW, Hu D, Lian BQ, Kleffel S, Yang Y, Neiswender J, Khorasani AJ, Fang R, Lezcano C, Duncan LM, Scolyer RA, Thompson JF, Kakavand H, Houvras Y, Zon LI, Mihm MC, Kaiser UB, Schatton T, Woda BA, Murphy GF, Shi YG. 2012. Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma. Cell. 150(6):1135-46. Pubmed: 22980977 DOI:S0092-8674(12)01012-4 DNA methylation at the 5 position of cytosine (5-mC) is a key epigenetic mark that is critical for various biological and pathological processes. 5-mC can be converted to 5-hydroxymethylcytosine (5-hmC) by the ten-eleven translocation (TET) family of DNA hydroxylases. Here, we report that "loss of 5-hmC" is an epigenetic hallmark of melanoma, with diagnostic and prognostic implications. Genome-wide mapping of 5-hmC reveals loss of the 5-hmC landscape in the melanoma epigenome. We show that downregulation of isocitrate dehydrogenase 2 (IDH2) and TET family enzymes is likely one of the mechanisms underlying 5-hmC loss in melanoma. Rebuilding the 5-hmC landscape in melanoma cells by reintroducing active TET2 or IDH2 suppresses melanoma growth and increases tumor-free survival in animal models. Thus, our study reveals a critical function of 5-hmC in melanoma development and directly links the IDH and TET activity-dependent epigenetic pathway to 5-hmC-mediated suppression of melanoma progression, suggesting a new strategy for epigenetic cancer therapy.Copyright © 2012 Elsevier Inc. All rights reserved. -
Shestopalov IA, Zon LI. 2012. Stem cells: The right neighbour. Nature. 481(7382):453-5. Pubmed: 22281591 DOI:10.1038/481453a Shestopalov IA, Zon LI. 2012. Stem cells: The right neighbour. Nature. 481(7382):453-5. Pubmed: 22281591 DOI:10.1038/481453a -
Bowman TV, Trompouki E, Zon LI. 2012. Linking hematopoietic regeneration to developmental signaling pathways: a story of BMP and Wnt. Cell cycle (Georgetown, Tex.). 11(3):424-5. Pubmed: 22262185 DOI:10.4161/cc.11.3.19161 Bowman TV, Trompouki E, Zon LI. 2012. Linking hematopoietic regeneration to developmental signaling pathways: a story of BMP and Wnt. Cell cycle (Georgetown, Tex.). 11(3):424-5. Pubmed: 22262185 DOI:10.4161/cc.11.3.19161 -
Yu M, Mazor T, Huang H, Huang HT, Kathrein KL, Woo AJ, Chouinard CR, Labadorf A, Akie TE, Moran TB, Xie H, Zacharek S, Taniuchi I, Roeder RG, Kim CF, Zon LI, Fraenkel E, Cantor AB. 2012. Direct recruitment of polycomb repressive complex 1 to chromatin by core binding transcription factors. Molecular cell. 45(3):330-43. Pubmed: 22325351 DOI:10.1016/j.molcel.2011.11.032 Yu M, Mazor T, Huang H, Huang HT, Kathrein KL, Woo AJ, Chouinard CR, Labadorf A, Akie TE, Moran TB, Xie H, Zacharek S, Taniuchi I, Roeder RG, Kim CF, Zon LI, Fraenkel E, Cantor AB. 2012. Direct recruitment of polycomb repressive complex 1 to chromatin by core binding transcription factors. Molecular cell. 45(3):330-43. Pubmed: 22325351 DOI:10.1016/j.molcel.2011.11.032 Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFβ transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFβ and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFβ deficiencies generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via PRC2-independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.Copyright © 2012 Elsevier Inc. All rights reserved. -
Tamplin OJ, White RM, Jing L, Kaufman CK, Lacadie SA, Li P, Taylor AM, Zon LI. 2012. Small molecule screening in zebrafish: swimming in potential drug therapies. Wiley interdisciplinary reviews. Developmental biology. 1(3):459-68. Pubmed: 23801494 DOI:10.1002/wdev.37 Tamplin OJ, White RM, Jing L, Kaufman CK, Lacadie SA, Li P, Taylor AM, Zon LI. 2012. Small molecule screening in zebrafish: swimming in potential drug therapies. Wiley interdisciplinary reviews. Developmental biology. 1(3):459-68. Pubmed: 23801494 DOI:10.1002/wdev.37 Phenotype-driven chemical genetic screens in zebrafish have become a proven approach for both dissection of developmental mechanisms and discovery of potential therapeutics. A library of small molecules can be arrayed into multiwell plates containing zebrafish embryos. The embryo becomes a whole organism in vivo bioassay that can produce a phenotype upon treatment. Screens have been performed that are based simply on the morphology of the embryo. Other screens have scored complex phenotypes using whole mount in situ hybridization, fluorescent transgenic reporters, and even tracking of embryo movement. The availability of many well-characterized zebrafish mutants has also enabled the discovery of chemical suppressors of genetic phenotypes. Importantly, the application of chemical libraries that already contain FDA-approved drugs has allowed the rapid translation of hits from zebrafish chemical screens to clinical trials.Copyright © 2012 Wiley Periodicals, Inc. -
Xu C, Fan ZP, Müller P, Fogley R, DiBiase A, Trompouki E, Unternaehrer J, Xiong F, Torregroza I, Evans T, Megason SG, Daley GQ, Schier AF, Young RA, Zon LI. 2012. Nanog-like regulates endoderm formation through the Mxtx2-Nodal pathway. Developmental cell. 22(3):625-38. Pubmed: 22421047 DOI:10.1016/j.devcel.2012.01.003 Xu C, Fan ZP, Müller P, Fogley R, DiBiase A, Trompouki E, Unternaehrer J, Xiong F, Torregroza I, Evans T, Megason SG, Daley GQ, Schier AF, Young RA, Zon LI. 2012. Nanog-like regulates endoderm formation through the Mxtx2-Nodal pathway. Developmental cell. 22(3):625-38. Pubmed: 22421047 DOI:10.1016/j.devcel.2012.01.003 In mammalian embryonic stem cells, the acquisition of pluripotency is dependent on Nanog, but the in vivo analysis of Nanog has been hampered by its requirement for early mouse development. In an effort to examine the role of Nanog in vivo, we identified a zebrafish Nanog ortholog and found that its knockdown impaired endoderm formation. Genome-wide transcription analysis revealed that nanog-like morphants fail to develop the extraembryonic yolk syncytial layer (YSL), which produces Nodal, required for endoderm induction. We examined the genes that were regulated by Nanog-like and identified the homeobox gene mxtx2, which is both necessary and sufficient for YSL induction. Chromatin immunoprecipitation assays and genetic studies indicated that Nanog-like directly activates mxtx2, which, in turn, specifies the YSL lineage by directly activating YSL genes. Our study identifies a Nanog-like-Mxtx2-Nodal pathway and establishes a role for Nanog-like in regulating the formation of the extraembryonic tissue required for endoderm induction.Copyright © 2012 Elsevier Inc. All rights reserved. -
Jing L, Durand EM, Ezzio C, Pagliuca SM, Zon LI. 2012. In situ hybridization assay-based small molecule screening in zebrafish. Current protocols in chemical biology. 4(2). Pubmed: 23001521 DOI:110236 Jing L, Durand EM, Ezzio C, Pagliuca SM, Zon LI. 2012. In situ hybridization assay-based small molecule screening in zebrafish. Current protocols in chemical biology. 4(2). Pubmed: 23001521 DOI:110236 In vitro biochemical and cell-based small molecule screens have been widely used to identify compounds that target specific signaling pathways. But the identified compounds frequently fail at the animal testing stage, largely due to the in vivo absorption, metabolism and toxicity of chemicals. Zebrafish has recently emerged as a vertebrate whole organism model for small molecule screening. The in vivo bioactivity and specificity of compounds are examined from the very beginning of zebrafish screens. In addition, zebrafish is suitable for chemical screens at a large scale similar to cellular assays. This protocol describes an approach for in situ hybridization (ISH)-based chemical screening in zebrafish, which, in principle, can be used to screen any gene product. The described protocol has been used to identify small molecules affecting specific molecular pathways and biological processes. It can also be adapted to zebrafish screens with different readouts. -
Ganis JJ, Hsia N, Trompouki E, de Jong JL, DiBiase A, Lambert JS, Jia Z, Sabo PJ, Weaver M, Sandstrom R, Stamatoyannopoulos JA, Zhou Y, Zon LI. 2012. Zebrafish globin switching occurs in two developmental stages and is controlled by the LCR. Developmental biology. 366(2):185-94. Pubmed: 22537494 DOI:10.1016/j.ydbio.2012.03.021 Ganis JJ, Hsia N, Trompouki E, de Jong JL, DiBiase A, Lambert JS, Jia Z, Sabo PJ, Weaver M, Sandstrom R, Stamatoyannopoulos JA, Zhou Y, Zon LI. 2012. Zebrafish globin switching occurs in two developmental stages and is controlled by the LCR. Developmental biology. 366(2):185-94. Pubmed: 22537494 DOI:10.1016/j.ydbio.2012.03.021 Globin gene switching is a complex, highly regulated process allowing expression of distinct globin genes at specific developmental stages. Here, for the first time, we have characterized all of the zebrafish globins based on the completed genomic sequence. Two distinct chromosomal loci, termed major (chromosome 3) and minor (chromosome 12), harbor the globin genes containing α/β pairs in a 5'-3' to 3'-5' orientation. Both these loci share synteny with the mammalian α-globin locus. Zebrafish globin expression was assayed during development and demonstrated two globin switches, similar to human development. A conserved regulatory element, the locus control region (LCR), was revealed by analyzing DNase I hypersensitive sites, H3K4 trimethylation marks and GATA1 binding sites. Surprisingly, the position of these sites with relation to the globin genes is evolutionarily conserved, despite a lack of overall sequence conservation. Motifs within the zebrafish LCR include CACCC, GATA, and NFE2 sites, suggesting functional interactions with known transcription factors but not the same LCR architecture. Functional homology to the mammalian α-LCR MCS-R2 region was confirmed by robust and specific reporter expression in erythrocytes of transgenic zebrafish. Our studies provide a comprehensive characterization of the zebrafish globin loci and clarify the regulation of globin switching.Copyright © 2012 Elsevier Inc. All rights reserved. -
Taylor AM, Humphries JM, White RM, Murphey RD, Burns CE, Zon LI. 2012. Hematopoietic defects in rps29 mutant zebrafish depend upon p53 activation. Experimental hematology. 40(3):228-237.e5. Pubmed: 22120640 Taylor AM, Humphries JM, White RM, Murphey RD, Burns CE, Zon LI. 2012. Hematopoietic defects in rps29 mutant zebrafish depend upon p53 activation. Experimental hematology. 40(3):228-237.e5. Pubmed: 22120640 Disruption of ribosomal proteins is associated with hematopoietic phenotypes in cell culture and animal models. Mutations in ribosomal proteins are seen in patients with Diamond Blackfan anemia, a rare congenital disease characterized by red cell aplasia and distinctive craniofacial anomalies. A zebrafish screen uncovered decreased hematopoietic stem cells in embryos with mutations in ribosomal protein rps29. Here, we determined that rps29(-/-) embryos also have red blood cell defects and increased apoptosis in the head. As the p53 pathway has been shown to play a role in other ribosomal protein mutants, we studied the genetic relationship of rps29 and p53. Transcriptional profiling revealed that genes upregulated in the rps29 mutant are enriched for genes upregulated by p53 after irradiation. p53 mutation near completely rescues the rps29 morphological and hematopoietic phenotypes, demonstrating that p53 mediates the effects of rps29 knockdown. We also identified neuronal gene orthopedia protein a (otpa) as one whose expression correlates with rps29 expression, suggesting that levels of expression of some genes are dependent on rps29 levels. Together, our studies demonstrate a role of p53 in mediating the cellular defects associated with rps29 and establish a role for rps29 and p53 in hematopoietic stem cells and red blood cell development.Copyright © 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved. -
Zhang L, Alt C, Li P, White RM, Zon LI, Wei X, Lin CP. 2012. An optical platform for cell tracking in adult zebrafish. Cytometry. Part A : the journal of the International Society for Analytical Cytology. 81(2):176-82. Pubmed: 22162445 DOI:10.1002/cyto.a.21167 Zhang L, Alt C, Li P, White RM, Zon LI, Wei X, Lin CP. 2012. An optical platform for cell tracking in adult zebrafish. Cytometry. Part A : the journal of the International Society for Analytical Cytology. 81(2):176-82. Pubmed: 22162445 DOI:10.1002/cyto.a.21167 Adult zebrafish are being increasingly used as a model in cancer and stem cell research. Here we describe an integrated optical system that combines a laser scanning confocal microscope (LSCM) and an in vivo flow cytometer (IVFC) for simultaneous visualization and cell quantification. The system is set up specifically for non-invasive tracking of both stationary and circulating cells in adult zebrafish (casper) that have been engineered to be optically transparent. Confocal imaging in this instrument serves the dual purpose of visualizing fish tissue microstructure and an imaging-based guide to locate a suitable vessel for quantitative analysis of circulating cells by IVFC. We demonstrate initial testing of this novel instrument by imaging the transparent adult zebrafish casper vasculature and tracking circulating cells in CD41-GFP/Gata1-DsRed transgenic fish whose thrombocytes/erythrocytes express the green and red fluorescent proteins. In vivo measurements allow cells to be tracked under physiological conditions in the same fish over time, without drawing blood samples or sacrificing animals. We also discuss the potential applications of this instrument in biomedical research.Copyright © 2011 International Society for Advancement of Cytometry. -
Brown KH, Dobrinski KP, Lee AS, Gokcumen O, Mills RE, Shi X, Chong WW, Chen JY, Yoo P, David S, Peterson SM, Raj T, Choy KW, Stranger BE, Williamson RE, Zon LI, Freeman JL, Lee C. 2012. Extensive genetic diversity and substructuring among zebrafish strains revealed through copy number variant analysis. Proceedings of the National Academy of Sciences of the United States of America. 109(2):529-34. Pubmed: 22203992 DOI:10.1073/pnas.1112163109 Brown KH, Dobrinski KP, Lee AS, Gokcumen O, Mills RE, Shi X, Chong WW, Chen JY, Yoo P, David S, Peterson SM, Raj T, Choy KW, Stranger BE, Williamson RE, Zon LI, Freeman JL, Lee C. 2012. Extensive genetic diversity and substructuring among zebrafish strains revealed through copy number variant analysis. Proceedings of the National Academy of Sciences of the United States of America. 109(2):529-34. Pubmed: 22203992 DOI:10.1073/pnas.1112163109 Copy number variants (CNVs) represent a substantial source of genomic variation in vertebrates and have been associated with numerous human diseases. Despite this, the extent of CNVs in the zebrafish, an important model for human disease, remains unknown. Using 80 zebrafish genomes, representing three commonly used laboratory strains and one native population, we constructed a genome-wide, high-resolution CNV map for the zebrafish comprising 6,080 CNV elements and encompassing 14.6% of the zebrafish reference genome. This amount of copy number variation is four times that previously observed in other vertebrates, including humans. Moreover, 69% of the CNV elements exhibited strain specificity, with the highest number observed for Tubingen. This variation likely arose, in part, from Tubingen's large founding size and composite population origin. Additional population genetic studies also provided important insight into the origins and substructure of these commonly used laboratory strains. This extensive variation among and within zebrafish strains may have functional effects that impact phenotype and, if not properly addressed, such extensive levels of germ-line variation and population substructure in this commonly used model organism can potentially confound studies intended for translation to human diseases. -
Zon LI. 2012. A great match. Cell stem cell. 10(6):638-639. Pubmed: 22704496 DOI:S1934-5909(12)00234-2 Zon LI. 2012. A great match. Cell stem cell. 10(6):638-639. Pubmed: 22704496 DOI:S1934-5909(12)00234-2 2011
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Mosimann C, Zon LI. 2011. Advanced zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments. Methods in cell biology. 104:173-94. Pubmed: 21924163 DOI:10.1016/B978-0-12-374814-0.00010-0 Mosimann C, Zon LI. 2011. Advanced zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments. Methods in cell biology. 104:173-94. Pubmed: 21924163 DOI:10.1016/B978-0-12-374814-0.00010-0 Spatio-temporal transgene regulation by transgenic DNA recombinases is a central tool for genetic research in multicellular organisms, with excellent applications for misexpression and lineage tracing experiments. Cre recombinase-controlled lox site recombination is a cornerstone of contemporary mouse genetics, and Cre/lox techniques therefore attract increasing interest in the zebrafish field. Tol2-mediated zebrafish transgenesis now provides a stable platform for lox cassette transgenes, while the ease of drug treatments in zebrafish makes the model an ideal candidate for Tamoxifen/4-hydroxytamoxifen-inducible CreER(T2) experiments. In this chapter, we will first introduce the basics of Cre/lox methodology, CreER(T2) regulation by Tamoxifen/4-hydroxytamoxifen, as well as the benefits of Tol2 transgenesis for Cre/lox experiments. We will then in detail outline practical experimental steps for Tol2 transgenesis toward the creation of single-insertion transgenes. Lastly, we will introduce protocols for 4-hydroxytamoxifen-mediated CreER(T2) induction to perform spatio-temporal lox transgene regulation experiments in zebrafish embryos.Copyright © 2011 Elsevier Inc. All rights reserved. -
Mosimann C, Kaufman CK, Li P, Pugach EK, Tamplin OJ, Zon LI. 2011. Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish. Development (Cambridge, England). 138(1):169-77. Pubmed: 21138979 DOI:10.1242/dev.059345 Mosimann C, Kaufman CK, Li P, Pugach EK, Tamplin OJ, Zon LI. 2011. Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish. Development (Cambridge, England). 138(1):169-77. Pubmed: 21138979 DOI:10.1242/dev.059345 Molecular genetics approaches in zebrafish research are hampered by the lack of a ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the zebrafish ubiquitin (ubi) promoter, which drives constitutive transgene expression during all developmental stages and analyzed adult organs. Notably, ubi expresses in all blood cell lineages, and we demonstrate the application of ubi-driven fluorophore transgenics in hematopoietic transplantation experiments to assess true multilineage potential of engrafted cells. We further generated transgenic zebrafish that express ubiquitous 4-hydroxytamoxifen-controlled Cre recombinase activity from a ubi:cre(ERt2) transgene, as well as ubi:loxP-EGFP-loxP-mCherry (ubi:Switch) transgenics and show their use as a constitutive fluorescent lineage tracing reagent. The ubi promoter and the transgenic lines presented here thus provide a broad resource and important advancement for transgenic applications in zebrafish. -
Li P, White RM, Zon LI. 2011. Transplantation in zebrafish. Methods in cell biology. 105:403-17. Pubmed: 21951540 DOI:10.1016/B978-0-12-381320-6.00017-5 Li P, White RM, Zon LI. 2011. Transplantation in zebrafish. Methods in cell biology. 105:403-17. Pubmed: 21951540 DOI:10.1016/B978-0-12-381320-6.00017-5 Tissue or cell transplantation has been an extremely valuable technique for studying developmental potential of certain cell population, dissecting cell-environment interaction relationship, identifying stem cells, and many other applications. One key technical requirement for performing transplantation assay is the capability of distinguishing the transplanted donor cells from the endogenous host cells, and tracing the donor cells over time. Zebrafish has emerged as an excellent model organism for performing transplantation assay, thanks to the transparency of embryos during development and even certain adults. Using transgenic techniques and fast-evolving imaging technology, fluorescence-labeled donor cells can be easily identified and studied in vivo. In this chapter, we will first discuss the rationale of different types of zebrafish transplantation in both embryos and adults, and then focus on detailed methods of three types of transplantation: blastula/gastrula transplantation for mosaic analysis, stem cell transplantation, and tumor transplantation.Copyright © 2011 Elsevier Inc. All rights reserved. -
Tan JL, Zon LI. 2011. Chemical screening in zebrafish for novel biological and therapeutic discovery. Methods in cell biology. 105:493-516. Pubmed: 21951544 DOI:10.1016/B978-0-12-381320-6.00021-7 Tan JL, Zon LI. 2011. Chemical screening in zebrafish for novel biological and therapeutic discovery. Methods in cell biology. 105:493-516. Pubmed: 21951544 DOI:10.1016/B978-0-12-381320-6.00021-7 Zebrafish chemical screening allows for an in vivo assessment of small molecule modulation of biological processes. Compound toxicities, chemical alterations by metabolism, pharmacokinetic and pharmacodynamic properties, and modulation of cell niches can be studied with this method. Furthermore, zebrafish screening is straightforward and cost-effective. Zebrafish provide an invaluable platform for novel therapeutic discovery through chemical screening.Copyright © 2011 Elsevier Inc. All rights reserved. -
Gupta V, Kawahara G, Gundry SR, Chen AT, Lencer WI, Zhou Y, Zon LI, Kunkel LM, Beggs AH. 2011. The zebrafish dag1 mutant: a novel genetic model for dystroglycanopathies. Human molecular genetics. 20(9):1712-25. Pubmed: 21296866 DOI:10.1093/hmg/ddr047 Gupta V, Kawahara G, Gundry SR, Chen AT, Lencer WI, Zhou Y, Zon LI, Kunkel LM, Beggs AH. 2011. The zebrafish dag1 mutant: a novel genetic model for dystroglycanopathies. Human molecular genetics. 20(9):1712-25. Pubmed: 21296866 DOI:10.1093/hmg/ddr047 In a forward genetic approach to identify novel genes for congenital muscle diseases, a zebrafish mutant, designated patchytail, was identified that exhibits degenerating muscle fibers with impaired motility behavior. Genetic mapping identified a genomic locus containing the zebrafish ortholog of the dystroglycan gene (DAG1). Patchytail fish contain a point mutation (c.1700T>A) in dag1, resulting in a missense change p.V567D. This change is associated with reduced transcripts and a complete absence of protein. The absence of α-dystroglycan and β-dystroglycan caused destabilization of dystroglycan complex, resulting in membrane damages. Membrane damage was localized on the extracellular matrix at myosepta as well as basement membrane between adjacent myofibers. These studies also identified structural abnormalities in triads at 3 days post fertilization (dpf) of dystroglycan-deficient muscles, significantly preceding sarcolemmal damage that becomes evident at 7 dpf. Immunofluorescence studies identified a subpopulation of dystroglycan that is expressed at t-tubules in normal skeletal muscles. In dag1-mutated fish, smaller and irregular-shaped t-tubule vesicles, as well as highly disorganized terminal cisternae of sarcoplasmic reticulum, were common. In addition to skeletal muscle defects, dag1-mutated fish have brain abnormalities and ocular defects in posterior as well as anterior chambers. These phenotypes of dystroglycan-deficient fish are highly reminiscent of the phenotypes observed in the human conditions muscle-eye-brain disease and Walker-Warburg syndrome. This animal model will provide unique opportunities in the understanding of biological functions of dystroglycan in a wide range of dystroglycanopathies, as disruption of this gene in higher vertebrates results in early embryonic lethality. -
Detrich HW, Westerfield M, Zon LI. 2011. The zebrafish. Preface. Methods in cell biology. 105:xxi-xxii. Pubmed: 22043535 Detrich HW, Westerfield M, Zon LI. 2011. The zebrafish. Preface. Methods in cell biology. 105:xxi-xxii. Pubmed: 22043535 -
Li P, Zon LI. 2011. Stem cell migration: a zebrafish model. Methods in molecular biology (Clifton, N.J.). 750:157-68. Pubmed: 21618090 DOI:10.1007/978-1-61779-145-1_11 Li P, Zon LI. 2011. Stem cell migration: a zebrafish model. Methods in molecular biology (Clifton, N.J.). 750:157-68. Pubmed: 21618090 DOI:10.1007/978-1-61779-145-1_11 Compared with other vertebrate animal models, zebrafish (Danio rerio) has its superior advantages for studying stem cell migration. Zebrafish have similar tissues and organs as mammals, where tissue-specific stem cells reside in. Zebrafish eggs are externally fertilized and remain transparent until most of the organs are fully developed. This allows imaging stem cells in vivo very easily. Recently, a zebrafish double pigmentation mutant, casper, became a new popular imaging model in the zebrafish field due to its completely transparent bodies in adulthood. It has been used as an excellent model to study adult hematopoietic stem cell (HSC) in the transplantation setting. The unparalleled imaging power of zebrafish provides great opportunities of tracing stem cells in vivo in the developmental and regenerative context. In this chapter, we use HSC as an example and combine the powerful imaging techniques in zebrafish, to provide protocols for in vivo imaging fluorescence-labeled stem cell migration, stem cell fate tracing in zebrafish embryos, HSC transplantation, and in vivo imaging in both zebrafish embryos and adults. These techniques can also be applied to other types of stem cells in zebrafish embryos and adults. -
Stachura DL, Svoboda O, Lau RP, Balla KM, Zon LI, Bartunek P, Traver D. 2011. Clonal analysis of hematopoietic progenitor cells in the zebrafish. Blood. 118(5):1274-82. Pubmed: 21415264 DOI:10.1182/blood-2011-01-331199 Stachura DL, Svoboda O, Lau RP, Balla KM, Zon LI, Bartunek P, Traver D. 2011. Clonal analysis of hematopoietic progenitor cells in the zebrafish. Blood. 118(5):1274-82. Pubmed: 21415264 DOI:10.1182/blood-2011-01-331199 Identification of hematopoietic progenitor cells in the zebrafish (Danio rerio) has been hindered by a lack of functional assays to gauge proliferative potential and differentiation capacity. To investigate the nature of myeloerythroid progenitor cells, we developed clonal methylcellulose assays by using recombinant zebrafish erythropoietin and granulocyte colony-stimulating factor. From adult whole kidney marrow, erythropoietin was required to support erythroid colony formation, and granulocyte colony-stimulating factor was required to support the formation of colonies containing neutrophils, monocytes, and macrophages. Myeloid and erythroid colonies showed distinct morphologies and were easily visualized and scored by their expression of lineage-specific fluorescent transgenes. Analysis of the gene-expression profiles after isolation of colonies marked by gata1:DsRed or mpx:eGFP transgenes confirmed our morphological erythroid and myeloid lineage designations, respectively. The majority of progenitor activity was contained within the precursor light scatter fraction, and more immature precursors were present within the lymphoid fraction. Finally, we performed kinetic analyses of progenitor activity after sublethal irradiation and demonstrated that recovery to preirradiation levels occurred by 14 days after irradiation. Together, these experiments provide the first report of clonal hematopoietic progenitor assays in the zebrafish and establish the number, characteristics, and kinetics of myeloerythroid progenitors during both steady-state and stress hematopoiesis. -
de Jong JL, Burns CE, Chen AT, Pugach E, Mayhall EA, Smith AC, Feldman HA, Zhou Y, Zon LI. 2011. Characterization of immune-matched hematopoietic transplantation in zebrafish. Blood. 117(16):4234-42. Pubmed: 21346254 DOI:10.1182/blood-2010-09-307488 de Jong JL, Burns CE, Chen AT, Pugach E, Mayhall EA, Smith AC, Feldman HA, Zhou Y, Zon LI. 2011. Characterization of immune-matched hematopoietic transplantation in zebrafish. Blood. 117(16):4234-42. Pubmed: 21346254 DOI:10.1182/blood-2010-09-307488 Evaluating hematopoietic stem cell (HSC) function in vivo requires a long-term transplantation assay. Although zebrafish are a powerful model for discovering the genetics of hematopoiesis, hematopoietic transplantation approaches have been underdeveloped. Here we established a long-term reconstitution assay in adult zebrafish. Primary and secondary recipients showed multilineage engraftment at 3 months after transplantation. Limiting dilution data suggest that at least 1 in 65 000 zebrafish marrow cells contain repopulating activity, consistent with mammalian HSC frequencies. We defined zebrafish haplotypes at the proposed major histocompatibility complex locus on chromosome 19 and tested functional significance through hematopoietic transplantation. Matching donors and recipients dramatically increased engraftment and percentage donor chimerism compared with unmatched fish. These data constitute the first functional test of zebrafish histocompatibility genes, enabling the development of matched hematopoietic transplantations. This lays the foundation for competitive transplantation experiments with mutant zebrafish HSCs and chemicals to test for effects on engraftment, thereby providing a model for human hematopoietic diseases and treatments not previously available. -
Trompouki E, Bowman TV, Dibiase A, Zhou Y, Zon LI. 2011. Chromatin immunoprecipitation in adult zebrafish red cells. Methods in cell biology. 104:341-52. Pubmed: 21924172 DOI:10.1016/B978-0-12-374814-0.00019-7 Trompouki E, Bowman TV, Dibiase A, Zhou Y, Zon LI. 2011. Chromatin immunoprecipitation in adult zebrafish red cells. Methods in cell biology. 104:341-52. Pubmed: 21924172 DOI:10.1016/B978-0-12-374814-0.00019-7 Zebrafish has been used for many years as a model to study development and disease. The ability of zebrafish to produce thousand of embryos in a synchronous manner has made zebrafish an invaluable tool for genetic and chemical screens. Since its emergence as an important model organism the molecular tools for studying zebrafish have been limited. In this chapter, we describe a simple method to identify DNA binding sites and chromatin architecture in erythrocytes from adult zebrafish using chromatin immunoprecipitation coupled with next generation sequencing. This technique has been used extensively and successfully in other systems and it will be a useful tool for studying epigenetics in zebrafish.Copyright © 2011 Elsevier Inc. All rights reserved. -
Zhou Y, Zon LI. 2011. The zon laboratory guide to positional cloning in zebrafish. Methods in cell biology. 104:287-309. Pubmed: 21924169 DOI:10.1016/B978-0-12-374814-0.00016-1 Zhou Y, Zon LI. 2011. The zon laboratory guide to positional cloning in zebrafish. Methods in cell biology. 104:287-309. Pubmed: 21924169 DOI:10.1016/B978-0-12-374814-0.00016-1 Zebrafish genome sequencing project has improved efficiency of positional cloning in zebrafish and traditional chromosome walking by isolating large insert genomic libraries has become a past. However, the genetic principles underlying the positional cloning still form the foundation for current chromosome walking using the genome sequence assemblies and related genomic sequence and clone information. This guide intends to summarize our accumulated experience in positional cloning using the current genomic resources and tools, and provide a practical guide to positional and/or candidate cloning of mutants of interest.Copyright © 2011 Elsevier Inc. All rights reserved. -
Bai X, Yang Z, Jiang H, Lin S, Zon LI. 2011. Genetic suppressor screens in haploids. Methods in cell biology. 104:129-36. Pubmed: 21924160 DOI:10.1016/B978-0-12-374814-0.00007-0 Bai X, Yang Z, Jiang H, Lin S, Zon LI. 2011. Genetic suppressor screens in haploids. Methods in cell biology. 104:129-36. Pubmed: 21924160 DOI:10.1016/B978-0-12-374814-0.00007-0 As a vertebrate genetic model, the zebrafish has been well recognized for its strength in studying a variety of biological processes and human diseases. Traditional forward genetic screens in zebrafish have generated a large pool of mutants with interesting phenotypes resembling human diseases but the underlying mechanisms are not well understood. A powerful approach to elucidate the mechanisms of these mutants is the modifier screen, which identifies 2(nd)-site mutations that specifically enhance or block the phenotype of a given mutant. Here we described the first genetic suppressor screen in zebrafish, which identifies a novel transcriptional mechanism regulating erythropoiesis. In combination with the haploid genetics in zebrafish, we have shown the feasibility and strength of a modifier screen in zebrafish. This strategy will greatly broaden the utility of the zebrafish as a model for making original discoveries and establishing novel paradigms for understanding vertebrate biology.Copyright © 2011 Elsevier Inc. All rights reserved. -
Ceol CJ, Houvras Y, Jane-Valbuena J, Bilodeau S, Orlando DA, Battisti V, Fritsch L, Lin WM, Hollmann TJ, Ferré F, Bourque C, Burke CJ, Turner L, Uong A, Johnson LA, Beroukhim R, Mermel CH, Loda M, Ait-Si-Ali S, Garraway LA, Young RA, Zon LI. 2011. The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset. Nature. 471(7339):513-7. Pubmed: 21430779 DOI:10.1038/nature09806 Ceol CJ, Houvras Y, Jane-Valbuena J, Bilodeau S, Orlando DA, Battisti V, Fritsch L, Lin WM, Hollmann TJ, Ferré F, Bourque C, Burke CJ, Turner L, Uong A, Johnson LA, Beroukhim R, Mermel CH, Loda M, Ait-Si-Ali S, Garraway LA, Young RA, Zon LI. 2011. The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset. Nature. 471(7339):513-7. Pubmed: 21430779 DOI:10.1038/nature09806 The most common mutation in human melanoma, BRAF(V600E), activates the serine/threonine kinase BRAF and causes excessive activity in the mitogen-activated protein kinase pathway. BRAF(V600E) mutations are also present in benign melanocytic naevi, highlighting the importance of additional genetic alterations in the genesis of malignant tumours. Such changes include recurrent copy number variations that result in the amplification of oncogenes. For certain amplifications, the large number of genes in the interval has precluded an understanding of the cooperating oncogenic events. Here we have used a zebrafish melanoma model to test genes in a recurrently amplified region of chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma. SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to accelerate melanoma formation significantly in zebrafish. Chromatin immunoprecipitation coupled with massively parallel DNA sequencing and gene expression analyses uncovered genes, including HOX genes, that are transcriptionally dysregulated in response to increased levels of SETDB1. Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis. -
Bolli N, Payne EM, Rhodes J, Gjini E, Johnston AB, Guo F, Lee JS, Stewart RA, Kanki JP, Chen AT, Zhou Y, Zon LI, Look AT. 2011. cpsf1 is required for definitive HSC survival in zebrafish. Blood. 117(15):3996-4007. Pubmed: 21330472 DOI:10.1182/blood-2010-08-304030 Bolli N, Payne EM, Rhodes J, Gjini E, Johnston AB, Guo F, Lee JS, Stewart RA, Kanki JP, Chen AT, Zhou Y, Zon LI, Look AT. 2011. cpsf1 is required for definitive HSC survival in zebrafish. Blood. 117(15):3996-4007. Pubmed: 21330472 DOI:10.1182/blood-2010-08-304030 A comprehensive understanding of the genes and pathways regulating hematopoiesis is needed to identify genes causally related to bone marrow failure syndromes, myelodysplastic syndromes, and hematopoietic neoplasms. To identify novel genes involved in hematopoiesis, we performed an ethyl-nitrosourea mutagenesis screen in zebrafish (Danio rerio) to search for mutants with defective definitive hematopoiesis. We report the recovery and analysis of the grechetto mutant, which harbors an inactivating mutation in cleavage and polyadenylation specificity factor 1 (cpsf1), a gene ubiquitously expressed and required for 3' untranslated region processing of a subset of pre-mRNAs. grechetto mutants undergo normal primitive hematopoiesis and specify appropriate numbers of definitive HSCs at 36 hours postfertilization. However, when HSCs migrate to the caudal hematopoietic tissue at 3 days postfertilization, their numbers start decreasing as a result of apoptotic cell death. Consistent with Cpsf1 function, c-myb:EGFP(+) cells in grechetto mutants also show defective polyadenylation of snrnp70, a gene required for HSC development. By 5 days postfertilization, definitive hematopoiesis is compromised and severely decreased blood cell numbers are observed across the myeloid, erythroid, and lymphoid cell lineages. These studies show that cpsf1 is essential for HSC survival and differentiation in caudal hematopoietic tissue. -
Alexander MS, Kawahara G, Kho AT, Howell MH, Pusack TJ, Myers JA, Montanaro F, Zon LI, Guyon JR, Kunkel LM. 2011. Isolation and transcriptome analysis of adult zebrafish cells enriched for skeletal muscle progenitors. Muscle & nerve. 43(5):741-50. Pubmed: 21337346 DOI:10.1002/mus.21972 Alexander MS, Kawahara G, Kho AT, Howell MH, Pusack TJ, Myers JA, Montanaro F, Zon LI, Guyon JR, Kunkel LM. 2011. Isolation and transcriptome analysis of adult zebrafish cells enriched for skeletal muscle progenitors. Muscle & nerve. 43(5):741-50. Pubmed: 21337346 DOI:10.1002/mus.21972 ArrayCopyright © 2011 Wiley Periodicals, Inc. -
Lacadie SA, Zon LI. 2011. The ERGonomics of hematopoietic stem cell self-renewal. Genes & development. 25(4):289-93. Pubmed: 21325129 DOI:10.1101/gad.2031511 Lacadie SA, Zon LI. 2011. The ERGonomics of hematopoietic stem cell self-renewal. Genes & development. 25(4):289-93. Pubmed: 21325129 DOI:10.1101/gad.2031511 Stem cells make more of themselves by self-renewing cell divisions. In the February 1, 2011, issue of Genes & Development, Taoudi and colleagues (pp. 251-262) show an essential role for the ETS transcription factor ERG in the self-renewal of embryonic hematopoietic stem cells. A model is presented in which the redundant functions of GATA2 and RUNX1 in self-renewal are under direct control of ERG. -
White RM, Cech J, Ratanasirintrawoot S, Lin CY, Rahl PB, Burke CJ, Langdon E, Tomlinson ML, Mosher J, Kaufman C, Chen F, Long HK, Kramer M, Datta S, Neuberg D, Granter S, Young RA, Morrison S, Wheeler GN, Zon LI. 2011. DHODH modulates transcriptional elongation in the neural crest and melanoma. Nature. 471(7339):518-22. Pubmed: 21430780 DOI:10.1038/nature09882 White RM, Cech J, Ratanasirintrawoot S, Lin CY, Rahl PB, Burke CJ, Langdon E, Tomlinson ML, Mosher J, Kaufman C, Chen F, Long HK, Kramer M, Datta S, Neuberg D, Granter S, Young RA, Morrison S, Wheeler GN, Zon LI. 2011. DHODH modulates transcriptional elongation in the neural crest and melanoma. Nature. 471(7339):518-22. Pubmed: 21430780 DOI:10.1038/nature09882 Melanoma is a tumour of transformed melanocytes, which are originally derived from the embryonic neural crest. It is unknown to what extent the programs that regulate neural crest development interact with mutations in the BRAF oncogene, which is the most commonly mutated gene in human melanoma. We have used zebrafish embryos to identify the initiating transcriptional events that occur on activation of human BRAF(V600E) (which encodes an amino acid substitution mutant of BRAF) in the neural crest lineage. Zebrafish embryos that are transgenic for mitfa:BRAF(V600E) and lack p53 (also known as tp53) have a gene signature that is enriched for markers of multipotent neural crest cells, and neural crest progenitors from these embryos fail to terminally differentiate. To determine whether these early transcriptional events are important for melanoma pathogenesis, we performed a chemical genetic screen to identify small-molecule suppressors of the neural crest lineage, which were then tested for their effects on melanoma. One class of compound, inhibitors of dihydroorotate dehydrogenase (DHODH), for example leflunomide, led to an almost complete abrogation of neural crest development in zebrafish and to a reduction in the self-renewal of mammalian neural crest stem cells. Leflunomide exerts these effects by inhibiting the transcriptional elongation of genes that are required for neural crest development and melanoma growth. When used alone or in combination with a specific inhibitor of the BRAF(V600E) oncogene, DHODH inhibition led to a marked decrease in melanoma growth both in vitro and in mouse xenograft studies. Taken together, these studies highlight developmental pathways in neural crest cells that have a direct bearing on melanoma formation. -
Taylor AM, Zon LI. 2011. Modeling Diamond Blackfan anemia in the zebrafish. Seminars in hematology. 48(2):81-8. Pubmed: 21435504 DOI:10.1053/j.seminhematol.2011.02.002 Taylor AM, Zon LI. 2011. Modeling Diamond Blackfan anemia in the zebrafish. Seminars in hematology. 48(2):81-8. Pubmed: 21435504 DOI:10.1053/j.seminhematol.2011.02.002 Diamond Blackfan anemia (DBA) is a rare congenital anemia, with more than 50% of patients having mutations in a ribosomal protein. Evidence suggests that both translation and p53 activation play roles in mediating the hematopoietic phenotype. The reason for erythroid specificity of DBA is unclear. Several zebrafish models of DBA have been generated, and these models have already provided key information about disease pathogenesis. The zebrafish model is particularly amenable for studying blood development, allows for advanced imaging techniques, can be manipulated genetically, and is useful for high-throughput screening. By applying zebrafish approaches to the existing DBA models, we will be able to better understand the role of the ribosomal protein mutation in DBA and develop better treatments for this disease.Copyright © 2011 Elsevier Inc. All rights reserved. -
Martin CS, Moriyama A, Zon LI. 2011. Hematopoietic stem cells, hematopoiesis and disease: lessons from the zebrafish model. Genome medicine. 3(12):83. Pubmed: 22206610 DOI:10.1186/gm299 Martin CS, Moriyama A, Zon LI. 2011. Hematopoietic stem cells, hematopoiesis and disease: lessons from the zebrafish model. Genome medicine. 3(12):83. Pubmed: 22206610 DOI:10.1186/gm299 The zebrafish model is rapidly gaining prominence in the study of development, hematopoiesis, and disease. The zebrafish provides distinct advantages over other vertebrate models during early embryonic development by producing transparent, externally fertilized embryos. Embryonic zebrafish are easily visualized and manipulated through microinjection, chemical treatment, and mutagenesis. These procedures have contributed to large-scale chemical, suppressor, and genetic screens to identify hematopoietic gene mutations. Genomic conservation and local synteny between the human and zebrafish genomes make genome-scale and epigenetic analysis of these mutations (by microarray, chromatin immunoprecipitation sequencing, and RNA sequencing procedures) powerful methods for translational research and medical discovery. In addition, large-scale screening techniques have resulted in the identification of several small molecules capable of rescuing hematopoietic defects and inhibiting disease. Here, we discuss the contributions of the zebrafish model to the understanding of hematopoiesis, hematopoietic stem cell development, and disease-related discovery. We also highlight the recent discovery of small molecules with clinical promise, such as dimethyl prostaglandin E2, 3F8, and thiazole-carboxamide 10A. -
Trompouki E, Bowman TV, Lawton LN, Fan ZP, Wu DC, DiBiase A, Martin CS, Cech JN, Sessa AK, Leblanc JL, Li P, Durand EM, Mosimann C, Heffner GC, Daley GQ, Paulson RF, Young RA, Zon LI. 2011. Lineage regulators direct BMP and Wnt pathways to cell-specific programs during differentiation and regeneration. Cell. 147(3):577-89. Pubmed: 22036566 DOI:10.1016/j.cell.2011.09.044 Trompouki E, Bowman TV, Lawton LN, Fan ZP, Wu DC, DiBiase A, Martin CS, Cech JN, Sessa AK, Leblanc JL, Li P, Durand EM, Mosimann C, Heffner GC, Daley GQ, Paulson RF, Young RA, Zon LI. 2011. Lineage regulators direct BMP and Wnt pathways to cell-specific programs during differentiation and regeneration. Cell. 147(3):577-89. Pubmed: 22036566 DOI:10.1016/j.cell.2011.09.044 BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, whereas expression of the erythroid regulator GATA1 directs SMAD1 loss on nonerythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity.Copyright © 2011 Elsevier Inc. All rights reserved. -
Bugeon L, Taylor HB, Progatzky F, Lin MI, Ellis CD, Welsh N, Smith E, Vargesson N, Gray C, Renshaw SA, Chico TJ, Zon LI, Lamb J, Dallman MJ. 2011. The NOTCH pathway contributes to cell fate decision in myelopoiesis. Haematologica. 96(12):1753-60. Pubmed: 21933862 DOI:10.3324/haematol.2011.044115 Bugeon L, Taylor HB, Progatzky F, Lin MI, Ellis CD, Welsh N, Smith E, Vargesson N, Gray C, Renshaw SA, Chico TJ, Zon LI, Lamb J, Dallman MJ. 2011. The NOTCH pathway contributes to cell fate decision in myelopoiesis. Haematologica. 96(12):1753-60. Pubmed: 21933862 DOI:10.3324/haematol.2011.044115 Array -
Goessling W, Allen RS, Guan X, Jin P, Uchida N, Dovey M, Harris JM, Metzger ME, Bonifacino AC, Stroncek D, Stegner J, Armant M, Schlaeger T, Tisdale JF, Zon LI, Donahue RE, North TE. 2011. Prostaglandin E2 enhances human cord blood stem cell xenotransplants and shows long-term safety in preclinical nonhuman primate transplant models. Cell stem cell. 8(4):445-58. Pubmed: 21474107 DOI:10.1016/j.stem.2011.02.003 Goessling W, Allen RS, Guan X, Jin P, Uchida N, Dovey M, Harris JM, Metzger ME, Bonifacino AC, Stroncek D, Stegner J, Armant M, Schlaeger T, Tisdale JF, Zon LI, Donahue RE, North TE. 2011. Prostaglandin E2 enhances human cord blood stem cell xenotransplants and shows long-term safety in preclinical nonhuman primate transplant models. Cell stem cell. 8(4):445-58. Pubmed: 21474107 DOI:10.1016/j.stem.2011.02.003 Hematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable; however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2 treatment by using human and nonhuman primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in vitro and enhanced engraftment of unfractionated and CD34(+) hCB after xenotransplantation. In nonhuman primate autologous transplantation, dmPGE2-treated CD34(+) MPBSCs showed stable multilineage engraftment over 1 year postinfusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and nonhuman primates and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial.Copyright © 2011 Elsevier Inc. All rights reserved. -
Glass TJ, Lund TC, Patrinostro X, Tolar J, Bowman TV, Zon LI, Blazar BR. 2011. Stromal cell-derived factor-1 and hematopoietic cell homing in an adult zebrafish model of hematopoietic cell transplantation. Blood. 118(3):766-74. Pubmed: 21622651 DOI:10.1182/blood-2011-01-328476 Glass TJ, Lund TC, Patrinostro X, Tolar J, Bowman TV, Zon LI, Blazar BR. 2011. Stromal cell-derived factor-1 and hematopoietic cell homing in an adult zebrafish model of hematopoietic cell transplantation. Blood. 118(3):766-74. Pubmed: 21622651 DOI:10.1182/blood-2011-01-328476 In mammals, stromal cell-derived factor-1 (SDF-1) promotes hematopoietic cell mobilization and migration. Although the zebrafish, Danio rerio, is an emerging model for studying hematopoietic cell transplantation (HCT), the role of SDF-1 in the adult zebrafish has yet to be determined. We sought to characterize sdf-1 expression and function in the adult zebrafish in the context of HCT. In situ hybridization of adult zebrafish organs shows sdf-1 expression in kidney tubules, gills, and skin. Radiation up-regulates sdf-1 expression in kidney to nearly 4-fold after 40 Gy. Assays indicate that zebrafish hematopoietic cells migrate toward sdf-1, with a migration ratio approaching 1.5 in vitro. A sdf-1a:DsRed2 transgenic zebrafish allows in vivo detection of sdf-1a expression in the adult zebrafish. Matings with transgenic reporters localized sdf-1a expression to the putative hematopoietic cell niche in proximal and distal renal tubules and collecting ducts. Importantly, transplant of hematopoietic cells into myelosuppressed recipients indicated migration of hematopoietic cells to sdf-1a-expressing sites in the kidney and skin. We conclude that sdf-1 expression and function in the adult zebrafish have important similarities to mammals, and this sdf-1 transgenic vertebrate will be useful in characterizing the hematopoietic cell niche and its interactions with hematopoietic cells. -
Adatto I, Lawrence C, Thompson M, Zon LI. 2011. A new system for the rapid collection of large numbers of developmentally staged zebrafish embryos. PloS one. 6(6):e21715. Pubmed: 21738776 DOI:10.1371/journal.pone.0021715 Adatto I, Lawrence C, Thompson M, Zon LI. 2011. A new system for the rapid collection of large numbers of developmentally staged zebrafish embryos. PloS one. 6(6):e21715. Pubmed: 21738776 DOI:10.1371/journal.pone.0021715 The zebrafish is an excellent genetic and developmental model system used to study biology and disease. While the zebrafish model is associated with high fecundity, its reproductive potential has not been completely realized by scientists. One major issue is that embryo collection is inefficient. Here, we have developed an innovative breeding vessel designed to stimulate the natural reproductive behavior of the fish. This novel apparatus allows us to collect large numbers of developmentally synchronized embryos in brief and defined windows of time, and with minimal investments in labor and space. To demonstrate the efficacy of this approach, we placed three separate groups (n = 180) of fish in the vessel and allowed them to spawn for 10-minute intervals. During these trials, which were repeated three times, the fish produced 8600±917, 8400±794, and 6800±1997 embryos, respectively. This level of embryo production is nearly twice what we were able to achieve when using conventional crossing equipment with some of the same fish, and it required significantly less room and time to set up and break down. This system overcomes major space and labor restrictions inherent in spawning equipment currently used in the field, and will greatly accelerate efforts to improve the scale and throughput of experiments. -
Jing L, Zon LI. 2011. Zebrafish as a model for normal and malignant hematopoiesis. Disease models & mechanisms. 4(4):433-8. Pubmed: 21708900 DOI:10.1242/dmm.006791 Jing L, Zon LI. 2011. Zebrafish as a model for normal and malignant hematopoiesis. Disease models & mechanisms. 4(4):433-8. Pubmed: 21708900 DOI:10.1242/dmm.006791 Zebrafish studies in the past two decades have made major contributions to our understanding of hematopoiesis and its associated disorders. The zebrafish has proven to be a powerful organism for studies in this area owing to its amenability to large-scale genetic and chemical screening. In addition, the externally fertilized and transparent embryos allow convenient genetic manipulation and in vivo imaging of normal and aberrant hematopoiesis. This review discusses available methods for studying hematopoiesis in zebrafish, summarizes key recent advances in this area, and highlights the current and potential contributions of zebrafish to the discovery and development of drugs to treat human blood disorders. 2010
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Wang Y, Krivtsov AV, Sinha AU, North TE, Goessling W, Feng Z, Zon LI, Armstrong SA. 2010. The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML. Science (New York, N.Y.). 327(5973):1650-3. Pubmed: 20339075 DOI:10.1126/science.1186624 Wang Y, Krivtsov AV, Sinha AU, North TE, Goessling W, Feng Z, Zon LI, Armstrong SA. 2010. The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML. Science (New York, N.Y.). 327(5973):1650-3. Pubmed: 20339075 DOI:10.1126/science.1186624 Leukemia stem cells (LSCs) are capable of limitless self-renewal and are responsible for the maintenance of leukemia. Because selective eradication of LSCs could offer substantial therapeutic benefit, there is interest in identifying the signaling pathways that control their development. We studied LSCs in mouse models of acute myelogenous leukemia (AML) induced either by coexpression of the Hoxa9 and Meis1a oncogenes or by the fusion oncoprotein MLL-AF9. We show that the Wnt/beta-catenin signaling pathway is required for self-renewal of LSCs that are derived from either hematopoietic stem cells (HSC) or more differentiated granulocyte-macrophage progenitors (GMP). Because the Wnt/beta-catenin pathway is normally active in HSCs but not in GMP, these results suggest that reactivation of beta-catenin signaling is required for the transformation of progenitor cells by certain oncogenes. beta-catenin is not absolutely required for self-renewal of adult HSCs; thus, targeting the Wnt/beta-catenin pathway may represent a new therapeutic opportunity in AML. -
Li P, Zon LI. 2010. Resolving the controversy about N-cadherin and hematopoietic stem cells. Cell stem cell. 6(3):199-202. Pubmed: 20207222 DOI:10.1016/j.stem.2010.02.007 Li P, Zon LI. 2010. Resolving the controversy about N-cadherin and hematopoietic stem cells. Cell stem cell. 6(3):199-202. Pubmed: 20207222 DOI:10.1016/j.stem.2010.02.007 Discrepancies in published results about the role of N-cadherin in hematopoietic stem cells have led to confusion in the field. Attempting to settle the disagreements and reach a consensus, we undertook a collective discussion approach. This process clarified a number of issues but left some questions still unresolved.2010 Elsevier Inc. All rights reserved. -
Zon LI, Peterson R. 2010. The new age of chemical screening in zebrafish. Zebrafish. 7(1):1. Pubmed: 20415642 DOI:10.1089/zeb.2010.9996 Zon LI, Peterson R. 2010. The new age of chemical screening in zebrafish. Zebrafish. 7(1):1. Pubmed: 20415642 DOI:10.1089/zeb.2010.9996 -
North TE, Babu IR, Vedder LM, Lord AM, Wishnok JS, Tannenbaum SR, Zon LI, Goessling W. 2010. PGE2-regulated wnt signaling and N-acetylcysteine are synergistically hepatoprotective in zebrafish acetaminophen injury. Proceedings of the National Academy of Sciences of the United States of America. 107(40):17315-20. Pubmed: 20855591 DOI:10.1073/pnas.1008209107 North TE, Babu IR, Vedder LM, Lord AM, Wishnok JS, Tannenbaum SR, Zon LI, Goessling W. 2010. PGE2-regulated wnt signaling and N-acetylcysteine are synergistically hepatoprotective in zebrafish acetaminophen injury. Proceedings of the National Academy of Sciences of the United States of America. 107(40):17315-20. Pubmed: 20855591 DOI:10.1073/pnas.1008209107 Acetaminophen (APAP) toxicity is the most common drug-induced cause of acute liver failure in the United States. The only available treatment, N-acetylcysteine (NAC), has a limited time window of efficacy, indicating a need for additional therapeutic options. Zebrafish have emerged as a powerful tool for drug discovery. Here, we developed a clinically relevant zebrafish model of APAP toxicity. APAP depleted glutathione stores, elevated aminotransferase levels, increased apoptosis, and caused dose-dependent hepatocyte necrosis. These outcomes were limited by NAC and conserved in zebrafish embryos. In a targeted embryonic chemical screen, prostaglandin E2 (PGE2) was identified as a potential therapeutic agent; in the adult, PGE2 similarly decreased APAP-associated toxicity. Significantly, when combined with NAC, PGE2 extended the time window for a successful intervention, synergistically reducing apoptosis, improving liver enzymes, and preventing death. Use of a wnt reporter zebrafish line and chemical genetic epistasis showed that the effects of PGE2 are mediated through the wnt signaling pathway. Zebrafish can be used as a clinically relevant toxicological model amenable to the identification of additional therapeutics and biomarkers of APAP injury; our data suggest combinatorial PGE2 and NAC treatment would be beneficial for patients with APAP-induced liver damage. -
Durand EM, Zon LI. 2010. Stem cells: The blood balance. Nature. 468(7324):644-5. Pubmed: 21124447 DOI:10.1038/468644a Durand EM, Zon LI. 2010. Stem cells: The blood balance. Nature. 468(7324):644-5. Pubmed: 21124447 DOI:10.1038/468644a -
Feng H, Stachura DL, White RM, Gutierrez A, Zhang L, Sanda T, Jette CA, Testa JR, Neuberg DS, Langenau DM, Kutok JL, Zon LI, Traver D, Fleming MD, Kanki JP, Look AT. 2010. T-lymphoblastic lymphoma cells express high levels of BCL2, S1P1, and ICAM1, leading to a blockade of tumor cell intravasation. Cancer cell. 18(4):353-66. Pubmed: 20951945 DOI:10.1016/j.ccr.2010.09.009 Feng H, Stachura DL, White RM, Gutierrez A, Zhang L, Sanda T, Jette CA, Testa JR, Neuberg DS, Langenau DM, Kutok JL, Zon LI, Traver D, Fleming MD, Kanki JP, Look AT. 2010. T-lymphoblastic lymphoma cells express high levels of BCL2, S1P1, and ICAM1, leading to a blockade of tumor cell intravasation. Cancer cell. 18(4):353-66. Pubmed: 20951945 DOI:10.1016/j.ccr.2010.09.009 The molecular events underlying the progression of T-lymphoblastic lymphoma (T-LBL) to acute T-lymphoblastic leukemia (T-ALL) remain elusive. In our zebrafish model, concomitant overexpression of bcl-2 with Myc accelerated T-LBL onset while inhibiting progression to T-ALL. The T-LBL cells failed to invade the vasculature and showed evidence of increased homotypic cell-cell adhesion and autophagy. Further analysis using clinical biopsy specimens revealed autophagy and increased levels of BCL2, S1P1, and ICAM1 in human T-LBL compared with T-ALL. Inhibition of S1P1 signaling in T-LBL cells led to decreased homotypic adhesion in vitro and increased tumor cell intravasation in vivo. Thus, blockade of intravasation and hematologic dissemination in T-LBL is due to elevated S1P1 signaling, increased expression of ICAM1, and augmented homotypic cell-cell adhesion.Copyright © 2010 Elsevier Inc. All rights reserved. -
Tamplin OJ, Zon LI. 2010. Fishing at the cellular level. Nature methods. 7(8):600-1. Pubmed: 20676080 DOI:10.1038/nmeth0810-600 Tamplin OJ, Zon LI. 2010. Fishing at the cellular level. Nature methods. 7(8):600-1. Pubmed: 20676080 DOI:10.1038/nmeth0810-600 A platform for automated screening of zebrafish larvae in high throughput should allow detection of phenotypic changes in single cells. -
Tamplin OJ, Zon LI. 2010. Blood flow: metalloproteases cut loose in primitive erythrocytes. Current biology : CB. 20(13):R561-2. Pubmed: 20619808 DOI:10.1016/j.cub.2010.05.014 Tamplin OJ, Zon LI. 2010. Blood flow: metalloproteases cut loose in primitive erythrocytes. Current biology : CB. 20(13):R561-2. Pubmed: 20619808 DOI:10.1016/j.cub.2010.05.014 Little is known about how blood begins to flow during development. A new study shows that release of primitive blood cells in the zebrafish embryo is synchronized and mediated by a metalloprotease.Copyright 2010 Elsevier Ltd. All rights reserved. -
Storer NY, Zon LI. 2010. Zebrafish models of p53 functions. Cold Spring Harbor perspectives in biology. 2(8):a001123. Pubmed: 20679337 DOI:10.1101/cshperspect.a001123 Storer NY, Zon LI. 2010. Zebrafish models of p53 functions. Cold Spring Harbor perspectives in biology. 2(8):a001123. Pubmed: 20679337 DOI:10.1101/cshperspect.a001123 Zebrafish models have significantly contributed to our understanding of vertebrate development and, more recently, human disease. The growing number of genetic tools available in zebrafish research has resulted in the identification of many genes involved in developmental and disease processes. In particular, studies in the zebrafish have clarified roles of the p53 tumor suppressor in the formation of specific tumor types, as well as roles of p53 family members during embryonic development. The zebrafish has also been instrumental in identifying novel mechanisms of p53 regulation and highlighting the importance of these mechanisms in vivo. This article will summarize how zebrafish models have been used to reveal numerous, important aspects of p53 function. -
de Jong JL, Davidson AJ, Wang Y, Palis J, Opara P, Pugach E, Daley GQ, Zon LI. 2010. Interaction of retinoic acid and scl controls primitive blood development. Blood. 116(2):201-9. Pubmed: 20410509 DOI:10.1182/blood-2009-10-249557 de Jong JL, Davidson AJ, Wang Y, Palis J, Opara P, Pugach E, Daley GQ, Zon LI. 2010. Interaction of retinoic acid and scl controls primitive blood development. Blood. 116(2):201-9. Pubmed: 20410509 DOI:10.1182/blood-2009-10-249557 Hematopoietic development during embryogenesis involves the interaction of extrinsic signaling pathways coupled to an intrinsic cell fate that is regulated by cell-specific transcription factors. Retinoic acid (RA) has been linked to stem cell self-renewal in adults and also participates in yolk sac blood island formation. Here, we demonstrate that RA decreases gata1 expression and blocks primitive hematopoiesis in zebrafish (Danio rerio) embryos, while increasing expression of the vascular marker, fli1. Treatment with an inhibitor of RA biosynthesis or a retinoic acid receptor antagonist increases gata1(+) erythroid progenitors in the posterior mesoderm of wild-type embryos and anemic cdx4(-/-) mutants, indicating a link between the cdx-hox signaling pathway and RA. Overexpression of scl, a DNA binding protein necessary for hematopoietic development, rescues the block of hematopoiesis induced by RA. We show that these effects of RA and RA pathway inhibitors are conserved during primitive hematopoiesis in murine yolk sac explant cultures and embryonic stem cell assays. Taken together, these data indicate that RA inhibits the commitment of mesodermal cells to hematopoietic fates, functioning downstream of cdx4 and upstream of scl. Our studies establish a new connection between RA and scl during development that may participate in stem cell self-renewal and hematopoietic differentiation. -
Durand EM, Zon LI. 2010. Newly emerging roles for prostaglandin E2 regulation of hematopoiesis and hematopoietic stem cell engraftment. Current opinion in hematology. 17(4):308-12. Pubmed: 20473159 DOI:10.1097/MOH.0b013e32833a888c Durand EM, Zon LI. 2010. Newly emerging roles for prostaglandin E2 regulation of hematopoiesis and hematopoietic stem cell engraftment. Current opinion in hematology. 17(4):308-12. Pubmed: 20473159 DOI:10.1097/MOH.0b013e32833a888c Array -
Koo S, Huntly BJ, Wang Y, Chen J, Brumme K, Ball B, McKinney-Freeman SL, Yabuuchi A, Scholl C, Bansal D, Zon LI, Fröhling S, Daley GQ, Gilliland DG, Mercher T. 2010. Cdx4 is dispensable for murine adult hematopoietic stem cells but promotes MLL-AF9-mediated leukemogenesis. Haematologica. 95(10):1642-50. Pubmed: 20494928 DOI:10.3324/haematol.2010.023168 Koo S, Huntly BJ, Wang Y, Chen J, Brumme K, Ball B, McKinney-Freeman SL, Yabuuchi A, Scholl C, Bansal D, Zon LI, Fröhling S, Daley GQ, Gilliland DG, Mercher T. 2010. Cdx4 is dispensable for murine adult hematopoietic stem cells but promotes MLL-AF9-mediated leukemogenesis. Haematologica. 95(10):1642-50. Pubmed: 20494928 DOI:10.3324/haematol.2010.023168 Array -
Paik EJ, de Jong JL, Pugach E, Opara P, Zon LI. 2010. A chemical genetic screen in zebrafish for pathways interacting with cdx4 in primitive hematopoiesis. Zebrafish. 7(1):61-8. Pubmed: 20415644 DOI:10.1089/zeb.2009.0643 Paik EJ, de Jong JL, Pugach E, Opara P, Zon LI. 2010. A chemical genetic screen in zebrafish for pathways interacting with cdx4 in primitive hematopoiesis. Zebrafish. 7(1):61-8. Pubmed: 20415644 DOI:10.1089/zeb.2009.0643 cdx4, a caudal-related homeodomain-containing transcription factor, functions as a regulator of hox genes, thereby playing a critical role in anterior-posterior (A-P) patterning during embryogenesis. In zebrafish, homozygous deletion of the cdx4 gene results in a mutant phenotype known as kugelig, with aberrant A-P patterning and severe anemia characterized by decreased gata1 expression in the posterior lateral mesoderm. To identify pathways that interact with cdx4 during primitive hematopoiesis, we conducted a chemical genetic screen in the cdx4 mutant background for compounds that increase gata1 expression in cdx4 mutants. Among 2640 compounds that were tested, we discovered two compounds that rescued gata1 expression in the cdx4-mutant embryos. The strongest rescue was observed with bergapten, a psoralen compound found in bergamont oil. Another member of the psoralen family, 8-methoxypsoralen, was also found to rescue gata1 expression in cdx4-mutant embryos. The psoralen compounds also disrupted normal A-P patterning of embryos. These compounds modify the cdx4-mutant phenotype and will help elucidate signaling pathways that act downstream or parallel to the cdx4-hox pathway. -
Smith AC, Raimondi AR, Salthouse CD, Ignatius MS, Blackburn JS, Mizgirev IV, Storer NY, de Jong JL, Chen AT, Zhou Y, Revskoy S, Zon LI, Langenau DM. 2010. High-throughput cell transplantation establishes that tumor-initiating cells are abundant in zebrafish T-cell acute lymphoblastic leukemia. Blood. 115(16):3296-303. Pubmed: 20056790 DOI:10.1182/blood-2009-10-246488 Smith AC, Raimondi AR, Salthouse CD, Ignatius MS, Blackburn JS, Mizgirev IV, Storer NY, de Jong JL, Chen AT, Zhou Y, Revskoy S, Zon LI, Langenau DM. 2010. High-throughput cell transplantation establishes that tumor-initiating cells are abundant in zebrafish T-cell acute lymphoblastic leukemia. Blood. 115(16):3296-303. Pubmed: 20056790 DOI:10.1182/blood-2009-10-246488 Self-renewal is a feature of cancer and can be assessed by cell transplantation into immune-compromised or immune-matched animals. However, studies in zebrafish have been severely limited by lack of these reagents. Here, Myc-induced T-cell acute lymphoblastic leukemias (T-ALLs) have been made in syngeneic, clonal zebrafish and can be transplanted into sibling animals without the need for immune suppression. These studies show that self-renewing cells are abundant in T-ALL and comprise 0.1% to 15.9% of the T-ALL mass. Large-scale single-cell transplantation experiments established that T-ALLs can be initiated from a single cell and that leukemias exhibit wide differences in tumor-initiating potential. T-ALLs also can be introduced into clonal-outcrossed animals, and T-ALLs arising in mixed genetic backgrounds can be transplanted into clonal recipients without the need for major histocompatibility complex matching. Finally, high-throughput imaging methods are described that allow large numbers of fluorescent transgenic animals to be imaged simultaneously, facilitating the rapid screening of engrafted animals. Our experiments highlight the large numbers of zebrafish that can be experimentally assessed by cell transplantation and establish new high-throughput methods to functionally interrogate gene pathways involved in cancer self-renewal. -
Trompouki E, Zon LI. 2010. Small molecule screen in zebrafish and HSC expansion. Methods in molecular biology (Clifton, N.J.). 636:301-16. Pubmed: 20336531 DOI:10.1007/978-1-60761-691-7_19 Trompouki E, Zon LI. 2010. Small molecule screen in zebrafish and HSC expansion. Methods in molecular biology (Clifton, N.J.). 636:301-16. Pubmed: 20336531 DOI:10.1007/978-1-60761-691-7_19 The zebrafish (Danio rerio) has emerged as a valuable model organism that is amenable for large-scale chemical and genetic screens. The ability of zebrafish to produce large quantities of synchronized, externally fertilized, transparent embryos makes them ideal for screens, which often are not possible in mammalian models. Signaling pathways important for hematopoiesis are well conserved between zebrafish and mammals, making many targets identified in zebrafish screens applicable to mammals. Hematopoiesis in zebrafish occurs in two waves: the primitive or embryonic wave and the definitive or adult wave. Definitive hematopoietic stem cells arise in the aorta-gonad-mesonephros region (AGM) and express conserved markers such as runx1 and c-myb that allow for the detection of stem cells by whole-mount in situ hybridization (WISH). In this protocol, we will discuss a chemical screen in zebrafish embryos to detect compounds that expand or deplete hematopoietic stem cells (HSCs) in vivo. This type of screen represents a powerful tool to study HSCs in zebrafish. -
Uong A, Zon LI. 2010. Melanocytes in development and cancer. Journal of cellular physiology. 222(1):38-41. Pubmed: 19795394 DOI:10.1002/jcp.21935 Uong A, Zon LI. 2010. Melanocytes in development and cancer. Journal of cellular physiology. 222(1):38-41. Pubmed: 19795394 DOI:10.1002/jcp.21935 Melanocytes are pigment-producing cells in the skin of humans and other vertebrates. A number of genes involved in melanocyte development and vertebrate pigmentation have been characterized, largely through studies of a diversity of pigment mutations in a variety of species. Embryonic development of the melanocyte initiates with cell fate specification in the neural crest, which is then followed by cell migration and niche localization. Many genes involved in melanocyte development have also been implicated in the development of melanoma, an aggressive and fatal form of skin cancer that originates in the melanocyte. Although early stage melanomas that have not spread to the lymph nodes can be excised with little risk of recurrence, patients diagnosed with metastatic melanoma have a high mortality rate due to the resistance of most tumors to radiotherapy and chemotherapy. Transformed melanocytes that develop into melanomas proliferate abnormally and often begin to grow radially in the skin. Vertical growth can then follow this radial growth, leading to an invasion through the basement membrane into the underlying dermis and subsequent metastasis. It is still unclear, however, how a normal melanocyte becomes a melanoma cell, and how melanoma utilizes the properties of the normal melanocyte and its progenitors in its progression. The goal of this mini-review is to highlight the role of melanocyte developmental pathways in melanoma, and to discuss recent studies and tools being used to illuminate this connection. -
Bowman TV, Zon LI. 2010. Swimming into the future of drug discovery: in vivo chemical screens in zebrafish. ACS chemical biology. 5(2):159-61. Pubmed: 20166761 DOI:10.1021/cb100029t Bowman TV, Zon LI. 2010. Swimming into the future of drug discovery: in vivo chemical screens in zebrafish. ACS chemical biology. 5(2):159-61. Pubmed: 20166761 DOI:10.1021/cb100029t In recent years in vivo chemical screening in zebrafish has emerged as a rapid and efficient method to identify lead compounds that modulate specific biological processes. By performing primary screening in vivo, the bioactivity, toxicity, and off-target side effects are determined from the onset of drug development. A recent study demonstrates that in vivo screening can be used successfully to perform structure-activity relationship (SAR) studies. This work validates the zebrafish as an effective model for not only drug discovery but also drug optimization. -
Paik EJ, Zon LI. 2010. Hematopoietic development in the zebrafish. The International journal of developmental biology. 54(6-7):1127-37. Pubmed: 20711990 DOI:10.1387/ijdb.093042ep Paik EJ, Zon LI. 2010. Hematopoietic development in the zebrafish. The International journal of developmental biology. 54(6-7):1127-37. Pubmed: 20711990 DOI:10.1387/ijdb.093042ep The model organism Danio rerio, also known as the zebrafish, is an excellent system for studying the developmental process of hematopoiesis. It is an ideal model for in vivo imaging, and it is useful for large-scale genetic screens. These have led to the discovery of previously unknown players in hematopoiesis, as well as helped our understanding of hematopoietic development. In this review, we will summarize hematopoiesis in the zebrafish and discuss how genetic approaches using the zebrafish system have helped to build our current knowledge in the field of hematopoiesis. -
Tournoij E, Weber GJ, Akkerman JW, de Groot PG, Zon LI, Moll FL, Schulte-Merker S. 2010. Mlck1a is expressed in zebrafish thrombocytes and is an essential component of thrombus formation. Journal of thrombosis and haemostasis : JTH. 8(3):588-95. Pubmed: 20002541 DOI:10.1111/j.1538-7836.2009.03721.x Tournoij E, Weber GJ, Akkerman JW, de Groot PG, Zon LI, Moll FL, Schulte-Merker S. 2010. Mlck1a is expressed in zebrafish thrombocytes and is an essential component of thrombus formation. Journal of thrombosis and haemostasis : JTH. 8(3):588-95. Pubmed: 20002541 DOI:10.1111/j.1538-7836.2009.03721.x Array -
Bai X, Kim J, Yang Z, Jurynec MJ, Akie TE, Lee J, LeBlanc J, Sessa A, Jiang H, DiBiase A, Zhou Y, Grunwald DJ, Lin S, Cantor AB, Orkin SH, Zon LI. 2010. TIF1gamma controls erythroid cell fate by regulating transcription elongation. Cell. 142(1):133-43. Pubmed: 20603019 DOI:10.1016/j.cell.2010.05.028 Bai X, Kim J, Yang Z, Jurynec MJ, Akie TE, Lee J, LeBlanc J, Sessa A, Jiang H, DiBiase A, Zhou Y, Grunwald DJ, Lin S, Cantor AB, Orkin SH, Zon LI. 2010. TIF1gamma controls erythroid cell fate by regulating transcription elongation. Cell. 142(1):133-43. Pubmed: 20603019 DOI:10.1016/j.cell.2010.05.028 Recent genome-wide studies have demonstrated that pausing of RNA polymerase II (Pol II) occurred on many vertebrate genes. By genetic studies in the zebrafish tif1gamma mutant moonshine we found that loss of function of Pol II-associated factors PAF or DSIF rescued erythroid gene transcription in tif1gamma-deficient animals. Biochemical analysis established physical interactions among TIF1gamma, the blood-specific SCL transcription complex, and the positive elongation factors p-TEFb and FACT. Chromatin immunoprecipitation assays in human CD34(+) cells supported a TIF1gamma-dependent recruitment of positive elongation factors to erythroid genes to promote transcription elongation by counteracting Pol II pausing. Our study establishes a mechanism for regulating tissue cell fate and differentiation through transcription elongation.Copyright 2010 Elsevier Inc. All rights reserved. -
Detrich HW, Westerfield M, Zon LI. 2010. The zebrafish: cellular and developmental biology, part A. Preface. Methods in cell biology. 100:xiii. Pubmed: 21111211 DOI:10.1016/B978-0-12-384892-5.00018-9 Detrich HW, Westerfield M, Zon LI. 2010. The zebrafish: cellular and developmental biology, part A. Preface. Methods in cell biology. 100:xiii. Pubmed: 21111211 DOI:10.1016/B978-0-12-384892-5.00018-9 2009
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Fraenkel PG, Gibert Y, Holzheimer JL, Lattanzi VJ, Burnett SF, Dooley KA, Wingert RA, Zon LI. 2009. Transferrin-a modulates hepcidin expression in zebrafish embryos. Blood. 113(12):2843-50. Pubmed: 19047682 DOI:10.1182/blood-2008-06-165340 Fraenkel PG, Gibert Y, Holzheimer JL, Lattanzi VJ, Burnett SF, Dooley KA, Wingert RA, Zon LI. 2009. Transferrin-a modulates hepcidin expression in zebrafish embryos. Blood. 113(12):2843-50. Pubmed: 19047682 DOI:10.1182/blood-2008-06-165340 The iron regulatory hormone hepcidin is transcriptionally up-regulated in response to iron loading, but the mechanisms by which iron levels are sensed are not well understood. Large-scale genetic screens in the zebrafish have resulted in the identification of hypochromic anemia mutants with a range of mutations affecting conserved pathways in iron metabolism and heme synthesis. We hypothesized that transferrin plays a critical role both in iron transport and in regulating hepcidin expression in zebrafish embryos. Here we report the identification and characterization of the zebrafish hypochromic anemia mutant, gavi, which exhibits transferrin deficiency due to mutations in transferrin-a. Morpholino knockdown of transferrin-a in wild-type embryos reproduced the anemia phenotype and decreased somite and terminal gut iron staining, while coinjection of transferrin-a cRNA partially restored these defects. Embryos with transferrin-a or transferrin receptor 2 (TfR2) deficiency exhibited low levels of hepcidin expression, however anemia, in the absence of a defect in the transferrin pathway, failed to impair hepcidin expression. These data indicate that transferrin-a transports iron and that hepcidin expression is regulated by a transferrin-a-dependent pathway in the zebrafish embryo. -
de Jong-Curtain TA, Parslow AC, Trotter AJ, Hall NE, Verkade H, Tabone T, Christie EL, Crowhurst MO, Layton JE, Shepherd IT, Nixon SJ, Parton RG, Zon LI, Stainier DY, Lieschke GJ, Heath JK. 2009. Abnormal nuclear pore formation triggers apoptosis in the intestinal epithelium of elys-deficient zebrafish. Gastroenterology. 136(3):902-11. Pubmed: 19073184 DOI:10.1053/j.gastro.2008.11.012 de Jong-Curtain TA, Parslow AC, Trotter AJ, Hall NE, Verkade H, Tabone T, Christie EL, Crowhurst MO, Layton JE, Shepherd IT, Nixon SJ, Parton RG, Zon LI, Stainier DY, Lieschke GJ, Heath JK. 2009. Abnormal nuclear pore formation triggers apoptosis in the intestinal epithelium of elys-deficient zebrafish. Gastroenterology. 136(3):902-11. Pubmed: 19073184 DOI:10.1053/j.gastro.2008.11.012 Array -
Burns CE, Galloway JL, Smith AC, Keefe MD, Cashman TJ, Paik EJ, Mayhall EA, Amsterdam AH, Zon LI. 2009. A genetic screen in zebrafish defines a hierarchical network of pathways required for hematopoietic stem cell emergence. Blood. 113(23):5776-82. Pubmed: 19332767 DOI:10.1182/blood-2008-12-193607 Burns CE, Galloway JL, Smith AC, Keefe MD, Cashman TJ, Paik EJ, Mayhall EA, Amsterdam AH, Zon LI. 2009. A genetic screen in zebrafish defines a hierarchical network of pathways required for hematopoietic stem cell emergence. Blood. 113(23):5776-82. Pubmed: 19332767 DOI:10.1182/blood-2008-12-193607 Defining the genetic pathways essential for hematopoietic stem cell (HSC) development remains a fundamental goal impacting stem cell biology and regenerative medicine. To genetically dissect HSC emergence in the aorta-gonad-mesonephros (AGM) region, we screened a collection of insertional zebrafish mutant lines for expression of the HSC marker, c-myb. Nine essential genes were identified, which were subsequently binned into categories representing their proximity to HSC induction. Using overexpression and loss-of-function studies in zebrafish, we ordered these signaling pathways with respect to each other and to the Vegf, Notch, and Runx programs. Overexpression of vegf and notch is sufficient to induce HSCs in the tbx16 mutant, despite a lack of axial vascular organization. Although embryos deficient for artery specification, such as the phospholipase C gamma-1 (plcgamma1) mutant, fail to specify HSCs, overexpression of notch or runx1 can rescue their hematopoietic defect. The most proximal HSC mutants, such as hdac1, were found to have no defect in vessel or artery formation. Further analysis demonstrated that hdac1 acts downstream of Notch signaling but upstream or in parallel to runx1 to promote AGM hematopoiesis. Together, our results establish a hierarchy of signaling programs required and sufficient for HSC emergence in the AGM. -
Goessling W, North TE, Loewer S, Lord AM, Lee S, Stoick-Cooper CL, Weidinger G, Puder M, Daley GQ, Moon RT, Zon LI. 2009. Genetic interaction of PGE2 and Wnt signaling regulates developmental specification of stem cells and regeneration. Cell. 136(6):1136-47. Pubmed: 19303855 DOI:10.1016/j.cell.2009.01.015 Goessling W, North TE, Loewer S, Lord AM, Lee S, Stoick-Cooper CL, Weidinger G, Puder M, Daley GQ, Moon RT, Zon LI. 2009. Genetic interaction of PGE2 and Wnt signaling regulates developmental specification of stem cells and regeneration. Cell. 136(6):1136-47. Pubmed: 19303855 DOI:10.1016/j.cell.2009.01.015 Interactions between developmental signaling pathways govern the formation and function of stem cells. Prostaglandin (PG) E2 regulates vertebrate hematopoietic stem cells (HSC). Similarly, the Wnt signaling pathway controls HSC self-renewal and bone marrow repopulation. Here, we show that wnt reporter activity in zebrafish HSCs is responsive to PGE2 modulation, demonstrating a direct interaction in vivo. Inhibition of PGE2 synthesis blocked wnt-induced alterations in HSC formation. PGE2 modified the wnt signaling cascade at the level of beta-catenin degradation through cAMP/PKA-mediated stabilizing phosphorylation events. The PGE2/Wnt interaction regulated murine stem and progenitor populations in vitro in hematopoietic ES cell assays and in vivo following transplantation. The relationship between PGE2 and Wnt was also conserved during regeneration of other organ systems. Our work provides in vivo evidence that Wnt activation in stem cells requires PGE2, and suggests the PGE2/Wnt interaction is a master regulator of vertebrate regeneration and recovery. -
Freeman JL, Ceol C, Feng H, Langenau DM, Belair C, Stern HM, Song A, Paw BH, Look AT, Zhou Y, Zon LI, Lee C. 2009. Construction and application of a zebrafish array comparative genomic hybridization platform. Genes, chromosomes & cancer. 48(2):155-70. Pubmed: 18973135 DOI:10.1002/gcc.20623 Freeman JL, Ceol C, Feng H, Langenau DM, Belair C, Stern HM, Song A, Paw BH, Look AT, Zhou Y, Zon LI, Lee C. 2009. Construction and application of a zebrafish array comparative genomic hybridization platform. Genes, chromosomes & cancer. 48(2):155-70. Pubmed: 18973135 DOI:10.1002/gcc.20623 The zebrafish is emerging as a prominent model system for studying the genetics of human development and disease. Genetic alterations that underlie each mutant model can exist in the form of single base changes, balanced chromosomal rearrangements, or genetic imbalances. To detect genetic imbalances in an unbiased genome-wide fashion, array comparative genomic hybridization (CGH) can be used. We have developed a 5-Mb resolution array CGH platform specifically for the zebrafish. This platform contains 286 bacterial artificial chromosome (BAC) clones, enriched for orthologous sequences of human oncogenes and tumor suppressor genes. Each BAC clone has been end-sequenced and cytogenetically assigned to a specific location within the zebrafish genome, allowing for ease of integration of array CGH data with the current version of the genome assembly. This platform has been applied to three zebrafish cancer models. Significant genomic imbalances were detected in each model, identifying different regions that may potentially play a role in tumorigenesis. Hence, this platform should be a useful resource for genetic dissection of additional zebrafish developmental and disease models as well as a benchmark for future array CGH platform development. -
Dovey MC, Zon LI. 2009. Defining cancer stem cells by xenotransplantation in zebrafish. Methods in molecular biology (Clifton, N.J.). 568:1-5. Pubmed: 19582417 DOI:10.1007/978-1-59745-280-9_1 Dovey MC, Zon LI. 2009. Defining cancer stem cells by xenotransplantation in zebrafish. Methods in molecular biology (Clifton, N.J.). 568:1-5. Pubmed: 19582417 DOI:10.1007/978-1-59745-280-9_1 The zebrafish (Danio rerio) has become an increasingly utilized and relevant model organism in the study of cancer. The use of transgenic and reverse genetic approaches has yielded several strains that model a variety of human neoplasms. In addition to modeling human disease, these strains provide a platform for the analysis of tumor stem cells. Here we describe the basic technique for the isolation and transplantation of tumor tissue in the zebrafish. This technique was designed to study metastasis and invasive potential of zebrafish tumor cells. Additionally, the basic protocol can be modified in order to describe cancer stem cell characteristics, including proliferative capacity, self-renewal, and the minimum number of tumor cells required for engraftment. -
Bowman TV, Zon LI. 2009. Lessons from the Niche for Generation and Expansion of Hematopoietic Stem Cells. Drug discovery today. Therapeutic strategies. 6(4):135-140. Pubmed: 21212834 Bowman TV, Zon LI. 2009. Lessons from the Niche for Generation and Expansion of Hematopoietic Stem Cells. Drug discovery today. Therapeutic strategies. 6(4):135-140. Pubmed: 21212834 Hematopoietic Stem Cells (HSCs) are used clinically to treat human blood-related genetic diseases and leukemias, but the availability of this therapy is constrained by the limiting number of transplantable HSCs. Previous strategies to expand HSCs in vitro resulted in precocious differentiation and reduced transplant efficiency. In vivo analysis of the mechanisms utilized by fetal and adult niches to control HSCs can be mimicked for therapeutic use. This review summarizes the latest research on the in vivo HSC niche and the clinical applications of this knowledge to promote ex vivo expansion and direct de novo generation of HSCs. -
Pugach EK, Li P, White R, Zon L. 2009. Retro-orbital injection in adult zebrafish. Journal of visualized experiments : JoVE. Pubmed: 19997061 DOI:10.3791/1645 Pugach EK, Li P, White R, Zon L. 2009. Retro-orbital injection in adult zebrafish. Journal of visualized experiments : JoVE. Pubmed: 19997061 DOI:10.3791/1645 Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy. -
Novoa B, Bowman TV, Zon L, Figueras A. 2009. LPS response and tolerance in the zebrafish (Danio rerio). Fish & shellfish immunology. 26(2):326-31. Pubmed: 19110060 DOI:10.1016/j.fsi.2008.12.004 Novoa B, Bowman TV, Zon L, Figueras A. 2009. LPS response and tolerance in the zebrafish (Danio rerio). Fish & shellfish immunology. 26(2):326-31. Pubmed: 19110060 DOI:10.1016/j.fsi.2008.12.004 Zebrafish (Danio rerio) has been used in the present work to study the fish response to bacterial lipopolysaccharide (LPS) exposure and LPS tolerance. These mechanisms are not completely understood in mammals and, presently, are totally unknown in fish. Zebrafish larval survival was assessed following treatment with various types of LPS at a variety of concentrations to determine the sensitivity of zebrafish to LPS-induced immune activation. In addition, fish pretreated with a sublethal concentration of LPS did not die after exposure to a lethal concentration of LPS demonstrating, for the first time that LPS tolerance also happens in fish. The time interval between pretreatment and secondary exposure as well as the type of pretreatment dictated the strength of protection. Since zebrafish are in intimate contact with microorganisms, the high resistance of fish to LPS suggests that there must be a tight control of the LPS receptor cluster in order to avoid an excess of inflammation. One of these components is CXCR4, which has previously been shown to regulate the signal transduced by TLR4. Treating fish with AMD3100, a specific inhibitor of CXCR4, increased LPS treatment associated mortality. Blocking CXCR4 via chemical or genetic inhibition resulted in a reversion of LPS tolerance, thus further supporting the negative regulatory role of CXCR4 in this inflammatory response. In support of an inhibitory role for CXCR4 in the inflammatory cascade, IL-1 transcript levels were elevated in both unstimulated and LPS stimulated zebrafish Odysseus (CXCR4 deficient mutant) larvae. -
Dovey M, White RM, Zon LI. 2009. Oncogenic NRAS cooperates with p53 loss to generate melanoma in zebrafish. Zebrafish. 6(4):397-404. Pubmed: 19954345 DOI:10.1089/zeb.2009.0606 Dovey M, White RM, Zon LI. 2009. Oncogenic NRAS cooperates with p53 loss to generate melanoma in zebrafish. Zebrafish. 6(4):397-404. Pubmed: 19954345 DOI:10.1089/zeb.2009.0606 NRAS mutations are a common oncogenic event in skin cancer, occurring frequently in congenital nevi and malignant melanoma. To study the role of NRAS in zebrafish, a transgenic approach was applied to generate fish that express human oncogenic NRAS(Q61K) under the control of the melanocyte-restricted mitfa promoter. By screening the progeny of the injected animals, two strains stably expressing the NRAS transgene were identified: Tg(mitfa:EGFP:NRAS(Q61K))(1) and Tg(mitfa:EGFP:NRAS(Q61K))(2). Stable expression of this transgene results in hyperpigmented fish displaying a complete ablation of the normal pigment pattern. Although oncogenic NRAS expression alone was found to be insufficient to promote tumor formation, loss of functional p53 was found to collaborate with NRAS expression in the genesis of melanoma. The tumors derived from these animals are variably pigmented and closely resemble human melanoma. Underscoring the pathological similarities between these tumors and human disease and suggesting that common pathways are similar in these models and human disease, gene set enrichment analysis performed on microarray data found that the upregulated genes from zebrafish melanomas are highly enriched in human tumor samples. This work characterizes two zebrafish melanoma models that will be useful tools for the study of melanoma pathogenesis. -
Kaufman CK, White RM, Zon L. 2009. Chemical genetic screening in the zebrafish embryo. Nature protocols. 4(10):1422-32. Pubmed: 19745824 DOI:10.1038/nprot.2009.144 Kaufman CK, White RM, Zon L. 2009. Chemical genetic screening in the zebrafish embryo. Nature protocols. 4(10):1422-32. Pubmed: 19745824 DOI:10.1038/nprot.2009.144 Chemical genetic screening can be described as a discovery approach in which chemicals are assayed for their effects on a defined biological system. The zebrafish, Danio rerio, is a well-characterized and genetically tractable vertebrate model organism that produces large numbers of rapidly developing embryos that develop externally. These characteristics allow for flexible, rapid and scalable chemical screen design using the zebrafish. We describe a protocol for screening compounds from a chemical library for effects on early zebrafish development using an automated in situ based read-out. As screenings are carried out in the context of a complete, developing organism, this approach allows for a more comprehensive analysis of the range of a chemical's effects than that provided by, for example, a cell culture-based or in vitro biochemical assay. Using a 24-h chemical treatment, one can complete a round of screening in 6 d. -
Chen AT, Zon LI. 2009. Zebrafish blood stem cells. Journal of cellular biochemistry. 108(1):35-42. Pubmed: 19565566 DOI:10.1002/jcb.22251 Chen AT, Zon LI. 2009. Zebrafish blood stem cells. Journal of cellular biochemistry. 108(1):35-42. Pubmed: 19565566 DOI:10.1002/jcb.22251 Within the past two decades, the zebrafish (Danio rerio) has become an excellent model to study the development of hematopoietic stem cells (HSCs). All vertebrates including zebrafish have primitive and definitive waves of hematopoiesis, but self-renewing pluripotent HSCs are only produced by the definitive wave. The primitive wave occurs in two intraembryonic locations called the intermediate cell mass (ICM) and the anterior lateral mesoderm (ALM). Primitive erythropoiesis is in the ICM, whereas myelopoiesis initiates in the ALM. After circulation starts at 24 h post-fertilization, hematopoiesis shifts to the posterior blood island (PBI) for a brief period. The definitive wave starts in the aorta-gonad-mesonephros (AGM). There are three different HSC migration and colonization events that begin 2 days post-fertilization: AGM progenitor cells migrate to (1) the caudal hematopoietic tissue (CHT), which is an intermediate site of blood development; (2) the thymus, which is a site of lymphocyte maturation; and (3) the developing kidney marrow, which is the larval and adult location for production of all hematopoietic cell types, and is comparable to the bone marrow of mammals. Many of the transcription factors and signaling pathways that regulate the formation of HSCs in a zebrafish are conserved with mammals. Large-scale forward and reverse genetic screens have identified zebrafish blood and HSC mutants that represent models for known human diseases. Along with the technological advancements in the field of zebrafish research, future HSC studies in zebrafish will help us illuminate the genetic network controlling the development and function of stem cells in all vertebrates.(c) 2009 Wiley-Liss, Inc. -
North TE, Goessling W, Peeters M, Li P, Ceol C, Lord AM, Weber GJ, Harris J, Cutting CC, Huang P, Dzierzak E, Zon LI. 2009. Hematopoietic stem cell development is dependent on blood flow. Cell. 137(4):736-48. Pubmed: 19450519 DOI:10.1016/j.cell.2009.04.023 North TE, Goessling W, Peeters M, Li P, Ceol C, Lord AM, Weber GJ, Harris J, Cutting CC, Huang P, Dzierzak E, Zon LI. 2009. Hematopoietic stem cell development is dependent on blood flow. Cell. 137(4):736-48. Pubmed: 19450519 DOI:10.1016/j.cell.2009.04.023 During vertebrate embryogenesis, hematopoietic stem cells (HSCs) arise in the aorta-gonads-mesonephros (AGM) region. We report here that blood flow is a conserved regulator of HSC formation. In zebrafish, chemical blood flow modulators regulated HSC development, and silent heart (sih) embryos, lacking a heartbeat and blood circulation, exhibited severely reduced HSCs. Flow-modifying compounds primarily affected HSC induction after the onset of heartbeat; however, nitric oxide (NO) donors regulated HSC number even when treatment occurred before the initiation of circulation, and rescued HSCs in sih mutants. Morpholino knockdown of nos1 (nnos/enos) blocked HSC development, and its requirement was shown to be cell autonomous. In the mouse, Nos3 (eNos) was expressed in HSCs in the AGM. Intrauterine Nos inhibition or embryonic Nos3 deficiency resulted in a reduction of hematopoietic clusters and transplantable murine HSCs. This work links blood flow to AGM hematopoiesis and identifies NO as a conserved downstream regulator of HSC development. -
Stachura DL, Reyes JR, Bartunek P, Paw BH, Zon LI, Traver D. 2009. Zebrafish kidney stromal cell lines support multilineage hematopoiesis. Blood. 114(2):279-89. Pubmed: 19433857 DOI:10.1182/blood-2009-02-203638 Stachura DL, Reyes JR, Bartunek P, Paw BH, Zon LI, Traver D. 2009. Zebrafish kidney stromal cell lines support multilineage hematopoiesis. Blood. 114(2):279-89. Pubmed: 19433857 DOI:10.1182/blood-2009-02-203638 Studies of zebrafish hematopoiesis have been largely performed using mutagenesis approaches and retrospective analyses based upon gene expression patterns in whole embryos. We previously developed transplantation assays to test the repopulation potentials of candidate hematopoietic progenitor cells. We have been impaired, however, in determining cellular differentiation potentials by a lack of short-term functional assays. To enable more precise analyses of hematopoietic progenitor cells, we have created zebrafish kidney stromal (ZKS) cell lines. Culture of adult whole kidney marrow with ZKS cells results in the maintenance and expansion of hematopoietic precursor cells. Hematopoietic growth is dependent upon ZKS cells, and we show that ZKS cells express many growth factors and ligands previously demonstrated to be important in maintaining mammalian hematopoietic cells. In the absence of exogenous growth factors, ZKS cells maintain early hematopoietic precursors and support differentiation of lymphoid and myeloid cells. With the addition of zebrafish erythropoietin, ZKS cells also support the differentiation of erythroid precursors. These conditions have enabled the ability to ascertain more precisely the points at which hematopoietic mutants are defective. The development of robust in vitro assays now provide the means to track defined, functional outcomes for prospectively isolated blood cell subsets in the zebrafish. -
Taylor AM, Zon LI. 2009. Zebrafish tumor assays: the state of transplantation. Zebrafish. 6(4):339-46. Pubmed: 20047467 DOI:10.1089/zeb.2009.0607 Taylor AM, Zon LI. 2009. Zebrafish tumor assays: the state of transplantation. Zebrafish. 6(4):339-46. Pubmed: 20047467 DOI:10.1089/zeb.2009.0607 Tumor transplant studies are important tools for studying cancer biology in a model organism. Transplantation is especially important for assaying tumor cell malignancy and migration capabilities, and is critical for identifying putative cancer stem cell populations. In this review, we discuss the current state of tumor transplantation studies performed in the zebrafish. We address several zebrafish-specific considerations for development of the transplant assay, including choosing recipient animals, transplant methods, and post-transplant observation. We also examine how the zebrafish is an advantageous model for transplantation, particularly with development of the translucent fish. Transplantation has already been critical for characterizing zebrafish models of leukemia, rhabdomyosarcoma, and melanoma. With further development of imaging techniques and other tools, zebrafish tumor transplantation will continue to contribute to our understanding of tumor cell biology. -
Dovey M, Patton EE, Bowman T, North T, Goessling W, Zhou Y, Zon LI. 2009. Topoisomerase II alpha is required for embryonic development and liver regeneration in zebrafish. Molecular and cellular biology. 29(13):3746-53. Pubmed: 19380487 DOI:10.1128/MCB.01684-08 Dovey M, Patton EE, Bowman T, North T, Goessling W, Zhou Y, Zon LI. 2009. Topoisomerase II alpha is required for embryonic development and liver regeneration in zebrafish. Molecular and cellular biology. 29(13):3746-53. Pubmed: 19380487 DOI:10.1128/MCB.01684-08 Topoisomerases solve the topological problems encountered by DNA throughout the lifetime of a cell. Topoisomerase II alpha, which is highly conserved among eukaryotes, untangles replicated chromosomes during mitosis and is absolutely required for cell viability. A homozygous lethal mutant, can4, was identified in a screen to identify genes important for cell proliferation in zebrafish by utilizing an antibody against a mitosis-specific marker, phospho-histone H3. Mutant embryos have a decrease in the number of proliferating cells and display increases in DNA content and apoptosis, as well as mitotic spindle defects. Positional cloning revealed that the genetic defect underlying these phenotypes was the result of a mutation in the zebrafish topoisomerase II alpha (top2a) gene. top2a was found to be required for decatenation but not for condensation in embryonic mitoses. In addition to being required for development, top2a was found to be a haploinsufficient regulator of adult liver regrowth in zebrafish. Regeneration analysis of other adult tissues, including fins, revealed no heterozygous phenotype. Our results confirm a conserved role for TOP2A in vertebrates as well as a dose-sensitive requirement for top2a in adults. 2008
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Bussmann J, Lawson N, Zon L, Schulte-Merker S. 2008. Zebrafish VEGF receptors: a guideline to nomenclature. PLoS genetics. 4(5):e1000064. Pubmed: 18516225 DOI:10.1371/journal.pgen.1000064 Bussmann J, Lawson N, Zon L, Schulte-Merker S. 2008. Zebrafish VEGF receptors: a guideline to nomenclature. PLoS genetics. 4(5):e1000064. Pubmed: 18516225 DOI:10.1371/journal.pgen.1000064 -
Goessling W, North TE, Lord AM, Ceol C, Lee S, Weidinger G, Bourque C, Strijbosch R, Haramis AP, Puder M, Clevers H, Moon RT, Zon LI. 2008. APC mutant zebrafish uncover a changing temporal requirement for wnt signaling in liver development. Developmental biology. 320(1):161-74. Pubmed: 18585699 DOI:10.1016/j.ydbio.2008.05.526 Goessling W, North TE, Lord AM, Ceol C, Lee S, Weidinger G, Bourque C, Strijbosch R, Haramis AP, Puder M, Clevers H, Moon RT, Zon LI. 2008. APC mutant zebrafish uncover a changing temporal requirement for wnt signaling in liver development. Developmental biology. 320(1):161-74. Pubmed: 18585699 DOI:10.1016/j.ydbio.2008.05.526 Developmental signaling pathways hold the keys to unlocking the promise of adult tissue regeneration, and to inhibiting carcinogenesis. Patients with mutations in the Adenomatous Polyposis Coli (APC) gene are at increased risk of developing hepatoblastoma, an embryonal form of liver cancer, suggesting that Wnt affects hepatic progenitor cells. To elucidate the role of APC loss and enhanced Wnt activity in liver development, we examined APC mutant and wnt inducible transgenic zebrafish. APC(+/-) embryos developed enlarged livers through biased induction of hepatic gene programs and increased proliferation. Conversely, APC(-/-) embryos formed no livers. Blastula transplantations determined that the effects of APC loss were cell autonomous. Induction of wnt modulators confirmed biphasic consequences of wnt activation: endodermal pattern formation and gene expression required suppression of wnt signaling in early somitogenesis; later, increased wnt activity altered endodermal fate by enhancing liver growth at the expense of pancreas formation; these effects persisted into the larval stage. In adult APC(+/-) zebrafish, increased wnt activity significantly accelerated liver regeneration after partial hepatectomy. Similarly, liver regeneration was significantly enhanced in APC(Min/+) mice, indicating the conserved effect of Wnt pathway activation in liver regeneration across vertebrate species. These studies reveal an important and time-dependent role for wnt signaling during liver development and regeneration. -
Zon LI. 2008. Intrinsic and extrinsic control of haematopoietic stem-cell self-renewal. Nature. 453(7193):306-13. Pubmed: 18480811 DOI:10.1038/nature07038 Zon LI. 2008. Intrinsic and extrinsic control of haematopoietic stem-cell self-renewal. Nature. 453(7193):306-13. Pubmed: 18480811 DOI:10.1038/nature07038 When stem cells divide, they can generate progeny with the same developmental potential as the original cell, a process referred to as self-renewal. Self-renewal is driven intrinsically by gene expression in a cell-type-specific manner and is modulated through interactions with extrinsic cues from the environment, such as growth factors. However, despite the prevalence of the term self-renewal in the scientific literature, this process has not been defined at the molecular level. Haematopoietic stem cells are an excellent model for the study of self-renewal because they can be isolated prospectively, manipulated relatively easily and assessed by using well-defined assays. Establishing the principles of self-renewal in haematopoietic stem cells will lead to insights into the mechanisms of self-renewal in other tissues. -
Langenau DM, Keefe MD, Storer NY, Jette CA, Smith AC, Ceol CJ, Bourque C, Look AT, Zon LI. 2008. Co-injection strategies to modify radiation sensitivity and tumor initiation in transgenic Zebrafish. Oncogene. 27(30):4242-8. Pubmed: 18345029 DOI:10.1038/onc.2008.56 Langenau DM, Keefe MD, Storer NY, Jette CA, Smith AC, Ceol CJ, Bourque C, Look AT, Zon LI. 2008. Co-injection strategies to modify radiation sensitivity and tumor initiation in transgenic Zebrafish. Oncogene. 27(30):4242-8. Pubmed: 18345029 DOI:10.1038/onc.2008.56 The zebrafish has emerged as a powerful genetic model of cancer, but has been limited by the use of stable transgenic approaches to induce disease. Here, a co-injection strategy is described that capitalizes on both the numbers of embryos that can be microinjected and the ability of transgenes to segregate together and exert synergistic effects in forming tumors. Using this mosaic transgenic approach, gene pathways involved in tumor initiation and radiation sensitivity have been identified. -
Deutsch EW, Ball CA, Berman JJ, Bova GS, Brazma A, Bumgarner RE, Campbell D, Causton HC, Christiansen JH, Daian F, Dauga D, Davidson DR, Gimenez G, Goo YA, Grimmond S, Henrich T, Herrmann BG, Johnson MH, Korb M, Mills JC, Oudes AJ, Parkinson HE, Pascal LE, Pollet N, Quackenbush J, Ramialison M, Ringwald M, Salgado D, Sansone SA, Sherlock G, Stoeckert CJ, Swedlow J, Taylor RC, Walashek L, Warford A, Wilkinson DG, Zhou Y, Zon LI, Liu AY, True LD. 2008. Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). Nature biotechnology. 26(3):305-12. Pubmed: 18327244 DOI:10.1038/nbt1391 Deutsch EW, Ball CA, Berman JJ, Bova GS, Brazma A, Bumgarner RE, Campbell D, Causton HC, Christiansen JH, Daian F, Dauga D, Davidson DR, Gimenez G, Goo YA, Grimmond S, Henrich T, Herrmann BG, Johnson MH, Korb M, Mills JC, Oudes AJ, Parkinson HE, Pascal LE, Pollet N, Quackenbush J, Ramialison M, Ringwald M, Salgado D, Sansone SA, Sherlock G, Stoeckert CJ, Swedlow J, Taylor RC, Walashek L, Warford A, Wilkinson DG, Zhou Y, Zon LI, Liu AY, True LD. 2008. Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). Nature biotechnology. 26(3):305-12. Pubmed: 18327244 DOI:10.1038/nbt1391 One purpose of the biomedical literature is to report results in sufficient detail that the methods of data collection and analysis can be independently replicated and verified. Here we present reporting guidelines for gene expression localization experiments: the minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). MISFISHIE is modeled after the Minimum Information About a Microarray Experiment (MIAME) specification for microarray experiments. Both guidelines define what information should be reported without dictating a format for encoding that information. MISFISHIE describes six types of information to be provided for each experiment: experimental design, biomaterials and treatments, reporters, staining, imaging data and image characterizations. This specification has benefited the consortium within which it was developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal. -
Ceol CJ, Houvras Y, White RM, Zon LI. 2008. Melanoma biology and the promise of zebrafish. Zebrafish. 5(4):247-55. Pubmed: 19133823 DOI:10.1089/zeb.2008.0544 Ceol CJ, Houvras Y, White RM, Zon LI. 2008. Melanoma biology and the promise of zebrafish. Zebrafish. 5(4):247-55. Pubmed: 19133823 DOI:10.1089/zeb.2008.0544 Advantageous organismal and technical attributes of the zebrafish are being increasingly applied to study cancer biology. Along with other tumor models, zebrafish that develop melanomas have been generated. In both genetics and phenotype, zebrafish melanomas are strikingly similar to their human counterparts. For this reason, studies in the zebrafish are poised to make significant contributions to melanoma biology. In this review, we summarize important features of human melanoma and discuss how the zebrafish can be used to address many questions that remain unanswered about this devastating disease. -
Sessa AK, White R, Houvras Y, Burke C, Pugach E, Baker B, Gilbert R, Thomas Look A, Zon LI. 2008. The effect of a depth gradient on the mating behavior, oviposition site preference, and embryo production in the zebrafish, Danio rerio. Zebrafish. 5(4):335-9. Pubmed: 19133832 DOI:10.1089/zeb.2008.0535 Sessa AK, White R, Houvras Y, Burke C, Pugach E, Baker B, Gilbert R, Thomas Look A, Zon LI. 2008. The effect of a depth gradient on the mating behavior, oviposition site preference, and embryo production in the zebrafish, Danio rerio. Zebrafish. 5(4):335-9. Pubmed: 19133832 DOI:10.1089/zeb.2008.0535 Captive zebrafish (Danio rerio) exhibit a limited repertoire of mating behaviors, likely due to the somewhat unnatural environment of aquaria. Observations in their natural habitat led us to believe that a depth gradient within the mating setup would positively affect fish mating. By tilting the tank to produce a depth gradient, we observed novel behaviors along with a preference for oviposition in the shallow area. Although we did not see an increase in the likelihood of a pair of fish to mate, we did see an increase in the embryo output in both adults and juveniles. In the adults, tilting led to a significant increase in embryo production (436 +/- 35 tilted vs. 362 +/- 34 untilted; p < 0.05). A similar effect was seen in juvenile fish as they progressed through sexual maturity. These results suggest that tilting of mating cages in the laboratory setting will lead to demonstrable improvements in embryo production for zebrafish researchers, and highlights the possibility of other manipulations to increase fecundity. -
Huang HT, Zon LI. 2008. Regulation of stem cells in the zebra fish hematopoietic system. Cold Spring Harbor symposia on quantitative biology. 73:111-8. Pubmed: 19022765 DOI:10.1101/sqb.2008.73.029 Huang HT, Zon LI. 2008. Regulation of stem cells in the zebra fish hematopoietic system. Cold Spring Harbor symposia on quantitative biology. 73:111-8. Pubmed: 19022765 DOI:10.1101/sqb.2008.73.029 Hematopoietic stem cells (HSCs) have been used extensively as a model for stem cell biology. Stem cells share the ability to self-renew and differentiate into multiple cell types, making them ideal candidates for tissue regeneration or replacement therapies. Current applications of stem cell technology are limited by our knowledge of the molecular mechanisms that control their proliferation and differentiation, and various model organisms have been used to fill these gaps. This chapter focuses on the contributions of the zebra fish model to our understanding of stem cell regulation within the hematopoietic system. Studies in zebra fish have been valuable for identifying new genetic and signaling factors that affect HSC formation and development with important implications for humans, and new advances in the zebra fish toolbox will allow other aspects of HSC behavior to be investigated as well, including migration, homing, and engraftment. -
Imamura S, Uchiyama J, Koshimizu E, Hanai J, Raftopoulou C, Murphey RD, Bayliss PE, Imai Y, Burns CE, Masutomi K, Gagos S, Zon LI, Roberts TM, Kishi S. 2008. A non-canonical function of zebrafish telomerase reverse transcriptase is required for developmental hematopoiesis. PloS one. 3(10):e3364. Pubmed: 18846223 DOI:10.1371/journal.pone.0003364 Imamura S, Uchiyama J, Koshimizu E, Hanai J, Raftopoulou C, Murphey RD, Bayliss PE, Imai Y, Burns CE, Masutomi K, Gagos S, Zon LI, Roberts TM, Kishi S. 2008. A non-canonical function of zebrafish telomerase reverse transcriptase is required for developmental hematopoiesis. PloS one. 3(10):e3364. Pubmed: 18846223 DOI:10.1371/journal.pone.0003364 Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from "authentic" telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words). -
Wang L, Sewell WF, Kim SD, Shin JT, MacRae CA, Zon LI, Seidman JG, Seidman CE. 2008. Eya4 regulation of Na+/K+-ATPase is required for sensory system development in zebrafish. Development (Cambridge, England). 135(20):3425-34. Pubmed: 18799547 DOI:10.1242/dev.012237 Wang L, Sewell WF, Kim SD, Shin JT, MacRae CA, Zon LI, Seidman JG, Seidman CE. 2008. Eya4 regulation of Na+/K+-ATPase is required for sensory system development in zebrafish. Development (Cambridge, England). 135(20):3425-34. Pubmed: 18799547 DOI:10.1242/dev.012237 To investigate the mechanisms by which mutations in the human transcriptional co-activator EYA4 gene cause sensorineural hearing loss that can occur in association with dilated cardiomyopathy, we studied eya4 expression during zebrafish development and characterized eya4 deficiency. eya4 morphant fish embryos had reduced numbers of hair cells in the otic vesicle and lateral line neuromasts with impaired sensory responses. Analyses of candidate genes that are known to be expressed in a temporal and spatial pattern comparable to eya4 focused our analyses on atp1b2b, which encodes the beta2b subunit of the zebrafish Na+/K+-ATPase. We demonstrate atp1b2b levels are reduced in eya4 morphant fish and that morpholino oligonucleotides targeting the atp1b2b gene recapitulated the eya4 deficiency phenotypes, including heart failure, decreased sensory hair cell numbers in the otic vesicle and neuromasts, and abnormal sensory responses. Furthermore, atp1b2b overexpression rescued these phenotypes in eya4 morphant fish. We conclude that eya4 regulation of Na+/K+-ATPase is crucial for the development of mechanosensory cells and the maintenance of cardiac function in zebrafish. -
White RM, Zon LI. 2008. Melanocytes in development, regeneration, and cancer. Cell stem cell. 3(3):242-52. Pubmed: 18786412 DOI:10.1016/j.stem.2008.08.005 White RM, Zon LI. 2008. Melanocytes in development, regeneration, and cancer. Cell stem cell. 3(3):242-52. Pubmed: 18786412 DOI:10.1016/j.stem.2008.08.005 The genes required for stem cell specification and lineage restriction during embryogenesis also play fundamental roles in adult tissue regeneration and cancer. This "development-regeneration-cancer" axis is exemplified by the vertebrate pigmentation system. Melanocytes exhibit almost unlimited self-renewal capacity during regenerative processes such as mammalian hair recoloration and zebrafish fin regeneration. Melanoma utilizes many regulatory signals and pathways required during ontogeny and regeneration. A discussion of these interconnections highlights how studies of stem cell function in embryonic and regenerative contexts can yield insights into melanoma biology. -
Trede NS, Ota T, Kawasaki H, Paw BH, Katz T, Demarest B, Hutchinson S, Zhou Y, Hersey C, Zapata A, Amemiya CT, Zon LI. 2008. Zebrafish mutants with disrupted early T-cell and thymus development identified in early pressure screen. Developmental dynamics : an official publication of the American Association of Anatomists. 237(9):2575-84. Pubmed: 18729230 DOI:10.1002/dvdy.21683 Trede NS, Ota T, Kawasaki H, Paw BH, Katz T, Demarest B, Hutchinson S, Zhou Y, Hersey C, Zapata A, Amemiya CT, Zon LI. 2008. Zebrafish mutants with disrupted early T-cell and thymus development identified in early pressure screen. Developmental dynamics : an official publication of the American Association of Anatomists. 237(9):2575-84. Pubmed: 18729230 DOI:10.1002/dvdy.21683 Generation of mature T lymphocytes requires an intact hematopoietic stem cell compartment and functional thymic epithelium. We used the zebrafish (Danio rerio) to isolate mutations that affect the earliest steps in T lymphopoiesis and thymic organogenesis. Here we describe the results of a genetic screen in which gynogenetic diploid offspring from heterozygous females were analyzed by whole-mount in situ hybridization for the expression of rag-1. To assess immediately if a global defect in hematopoiesis resulted in the mutant phenotype, alpha-embryonic globin expression was simultaneously assayed for multilineage defects. In this report, we present the results obtained with this strategy and show representative mutant phenotypes affecting early steps in T-cell development and/or thymic epithelial cell development. We discuss the advantage of this strategy and the general usefulness of the zebrafish as a model system for vertebrate lymphopoiesis and thymic organogenesis. -
Zon LI, Le X. 2008. Potential of zebrafish for anticancer drug screening. Expert opinion on drug discovery. 3(12):1451-60. Pubmed: 23506108 DOI:10.1517/17460440802580171 Zon LI, Le X. 2008. Potential of zebrafish for anticancer drug screening. Expert opinion on drug discovery. 3(12):1451-60. Pubmed: 23506108 DOI:10.1517/17460440802580171 Array -
Zon LI. 2008. Self-renewal and differentiation at Cell Stem Cell. Cell stem cell. 2(6):510. Pubmed: 18522838 DOI:10.1016/j.stem.2008.05.016 Zon LI. 2008. Self-renewal and differentiation at Cell Stem Cell. Cell stem cell. 2(6):510. Pubmed: 18522838 DOI:10.1016/j.stem.2008.05.016 -
Dooley KA, Fraenkel PG, Langer NB, Schmid B, Davidson AJ, Weber G, Chiang K, Foott H, Dwyer C, Wingert RA, Zhou Y, Paw BH, Zon LI. 2008. montalcino, A zebrafish model for variegate porphyria. Experimental hematology. 36(9):1132-42. Pubmed: 18550261 DOI:10.1016/j.exphem.2008.04.008 Dooley KA, Fraenkel PG, Langer NB, Schmid B, Davidson AJ, Weber G, Chiang K, Foott H, Dwyer C, Wingert RA, Zhou Y, Paw BH, Zon LI. 2008. montalcino, A zebrafish model for variegate porphyria. Experimental hematology. 36(9):1132-42. Pubmed: 18550261 DOI:10.1016/j.exphem.2008.04.008 Array -
Upadhyay G, Goessling W, North TE, Xavier R, Zon LI, Yajnik V. 2008. Molecular association between beta-catenin degradation complex and Rac guanine exchange factor DOCK4 is essential for Wnt/beta-catenin signaling. Oncogene. 27(44):5845-55. Pubmed: 18641688 DOI:10.1038/onc.2008.202 Upadhyay G, Goessling W, North TE, Xavier R, Zon LI, Yajnik V. 2008. Molecular association between beta-catenin degradation complex and Rac guanine exchange factor DOCK4 is essential for Wnt/beta-catenin signaling. Oncogene. 27(44):5845-55. Pubmed: 18641688 DOI:10.1038/onc.2008.202 The canonical Wnt/beta-catenin pathway is a highly conserved signaling cascade that is involved in development and stem cell renewal. The deregulation of this pathway is often associated with increased cell growth and neoplasia. The small GTPase Rac has been shown to influence canonical Wnt signaling by regulating beta-catenin stability through an unknown mechanism. We report that DOCK4, a guanine nucleotide exchange factor (GEF) for Rac and a member of the CDM family of unconventional GEFs, mediates Wnt-induced Rac activation in the canonical Wnt/beta-catenin pathway. DOCK4 expression regulates cellular beta-catenin levels in response to the Wnt signal, in vitro. Biochemical studies demonstrate that DOCK4 interacts with the beta-catenin degradation complex, consisting of the proteins adenomatosis polyposis coli, Axin and glycogen synthase kinase 3beta (GSK3beta). This molecular interaction enhances beta-catenin stability and Axin degradation. Furthermore, we observe that DOCK4 is phosphorylated by GSK3beta, which enhances Wnt-induced Rac activation. Using a T-cell factor reporter zebrafish we confirm that DOCK4 is required for Wnt/beta-catenin activity, in vivo. These results elucidate a novel intracellular signaling mechanism in which a Rac GEF, DOCK4 acts as a scaffold protein in the Wnt/beta-catenin pathway. -
Orkin SH, Zon LI. 2008. Hematopoiesis: an evolving paradigm for stem cell biology. Cell. 132(4):631-44. Pubmed: 18295580 DOI:10.1016/j.cell.2008.01.025 Orkin SH, Zon LI. 2008. Hematopoiesis: an evolving paradigm for stem cell biology. Cell. 132(4):631-44. Pubmed: 18295580 DOI:10.1016/j.cell.2008.01.025 Establishment and maintenance of the blood system relies on self-renewing hematopoietic stem cells (HSCs) that normally reside in small numbers in the bone marrow niche of adult mammals. This Review describes the developmental origins of HSCs and the molecular mechanisms that regulate lineage-specific differentiation. Studies of hematopoiesis provide critical insights of general relevance to other areas of stem cell biology including the role of cellular interactions in development and tissue homeostasis, lineage programming and reprogramming by transcription factors, and stage- and age-specific differences in cellular phenotypes. -
White RM, Sessa A, Burke C, Bowman T, LeBlanc J, Ceol C, Bourque C, Dovey M, Goessling W, Burns CE, Zon LI. 2008. Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell stem cell. 2(2):183-9. Pubmed: 18371439 DOI:10.1016/j.stem.2007.11.002 White RM, Sessa A, Burke C, Bowman T, LeBlanc J, Ceol C, Bourque C, Dovey M, Goessling W, Burns CE, Zon LI. 2008. Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell stem cell. 2(2):183-9. Pubmed: 18371439 DOI:10.1016/j.stem.2007.11.002 The zebrafish is a useful model for understanding normal and cancer stem cells, but analysis has been limited to embryogenesis due to the opacity of the adult fish. To address this, we have created a transparent adult zebrafish in which we transplanted either hematopoietic stem/progenitor cells or tumor cells. In a hematopoiesis radiation recovery assay, transplantation of GFP-labeled marrow cells allowed for striking in vivo visual assessment of engraftment from 2 hr-5 weeks posttransplant. Using FACS analysis, both transparent and wild-type fish had equal engraftment, but this could only be visualized in the transparent recipient. In a tumor engraftment model, transplantation of RAS-melanoma cells allowed for visualization of tumor engraftment, proliferation, and distant metastases in as little as 5 days, which is not seen in wild-type recipients until 3 to 4 weeks. This transparent adult zebrafish serves as the ideal combination of both sensitivity and resolution for in vivo stem cell analyses. -
Galloway JL, Wingert RA, Thisse C, Thisse B, Zon LI. 2008. Combinatorial regulation of novel erythroid gene expression in zebrafish. Experimental hematology. 36(4):424-32. Pubmed: 18243489 DOI:10.1016/j.exphem.2007.11.015 Galloway JL, Wingert RA, Thisse C, Thisse B, Zon LI. 2008. Combinatorial regulation of novel erythroid gene expression in zebrafish. Experimental hematology. 36(4):424-32. Pubmed: 18243489 DOI:10.1016/j.exphem.2007.11.015 Array -
Lengerke C, Schmitt S, Bowman TV, Jang IH, Maouche-Chretien L, McKinney-Freeman S, Davidson AJ, Hammerschmidt M, Rentzsch F, Green JB, Zon LI, Daley GQ. 2008. BMP and Wnt specify hematopoietic fate by activation of the Cdx-Hox pathway. Cell stem cell. 2(1):72-82. Pubmed: 18371423 DOI:10.1016/j.stem.2007.10.022 Lengerke C, Schmitt S, Bowman TV, Jang IH, Maouche-Chretien L, McKinney-Freeman S, Davidson AJ, Hammerschmidt M, Rentzsch F, Green JB, Zon LI, Daley GQ. 2008. BMP and Wnt specify hematopoietic fate by activation of the Cdx-Hox pathway. Cell stem cell. 2(1):72-82. Pubmed: 18371423 DOI:10.1016/j.stem.2007.10.022 The formation of blood in the embryo is dependent on bone morphogenetic protein (BMP), but how BMP signaling intersects with other regulators of hematopoietic development is unclear. Using embryonic stem (ES) cells, we show that BMP4 first induces ventral-posterior (V-P) mesoderm and subsequently directs mesodermal cells toward blood fate by activating Wnt3a and upregulating Cdx and Hox genes. When BMP signaling is blocked during this latter phase, enforced expression of either Cdx1 or Cdx4 rescues hematopoietic development, thereby placing BMP4 signaling upstream of the Cdx-Hox pathway. Wnt signaling cooperates in BMP-induced hemogenesis, and the Wnt effector LEF1 mediates BMP4 activation of Cdx genes. Our data suggest that BMP signaling plays two distinct and sequential roles during blood formation, initially as an inducer of mesoderm, and later to specify blood via activation of Wnt signaling and the Cdx-Hox pathway. -
Orkin SH, Zon LI. 2008. SnapShot: hematopoiesis. Cell. 132(4):712. Pubmed: 18295585 DOI:10.1016/j.cell.2008.02.013 Orkin SH, Zon LI. 2008. SnapShot: hematopoiesis. Cell. 132(4):712. Pubmed: 18295585 DOI:10.1016/j.cell.2008.02.013 -
Burket CT, Montgomery JE, Thummel R, Kassen SC, LaFave MC, Langenau DM, Zon LI, Hyde DR. 2008. Generation and characterization of transgenic zebrafish lines using different ubiquitous promoters. Transgenic research. 17(2):265-79. Pubmed: 17968670 Burket CT, Montgomery JE, Thummel R, Kassen SC, LaFave MC, Langenau DM, Zon LI, Hyde DR. 2008. Generation and characterization of transgenic zebrafish lines using different ubiquitous promoters. Transgenic research. 17(2):265-79. Pubmed: 17968670 Two commonly used promoters to ubiquitously express transgenes in zebrafish are the Xenopus laevis elongation factor 1 alpha promoter (XlEef1a1) and the zebrafish histone variant H2A.F/Z (h2afv) promoter. Recently, transgenes utilizing these promoters were shown to be silenced in certain adult tissues, particularly the central nervous system. To overcome this limitation, we cloned the promoters of four zebrafish genes that likely are transcribed ubiquitously throughout development and into the adult. These four genes are the TATA box binding protein gene, the taube nuss-like gene, the eukaryotic elongation factor 1-gamma gene, and the beta-actin-1 gene. We PCR amplified approximately 2.5 kb upstream of the putative translational start site of each gene and cloned each into a Tol2 expression vector that contains the EGFP reporter transgene. We used these four Tol2 vectors to independently generate stable transgenic fish lines for analysis of transgene expression during development and in the adult. We demonstrated that all four promoters drive a very broad pattern of EGFP expression throughout development and the adult. Using the retina as a well-characterized component of the CNS, all four promoters appeared to drive EGFP expression in all neuronal and non-neuronal cells of the adult retina. In contrast, the h2afv promoter failed to express EGFP in the adult retina. When we examined EGFP expression in the various cells of the blood cell lineage, we observed that all four promoters exhibited a more heterogenous expression pattern than either the XlEef1a1 or h2afv promoters. While these four ubiquitous promoters did not express EGFP in all the adult blood cells, they did express EGFP throughout the CNS and in broader expression patterns in the adult than either the XlEef1a1 or h2afv promoters. For these reasons, these four promoters will be valuable tools for expressing transgenes in adult zebrafish. -
Flores MV, Hall CJ, Davidson AJ, Singh PP, Mahagaonkar AA, Zon LI, Crosier KE, Crosier PS. 2008. Intestinal differentiation in zebrafish requires Cdx1b, a functional equivalent of mammalian Cdx2. Gastroenterology. 135(5):1665-75. Pubmed: 18804112 DOI:10.1053/j.gastro.2008.07.024 Flores MV, Hall CJ, Davidson AJ, Singh PP, Mahagaonkar AA, Zon LI, Crosier KE, Crosier PS. 2008. Intestinal differentiation in zebrafish requires Cdx1b, a functional equivalent of mammalian Cdx2. Gastroenterology. 135(5):1665-75. Pubmed: 18804112 DOI:10.1053/j.gastro.2008.07.024 Array 2007
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Zon LI. 2007. Cell stem cell. Cell stem cell. 1(1):7. Pubmed: 18371324 DOI:S1934-5909(07)00015-X Zon LI. 2007. Cell stem cell. Cell stem cell. 1(1):7. Pubmed: 18371324 DOI:S1934-5909(07)00015-X -
Lensch MW, Schlaeger TM, Zon LI, Daley GQ. 2007. Teratoma formation assays with human embryonic stem cells: a rationale for one type of human-animal chimera. Cell stem cell. 1(3):253-8. Pubmed: 18371359 DOI:10.1016/j.stem.2007.07.019 Lensch MW, Schlaeger TM, Zon LI, Daley GQ. 2007. Teratoma formation assays with human embryonic stem cells: a rationale for one type of human-animal chimera. Cell stem cell. 1(3):253-8. Pubmed: 18371359 DOI:10.1016/j.stem.2007.07.019 Despite a long and valuable history, human-animal chimera research has often been questioned. Among the moral issues raised by chimeras is the concept that integration of human cells into anatomical locations such as the brain might endow animals with "human-like" capacities including self-awareness. We present a justification for one type of human-animal chimera experiment: the evaluation of hES cell developmental potency via teratoma formation in immunodeficient mice. We argue that this experiment raises no significant moral concerns and should be the jurisdiction of animal care and use committees and exempt from formal review by the stem cell research oversight process. -
Langenau DM, Keefe MD, Storer NY, Guyon JR, Kutok JL, Le X, Goessling W, Neuberg DS, Kunkel LM, Zon LI. 2007. Effects of RAS on the genesis of embryonal rhabdomyosarcoma. Genes & development. 21(11):1382-95. Pubmed: 17510286 Langenau DM, Keefe MD, Storer NY, Guyon JR, Kutok JL, Le X, Goessling W, Neuberg DS, Kunkel LM, Zon LI. 2007. Effects of RAS on the genesis of embryonal rhabdomyosarcoma. Genes & development. 21(11):1382-95. Pubmed: 17510286 Embryonal rhabdomyosarcoma (ERMS) is a devastating cancer with specific features of muscle differentiation that can result from mutational activation of RAS family members. However, to date, RAS pathway activation has not been reported in a majority of ERMS patients. Here, we have created a zebrafish model of RAS-induced ERMS, in which animals develop externally visible tumors by 10 d of life. Microarray analysis and cross-species comparisons identified two conserved gene signatures found in both zebrafish and human ERMS, one associated with tumor-specific and tissue-restricted gene expression in rhabdomyosarcoma and a second comprising a novel RAS-induced gene signature. Remarkably, our analysis uncovered that RAS pathway activation is exceedingly common in human RMS. We also created a new transgenic coinjection methodology to fluorescently label distinct subpopulations of tumor cells based on muscle differentiation status. In conjunction with fluorescent activated cell sorting, cell transplantation, and limiting dilution analysis, we were able to identify the cancer stem cell in zebrafish ERMS. When coupled with gene expression studies of this cell population, we propose that the zebrafish RMS cancer stem cell shares similar self-renewal programs as those found in activated satellite cells. -
Trede NS, Medenbach J, Damianov A, Hung LH, Weber GJ, Paw BH, Zhou Y, Hersey C, Zapata A, Keefe M, Barut BA, Stuart AB, Katz T, Amemiya CT, Zon LI, Bindereif A. 2007. Network of coregulated spliceosome components revealed by zebrafish mutant in recycling factor p110. Proceedings of the National Academy of Sciences of the United States of America. 104(16):6608-13. Pubmed: 17416673 Trede NS, Medenbach J, Damianov A, Hung LH, Weber GJ, Paw BH, Zhou Y, Hersey C, Zapata A, Keefe M, Barut BA, Stuart AB, Katz T, Amemiya CT, Zon LI, Bindereif A. 2007. Network of coregulated spliceosome components revealed by zebrafish mutant in recycling factor p110. Proceedings of the National Academy of Sciences of the United States of America. 104(16):6608-13. Pubmed: 17416673 The spliceosome cycle consists of assembly, catalysis, and recycling phases. Recycling of postspliceosomal U4 and U6 small nuclear ribonucleoproteins (snRNPs) requires p110/SART3, a general splicing factor. In this article, we report that the zebrafish earl grey (egy) mutation maps in the p110 gene and results in a phenotype characterized by thymus hypoplasia, other organ-specific defects, and death by 7 to 8 days postfertilization. U4/U6 snRNPs were disrupted in egy mutant embryos, demonstrating the importance of p110 for U4/U6 snRNP recycling in vivo. Surprisingly, expression profiling of the egy mutant revealed an extensive network of coordinately up-regulated components of the spliceosome cycle, providing a mechanism compensating for the recycling defect. Together, our data demonstrate that a mutation in a general splicing factor can lead to distinct defects in organ development and cause disease. -
Steffen LS, Guyon JR, Vogel ED, Beltre R, Pusack TJ, Zhou Y, Zon LI, Kunkel LM. 2007. Zebrafish orthologs of human muscular dystrophy genes. BMC genomics. 8:79. Pubmed: 17374169 Steffen LS, Guyon JR, Vogel ED, Beltre R, Pusack TJ, Zhou Y, Zon LI, Kunkel LM. 2007. Zebrafish orthologs of human muscular dystrophy genes. BMC genomics. 8:79. Pubmed: 17374169 Array -
Peng CY, Yajima H, Burns CE, Zon LI, Sisodia SS, Pfaff SL, Sharma K. 2007. Notch and MAML signaling drives Scl-dependent interneuron diversity in the spinal cord. Neuron. 53(6):813-27. Pubmed: 17359917 Peng CY, Yajima H, Burns CE, Zon LI, Sisodia SS, Pfaff SL, Sharma K. 2007. Notch and MAML signaling drives Scl-dependent interneuron diversity in the spinal cord. Neuron. 53(6):813-27. Pubmed: 17359917 The ventral spinal cord generates multiple inhibitory and excitatory interneuron subtypes from four cardinal progenitor domains (p0, p1, p2, p3). Here we show that cell-cell interactions mediated by the Notch receptor play a critical evolutionarily conserved role in the generation of excitatory v2aIN and inhibitory v2bIN interneurons. Lineage-tracing experiments show that the v2aIN and v2bIN develop from genetically identical p2 progenitors. The p2 daughter cell fate is controlled by Delta4 activation of Notch receptors together with MAML factors. Cells receiving Notch signals activate a transcription factor code that specifies the v2bIN fate, whereas cells deprived of Notch signaling express another code for v2aIN formation. Thus, our study provides insight into the cell-extrinsic signaling that controls combinatorial transcription factor profiles involved in regulating the process of interneuron subtype diversification. -
LeBlanc J, Bowman TV, Zon L. 2007. Transplantation of whole kidney marrow in adult zebrafish. Journal of visualized experiments : JoVE. Pubmed: 18830422 DOI:10.3791/159 LeBlanc J, Bowman TV, Zon L. 2007. Transplantation of whole kidney marrow in adult zebrafish. Journal of visualized experiments : JoVE. Pubmed: 18830422 DOI:10.3791/159 Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (<3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology. -
Shepard JL, Amatruda JF, Finkelstein D, Ziai J, Finley KR, Stern HM, Chiang K, Hersey C, Barut B, Freeman JL, Lee C, Glickman JN, Kutok JL, Aster JC, Zon LI. 2007. A mutation in separase causes genome instability and increased susceptibility to epithelial cancer. Genes & development. 21(1):55-9. Pubmed: 17210788 Shepard JL, Amatruda JF, Finkelstein D, Ziai J, Finley KR, Stern HM, Chiang K, Hersey C, Barut B, Freeman JL, Lee C, Glickman JN, Kutok JL, Aster JC, Zon LI. 2007. A mutation in separase causes genome instability and increased susceptibility to epithelial cancer. Genes & development. 21(1):55-9. Pubmed: 17210788 Proper chromosome segregation is essential for maintenance of genomic integrity and instability resulting from failure of this process may contribute to cancer. Here, we demonstrate that a mutation in the mitotic regulator separase is responsible for the cell cycle defects seen in the zebrafish mutant, cease&desist (cds). Analysis of cds homozygous mutant embryos reveals high levels of polyploidy and aneuploidy, spindle defects, and a mitotic exit delay. Carcinogenesis studies demonstrated that cds heterozygous adults have a shift in tumor spectrum with an eightfold increase in the percentage of fish bearing epithelial tumors, indicating that separase is a tumor suppressor gene in vertebrates. These data strongly support a conserved cross-species role for mitotic checkpoint genes in genetic stability and epithelial carcinogenesis. -
Le X, Langenau DM, Keefe MD, Kutok JL, Neuberg DS, Zon LI. 2007. Heat shock-inducible Cre/Lox approaches to induce diverse types of tumors and hyperplasia in transgenic zebrafish. Proceedings of the National Academy of Sciences of the United States of America. 104(22):9410-5. Pubmed: 17517602 Le X, Langenau DM, Keefe MD, Kutok JL, Neuberg DS, Zon LI. 2007. Heat shock-inducible Cre/Lox approaches to induce diverse types of tumors and hyperplasia in transgenic zebrafish. Proceedings of the National Academy of Sciences of the United States of America. 104(22):9410-5. Pubmed: 17517602 RAS family members are among the most frequently mutated oncogenes in human cancers. Given the utility of zebrafish in both chemical and genetic screens, developing RAS-induced cancer models will make large-scale screens possible to understand further the molecular mechanisms underlying malignancy. We developed a heat shock-inducible Cre/Lox-mediated transgenic approach in which activated human kRASG12D can be conditionally induced within transgenic animals by heat shock treatment. Specifically, double transgenic fish Tg(B-actin-LoxP-EGFP-LoxP-kRASG12D; hsp70-Cre) developed four types of tumors and hyperplasia after heat shock of whole zebrafish embryos, including rhabdomyosarcoma, myeloproliferative disorder, intestinal hyperplasia, and malignant peripheral nerve sheath tumor. Using ex vivo heat shock and transplantation of whole kidney marrow cells from double transgenic animals, we were able to generate specifically kRASG12D-induced myeloproliferative disorder in recipient fish. This heat shock-inducible recombination approach allowed for the generation of multiple types of RAS-induced tumors and hyperplasia without characterizing tissue-specific promoters. Moreover, these tumors and hyperplasia closely resemble human diseases at both the morphologic and molecular levels. -
Bahary N, Goishi K, Stuckenholz C, Weber G, Leblanc J, Schafer CA, Berman SS, Klagsbrun M, Zon LI. 2007. Duplicate VegfA genes and orthologues of the KDR receptor tyrosine kinase family mediate vascular development in the zebrafish. Blood. 110(10):3627-36. Pubmed: 17698971 Bahary N, Goishi K, Stuckenholz C, Weber G, Leblanc J, Schafer CA, Berman SS, Klagsbrun M, Zon LI. 2007. Duplicate VegfA genes and orthologues of the KDR receptor tyrosine kinase family mediate vascular development in the zebrafish. Blood. 110(10):3627-36. Pubmed: 17698971 Vascular endothelial growth factor A (VEGFA) and the type III receptor tyrosine kinase receptors (RTKs) are both required for the differentiation of endothelial cells (vasculogenesis) and for the sprouting of new capillaries (angiogenesis). We have isolated a duplicated zebrafish VegfA locus, termed VegfAb, and a duplicate RTK locus with homology to KDR/FLK1 (named Kdrb). Morpholino-disrupted VegfAb embryos develop a normal circulatory system until approximately 2 to 3 days after fertilization (dpf), when defects in angiogenesis permit blood to extravasate into many tissues. Unlike the VegfAa(121) and VegfAa(165) isoforms, the VegfAb isoforms VegfAb(171) and VegfAb(210) are not normally secreted when expressed in mammalian tissue culture cells. The Kdrb locus encodes a 1361-amino acid transmembrane receptor with strong homology to mammalian KDR. Combined knockdown of both RTKs leads to defects in vascular development, suggesting that they cooperate in mediating the vascular effects of VegfA in zebrafish development. Both VegfAa and VegfAb can individually bind and promote phosphorylation of both Flk1 (Kdra) and Kdrb proteins in vitro. Taken together, our data support a model in the zebrafish, in which duplicated VegfA and multiple type III RTKs mediate vascular development. -
Hultman KA, Bahary N, Zon LI, Johnson SL. 2007. Gene Duplication of the zebrafish kit ligand and partitioning of melanocyte development functions to kit ligand a. PLoS genetics. 3(1):e17. Pubmed: 17257055 DOI:e17 Hultman KA, Bahary N, Zon LI, Johnson SL. 2007. Gene Duplication of the zebrafish kit ligand and partitioning of melanocyte development functions to kit ligand a. PLoS genetics. 3(1):e17. Pubmed: 17257055 DOI:e17 The retention of particular genes after the whole genome duplication in zebrafish has given insights into how genes may evolve through partitioning of ancestral functions. We examine the partitioning of expression patterns and functions of two zebrafish kit ligands, kit ligand a (kitla) and kit ligand b (kitlb), and discuss their possible coevolution with the duplicated zebrafish kit receptors (kita and kitb). In situ hybridizations show that kitla mRNA is expressed in the trunk adjacent to the notochord in the middle of each somite during stages of melanocyte migration and later expressed in the skin, when the receptor is required for melanocyte survival. kitla is also expressed in other regions complementary to kita receptor expression, including the pineal gland, tail bud, and ear. In contrast, kitlb mRNA is expressed in brain ventricles, ear, and cardinal vein plexus, in regions generally not complementary to either zebrafish kit receptor ortholog. However, like kitla, kitlb is expressed in the skin during stages consistent with melanocyte survival. Thus, it appears that kita and kitla have maintained congruent expression patterns, while kitb and kitlb have evolved divergent expression patterns. We demonstrate the interaction of kita and kitla by morpholino knockdown analysis. kitla morphants, but not kitlb morphants, phenocopy the null allele of kita, with defects for both melanocyte migration and survival. Furthermore, kitla morpholino, but not kitlb morpholino, interacts genetically with a sensitized allele of kita, confirming that kitla is the functional ligand to kita. Last, we examine kitla overexpression in embryos, which results in hyperpigmentation caused by an increase in the number and size of melanocytes. This hyperpigmentation is dependent on kita function. We conclude that following genome duplication, kita and kitla have maintained their receptor-ligand relationship, coevolved complementary expression patterns, and that functional analysis reveals that most or all of the kita receptor's function in the embryo are promoted by its interaction with kitla. -
Steffen LS, Guyon JR, Vogel ED, Howell MH, Zhou Y, Weber GJ, Zon LI, Kunkel LM. 2007. The zebrafish runzel muscular dystrophy is linked to the titin gene. Developmental biology. 309(2):180-92. Pubmed: 17678642 Steffen LS, Guyon JR, Vogel ED, Howell MH, Zhou Y, Weber GJ, Zon LI, Kunkel LM. 2007. The zebrafish runzel muscular dystrophy is linked to the titin gene. Developmental biology. 309(2):180-92. Pubmed: 17678642 Titin (also called connectin) acts as a scaffold for signaling proteins in muscle and is responsible for establishing and maintaining the structure and elasticity of sarcomeres in striated muscle. Several human muscular dystrophies and cardiomyopathies have previously been linked to mutations in the titin gene. This study reports linkage of the runzel homozygous lethal muscular dystrophy in the zebrafish Danio rerio to a genomic interval containing the titin gene. Analysis of the genomic sequence suggests that zebrafish contain two adjacent titin loci. One titin locus lies within the genetic linkage interval and its expression is significantly reduced in runzel mutants by both immunofluorescence and protein electrophoresis. Morpholino downregulation of this same titin locus in wild-type embryos results in decreased muscle organization and mobility, phenocopying runzel mutants. Additional protein analysis demonstrates that, in wild-type zebrafish, titin isoform sizes are rapidly altered during the development of striated muscle, likely requiring a previously unrecognized need for vertebrate sarcomere remodeling to incorporate developmentally regulated titin isoforms. Decreases of affected titin isoforms in runzel mutants during this time correlate with a progressive loss of sarcomeric organization and suggest that the unaffected titin proteins are capable of sarcomerogenesis but not sarcomere maintenance. In addition, microarray analysis of the ruz transcriptome suggests a novel mechanism of dystrophy pathogenesis, involving mild increases in calpain-3 expression and upregulation of heat shock proteins. These studies should lead to a better understanding of titin's role in normal and diseased muscle. -
North TE, Goessling W, Walkley CR, Lengerke C, Kopani KR, Lord AM, Weber GJ, Bowman TV, Jang IH, Grosser T, Fitzgerald GA, Daley GQ, Orkin SH, Zon LI. 2007. Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature. 447(7147):1007-11. Pubmed: 17581586 North TE, Goessling W, Walkley CR, Lengerke C, Kopani KR, Lord AM, Weber GJ, Bowman TV, Jang IH, Grosser T, Fitzgerald GA, Daley GQ, Orkin SH, Zon LI. 2007. Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature. 447(7147):1007-11. Pubmed: 17581586 Haematopoietic stem cell (HSC) homeostasis is tightly controlled by growth factors, signalling molecules and transcription factors. Definitive HSCs derived during embryogenesis in the aorta-gonad-mesonephros region subsequently colonize fetal and adult haematopoietic organs. To identify new modulators of HSC formation and homeostasis, a panel of biologically active compounds was screened for effects on stem cell induction in the zebrafish aorta-gonad-mesonephros region. Here, we show that chemicals that enhance prostaglandin (PG) E2 synthesis increased HSC numbers, and those that block prostaglandin synthesis decreased stem cell numbers. The cyclooxygenases responsible for PGE2 synthesis were required for HSC formation. A stable derivative of PGE2 improved kidney marrow recovery following irradiation injury in the adult zebrafish. In murine embryonic stem cell differentiation assays, PGE2 caused amplification of multipotent progenitors. Furthermore, ex vivo exposure to stabilized PGE2 enhanced spleen colony forming units at day 12 post transplant and increased the frequency of long-term repopulating HSCs present in murine bone marrow after limiting dilution competitive transplantation. The conserved role for PGE2 in the regulation of vertebrate HSC homeostasis indicates that modulation of the prostaglandin pathway may facilitate expansion of HSC number for therapeutic purposes. -
Paffett-Lugassy N, Hsia N, Fraenkel PG, Paw B, Leshinsky I, Barut B, Bahary N, Caro J, Handin R, Zon LI. 2007. Functional conservation of erythropoietin signaling in zebrafish. Blood. 110(7):2718-26. Pubmed: 17579187 Paffett-Lugassy N, Hsia N, Fraenkel PG, Paw B, Leshinsky I, Barut B, Bahary N, Caro J, Handin R, Zon LI. 2007. Functional conservation of erythropoietin signaling in zebrafish. Blood. 110(7):2718-26. Pubmed: 17579187 Erythropoietin (Epo) and its cognate receptor (EpoR) are required for maintaining adequate levels of circulating erythrocytes during embryogenesis and adulthood. Here, we report the functional characterization of the zebrafish epo and epor genes. The expression of epo and epor was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization, revealing marked parallels between zebrafish and mammalian gene expression patterns. Examination of the hypochromic mutant, weissherbst, and adult hypoxia-treated hearts indicate that zebrafish epo expression is induced by anemia and hypoxia. Overexpression of epo mRNA resulted in severe polycythemia, characterized by a striking increase in the number of cells expressing scl, c-myb, gata1, ikaros, epor, and betae1-globin, suggesting that both the erythroid progenitor and mature erythrocyte compartments respond to epo. Morpholino-mediated knockdown of the epor caused a slight decrease in primitive and complete block of definitive erythropoiesis. Abrogation of STAT5 blocked the erythropoietic expansion by epo mRNA, consistent with a requirement for STAT5 in epo signaling. Together, the characterization of zebrafish epo and epor demonstrates the conservation of an ancient program that ensures proper red blood cell numbers during normal homeostasis and under hypoxic conditions. -
Pfaff KL, Straub CT, Chiang K, Bear DM, Zhou Y, Zon LI. 2007. The zebra fish cassiopeia mutant reveals that SIL is required for mitotic spindle organization. Molecular and cellular biology. 27(16):5887-97. Pubmed: 17576815 Pfaff KL, Straub CT, Chiang K, Bear DM, Zhou Y, Zon LI. 2007. The zebra fish cassiopeia mutant reveals that SIL is required for mitotic spindle organization. Molecular and cellular biology. 27(16):5887-97. Pubmed: 17576815 A critical step in cell division is formation of the mitotic spindle, which is a bipolar array of microtubules that mediates chromosome separation. Here, we report that the SCL-interrupting locus (SIL), a vertebrate-specific cytosolic protein, is necessary for proper mitotic spindle organization in zebrafish and human cells. A homozygous lethal zebrafish mutant, cassiopeia (csp), was identified by a genetic screen for mitotic mutant. csp mutant embryos have an increased mitotic index, have highly disorganized mitotic spindles, and often lack one or both centrosomes. These phenotypes are caused by a loss-of-function mutation in zebrafish sil. To determine if the requirement for SIL in mitotic spindle organization is conserved in mammals, we generated an antibody against human SIL, which revealed that SIL localizes to the poles of the mitotic spindle during metaphase. Furthermore, short hairpin RNA knockdown of SIL in human cells recapitulates the zebrafish csp mitotic spindle defects. These data, taken together, identify SIL as a novel, vertebrate-specific regulator of mitotic spindle assembly. -
Goessling W, North TE, Zon LI. 2007. Ultrasound biomicroscopy permits in vivo characterization of zebrafish liver tumors. Nature methods. 4(7):551-3. Pubmed: 17572681 Goessling W, North TE, Zon LI. 2007. Ultrasound biomicroscopy permits in vivo characterization of zebrafish liver tumors. Nature methods. 4(7):551-3. Pubmed: 17572681 Zebrafish are a valuable vertebrate model to study carcinogenesis, but noninvasive imaging is challenging because adult fish are not transparent. Here we show that tumors could be readily detected in vivo using high-resolution microscopic ultrasound in zebrafish. We successfully obtained tissue perfusion calculations and cellular aspirates, and analyzed tumor progression and response to treatment. Ultrasound biomicroscopy allows longitudinal studies of tumor development and real-time assessment of therapeutic effects in zebrafish. -
Goessling W, North TE, Zon LI. 2007. New waves of discovery: modeling cancer in zebrafish. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 25(17):2473-9. Pubmed: 17557959 Goessling W, North TE, Zon LI. 2007. New waves of discovery: modeling cancer in zebrafish. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 25(17):2473-9. Pubmed: 17557959 -
Ceol CJ, Pellman D, Zon LI. 2007. APC and colon cancer: two hits for one. Nature medicine. 13(11):1286-7. Pubmed: 17987022 Ceol CJ, Pellman D, Zon LI. 2007. APC and colon cancer: two hits for one. Nature medicine. 13(11):1286-7. Pubmed: 17987022 -
Lord AM, North TE, Zon LI. 2007. Prostaglandin E2: making more of your marrow. Cell cycle (Georgetown, Tex.). 6(24):3054-7. Pubmed: 18075310 Lord AM, North TE, Zon LI. 2007. Prostaglandin E2: making more of your marrow. Cell cycle (Georgetown, Tex.). 6(24):3054-7. Pubmed: 18075310 We have recently demonstrated through a chemical screen in the zebrafish embryo that prostaglandin E2 (PGE2) is an evolutionarily conserved regulator of hematopoietic stem cell (HSC) number. These results have further been confirmed by in vitro and in vivo studies in the murine model. Bioactive PGE2 derivatives have potential clinical application to accelerate recovery of the hematopoietic system following chemotherapy or irradiation. Ex vivo expansion of HSCs prior to stem cell transplantation may improve reconstitution of hematopoiesis and immune function. This article aims to summarize current knowledge of PGE2-mediated regulation of blood cell homeostasis as well as to discuss the proposed use of PGE2 to expand hematopoietic stem cells for transplantation in the clinical setting. -
Freeman JL, Adeniyi A, Banerjee R, Dallaire S, Maguire SF, Chi J, Ng BL, Zepeda C, Scott CE, Humphray S, Rogers J, Zhou Y, Zon LI, Carter NP, Yang F, Lee C. 2007. Definition of the zebrafish genome using flow cytometry and cytogenetic mapping. BMC genomics. 8:195. Pubmed: 17597531 Freeman JL, Adeniyi A, Banerjee R, Dallaire S, Maguire SF, Chi J, Ng BL, Zepeda C, Scott CE, Humphray S, Rogers J, Zhou Y, Zon LI, Carter NP, Yang F, Lee C. 2007. Definition of the zebrafish genome using flow cytometry and cytogenetic mapping. BMC genomics. 8:195. Pubmed: 17597531 Array 2006
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Murphey RD, Stern HM, Straub CT, Zon LI. 2006. A chemical genetic screen for cell cycle inhibitors in zebrafish embryos. Chemical biology & drug design. 68(4):213-9. Pubmed: 17105485 Murphey RD, Stern HM, Straub CT, Zon LI. 2006. A chemical genetic screen for cell cycle inhibitors in zebrafish embryos. Chemical biology & drug design. 68(4):213-9. Pubmed: 17105485 Chemical genetic screening is an effective strategy to identify compounds that alter a specific biological phenotype. As a complement to cell line screens, multicellular organism screens may reveal additional compounds. The zebrafish embryo is ideal for small molecule studies because of its small size and the ease of waterborne treatment. We first examined a broad range of known cell cycle compounds in embryos using the mitotic marker phospho-histone H3. The majority of the known compounds exhibited the predicted cell cycle effect in embryos. To determine whether we could identify novel compounds, we screened a 16 320-compound library for alterations of pH3. This screen revealed 14 compounds that had not been previously identified as having cell cycle activity despite numerous mitotic screens of the same library with mammalian cell lines. With six of the novel compounds, sensitivity was greater in embryos than cell lines, but activity was still detected in cell lines at higher doses. One compound had activity in zebrafish embryos and cell lines but not in mammalian cell lines. The remaining compounds exhibited activity only in embryos. These findings demonstrate that small molecule screens in zebrafish can identify compounds with novel activity and thus may be useful tools for chemical genetics and drug discovery. -
MacDonald BA, Sund M, Grant MA, Pfaff KL, Holthaus K, Zon LI, Kalluri R. 2006. Zebrafish to humans: evolution of the alpha3-chain of type IV collagen and emergence of the autoimmune epitopes associated with Goodpasture syndrome. Blood. 107(5):1908-15. Pubmed: 16254142 MacDonald BA, Sund M, Grant MA, Pfaff KL, Holthaus K, Zon LI, Kalluri R. 2006. Zebrafish to humans: evolution of the alpha3-chain of type IV collagen and emergence of the autoimmune epitopes associated with Goodpasture syndrome. Blood. 107(5):1908-15. Pubmed: 16254142 Goodpasture syndrome is an autoimmune vascular disease associated with kidney and lung failure, with pathogenic circulating autoantibodies targeted to a set of discontinuous epitope sequences within the noncollagenous domain-1 (NC1) of the alpha3 chain of type IV collagen (alpha3(IV)NC1), the Goodpasture autoantigen. We demonstrate that basement membrane extracted NC1 domain preparations from Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio do not bind Goodpasture autoantibodies, while Xenopus laevis, chicken, mouse and human alpha3(IV)NC1 domains bind autoantibodies. The alpha3(IV) chain is not present in C elegans and Drosophila melanogaster, but is first detected in the Danio rerio. Interestingly, native Danio rerio alpha3(IV)NC1 does not bind Goodpasture autoantibodies. Next, we cloned, sequenced, and generated recombinant Danio rerio alpha3(IV)NC1 domain. In contrast to recombinant human alpha3(IV)NC1 domain, there was complete absence of autoantibody binding to recombinant Danio rerio alpha3(IV)NC1. Three-dimensional molecular modeling from existing x-ray coordinates of human NC1 domain suggest that evolutionary alteration of electrostatic charge and polarity due to the emergence of critical serine, aspartic acid, and lysine residues, accompanied by the loss of asparagine and glutamine, contributes to the emergence of the 2 major Goodpasture epitopes on the human alpha3(IV)NC1 domain, as it evolved from the Danio rerio over 450 million years. -
Tang J, Hu G, Hanai J, Yadlapalli G, Lin Y, Zhang B, Galloway J, Bahary N, Sinha S, Thisse B, Thisse C, Jin JP, Zon LI, Sukhatme VP. 2006. A critical role for calponin 2 in vascular development. The Journal of biological chemistry. 281(10):6664-72. Pubmed: 16317011 Tang J, Hu G, Hanai J, Yadlapalli G, Lin Y, Zhang B, Galloway J, Bahary N, Sinha S, Thisse B, Thisse C, Jin JP, Zon LI, Sukhatme VP. 2006. A critical role for calponin 2 in vascular development. The Journal of biological chemistry. 281(10):6664-72. Pubmed: 16317011 Calponin 2 (h2 calponin, CNN2) is an actin-binding protein implicated in cytoskeletal organization. We have found that the expression of calponin 2 is relatively restricted to vasculature from 16 to 30 h post-fertilization during zebrafish (Danio rerio) development. Forty-eight hours after injecting antisense morpholino oligos against calponin 2 into embryos at the 1-4-cell stage, zebrafish demonstrated various cardiovascular defects, including sluggish axial and head circulation, absence of circulation in intersegmental vessels and in the dorsal longitudinal anastomotic vessel, enlarged cerebral ventricles, and pericardial edema, in addition to an excess bending, spiraling tail and twisting of the caudal fin. Knockdown of calponin 2 in the Tg(fli1:EGFP)(y1) zebrafish line (in which a fli1 promoter drives vascular-specific enhanced green fluorescent protein expression) indicated that diminished calponin 2 expression blocked the proper migration of endothelial cells during formation of intersegmental vessels. In vitro studies showed that basic fibroblast growth factor-induced human umbilical vein endothelial cell migration was down-regulated by knockdown of calponin 2 expression using an antisense adenovirus, and overexpression of calponin 2 enhanced migration and hastened wound healing. These events were correlated with activation of mitogen-activated protein kinase; moreover, inhibition of this pathway blocked the promigratory effect of calponin 2. Collectively, these data suggest that calponin 2 plays an important role in the migration of endothelial cells both in vivo and in vitro and that its expression is critical for proper vascular development. -
Hu G, Tang J, Zhang B, Lin Y, Hanai J, Galloway J, Bedell V, Bahary N, Han Z, Ramchandran R, Thisse B, Thisse C, Zon LI, Sukhatme VP. 2006. A novel endothelial-specific heat shock protein HspA12B is required in both zebrafish development and endothelial functions in vitro. Journal of cell science. 119(Pt 19):4117-26. Pubmed: 16968741 Hu G, Tang J, Zhang B, Lin Y, Hanai J, Galloway J, Bedell V, Bahary N, Han Z, Ramchandran R, Thisse B, Thisse C, Zon LI, Sukhatme VP. 2006. A novel endothelial-specific heat shock protein HspA12B is required in both zebrafish development and endothelial functions in vitro. Journal of cell science. 119(Pt 19):4117-26. Pubmed: 16968741 A zebrafish transcript dubbed GA2692 was initially identified via a whole-mount in situ hybridization screen for vessel specific transcripts. Its mRNA expression during embryonic development was detected in ventral hematopoietic and vasculogenic mesoderm and later throughout the vasculature up to 48 hours post fertilization. Morpholino-mediated knockdown of GA2692 in embryos resulted in multiple defects in vasculature, particularly, at sites undergoing active capillary sprouting: the intersegmental vessels, sub-intestinal vessels and the capillary sprouts of the pectoral fin vessel. During the course of these studies, a homology search indicated that GA2692 is the zebrafish orthologue of mammalian HspA12B, a distant member of the heat shock protein 70 (Hsp70) family. By a combination of northern blot and real-time PCR analysis, we showed that HspA12B is highly expressed in human endothelial cells in vitro. Knockdown of HspA12B by small interfering RNAs (siRNAs) in human umbilical vein endothelial cells blocked wound healing, migration and tube formation, whereas overexpression of HspA12B enhanced migration and accelerated wound healing - data that are consistent with the in vivo fish phenotype obtained in the morpholino-knockdown studies. Phosphorylation of Akt was consistently reduced by siRNAs against HspA12B. Overexpression of a constitutively active form of Akt rescued the inhibitory effects of knockdown of HspA12B on migration of human umbilical vein endothelial cells. Collectively, our data suggests that HspA12B is a highly endothelial-cell-specific distant member of the Hsp70 family and plays a significant role in endothelial cells during development and angiogenesis in vitro, partially attributable to modulation of Akt phosphorylation. -
Murphey RD, Zon LI. 2006. Small molecule screening in the zebrafish. Methods (San Diego, Calif.). 39(3):255-61. Pubmed: 16877005 Murphey RD, Zon LI. 2006. Small molecule screening in the zebrafish. Methods (San Diego, Calif.). 39(3):255-61. Pubmed: 16877005 The zebrafish is an ideal organism for small molecule studies. The ability to use the whole organism allows complex in vivo phenotypes to be assayed and combines animal testing with screening. Embryos are easily treatable by waterborne exposure. The small size and abundance of embryos make zebrafish suitable for screening in a high-throughput manner in 96- or 48-well plates. Zebrafish embryos have successfully been used in chemical genetic screens to elucidate biological pathways and find chemical suppressors. Small molecules discovered by screening zebrafish disease models may also be useful as lead compounds for drug development as there appears to be a high level of conservation of drug activity between mammals and zebrafish. Here we provide the technical aspects of treating embryos with small molecules and performing chemical screens with zebrafish. -
Protas ME, Hersey C, Kochanek D, Zhou Y, Wilkens H, Jeffery WR, Zon LI, Borowsky R, Tabin CJ. 2006. Genetic analysis of cavefish reveals molecular convergence in the evolution of albinism. Nature genetics. 38(1):107-11. Pubmed: 16341223 Protas ME, Hersey C, Kochanek D, Zhou Y, Wilkens H, Jeffery WR, Zon LI, Borowsky R, Tabin CJ. 2006. Genetic analysis of cavefish reveals molecular convergence in the evolution of albinism. Nature genetics. 38(1):107-11. Pubmed: 16341223 The genetic basis of vertebrate morphological evolution has traditionally been very difficult to examine in naturally occurring populations. Here we describe the generation of a genome-wide linkage map to allow quantitative trait analysis of evolutionarily derived morphologies in the Mexican cave tetra, a species that has, in a series of independent caves, repeatedly evolved specialized characteristics adapted to a unique and well-studied ecological environment. We focused on the trait of albinism and discovered that it is linked to Oca2, a known pigmentation gene, in two cave populations. We found different deletions in Oca2 in each population and, using a cell-based assay, showed that both cause loss of function of the corresponding protein, OCA2. Thus, the two cave populations evolved albinism independently, through similar mutational events. -
Shaw GC, Cope JJ, Li L, Corson K, Hersey C, Ackermann GE, Gwynn B, Lambert AJ, Wingert RA, Traver D, Trede NS, Barut BA, Zhou Y, Minet E, Donovan A, Brownlie A, Balzan R, Weiss MJ, Peters LL, Kaplan J, Zon LI, Paw BH. 2006. Mitoferrin is essential for erythroid iron assimilation. Nature. 440(7080):96-100. Pubmed: 16511496 Shaw GC, Cope JJ, Li L, Corson K, Hersey C, Ackermann GE, Gwynn B, Lambert AJ, Wingert RA, Traver D, Trede NS, Barut BA, Zhou Y, Minet E, Donovan A, Brownlie A, Balzan R, Weiss MJ, Peters LL, Kaplan J, Zon LI, Paw BH. 2006. Mitoferrin is essential for erythroid iron assimilation. Nature. 440(7080):96-100. Pubmed: 16511496 Iron has a fundamental role in many metabolic processes, including electron transport, deoxyribonucleotide synthesis, oxygen transport and many essential redox reactions involving haemoproteins and Fe-S cluster proteins. Defective iron homeostasis results in either iron deficiency or iron overload. Precise regulation of iron transport in mitochondria is essential for haem biosynthesis, haemoglobin production and Fe-S cluster protein assembly during red cell development. Here we describe a zebrafish mutant, frascati (frs), that shows profound hypochromic anaemia and erythroid maturation arrest owing to defects in mitochondrial iron uptake. Through positional cloning, we show that the gene mutated in the frs mutant is a member of the vertebrate mitochondrial solute carrier family (SLC25) that we call mitoferrin (mfrn). mfrn is highly expressed in fetal and adult haematopoietic tissues of zebrafish and mouse. Erythroblasts generated from murine embryonic stem cells null for Mfrn (also known as Slc25a37) show maturation arrest with severely impaired incorporation of 55Fe into haem. Disruption of the yeast mfrn orthologues, MRS3 and MRS4, causes defects in iron metabolism and mitochondrial Fe-S cluster biogenesis. Murine Mfrn rescues the defects in frs zebrafish, and zebrafish mfrn complements the yeast mutant, indicating that the function of the gene may be highly conserved. Our data show that mfrn functions as the principal mitochondrial iron importer essential for haem biosynthesis in vertebrate erythroblasts. -
Davidson AJ, Zon LI. 2006. The caudal-related homeobox genes cdx1a and cdx4 act redundantly to regulate hox gene expression and the formation of putative hematopoietic stem cells during zebrafish embryogenesis. Developmental biology. 292(2):506-18. Pubmed: 16457800 Davidson AJ, Zon LI. 2006. The caudal-related homeobox genes cdx1a and cdx4 act redundantly to regulate hox gene expression and the formation of putative hematopoietic stem cells during zebrafish embryogenesis. Developmental biology. 292(2):506-18. Pubmed: 16457800 The hox genes play a central role in organogenesis and are implicated in the formation of hematopoietic stem cells (HSCs). The cdx genes encode homeodomain transcription factors that act as master regulators of the hox genes. In zebrafish, mutations in cdx4 cause a severe, but not complete, deficit in embryonic blood cells. Here, we report the expression and function of cdx1a, a zebrafish Cdx1 paralogue. Using morpholino-mediated knockdown of cdx1a in a cdx4 mutant background, we show that a deficiency in both cdx genes causes a severe perturbation of hox gene expression and a complete failure to specify blood. The hematopoietic defect in cdx-deficient embryos does not result from a general block in posterior mesoderm differentiation as endothelial cells and kidney progenitors are still formed in the doubly deficient embryos. In addition, cdx-deficient embryos display a significant reduction in runx1a(+) putative HSCs in the zebrafish equivalent to the aorta-gonad-mesonephros (AGM) region. Overexpressing hoxa9a in cdx-deficient embryos rescues embryonic erythropoiesis in the posterior mesoderm as well as the formation of HSCs in the AGM region. Taken together, these results suggest that the cdx-hox pathway plays an essential role in the formation of both embryonic erythroid cells and definitive HSCs during vertebrate embryogenesis. -
Gupta S, Zhu H, Zon LI, Evans T. 2006. BMP signaling restricts hemato-vascular development from lateral mesoderm during somitogenesis. Development (Cambridge, England). 133(11):2177-87. Pubmed: 16672337 Gupta S, Zhu H, Zon LI, Evans T. 2006. BMP signaling restricts hemato-vascular development from lateral mesoderm during somitogenesis. Development (Cambridge, England). 133(11):2177-87. Pubmed: 16672337 The bone morphogenetic protein (BMP) signaling pathway is essential during gastrulation for the generation of ventral mesoderm, which makes it a challenge to define functions for this pathway at later stages of development. We have established an approach to disrupt BMP signaling specifically in lateral mesoderm during somitogenesis, by targeting a dominant-negative BMP receptor to Lmo2+ cells in developing zebrafish embryos. This results in expansion of hematopoietic and endothelial cells, while restricting the expression domain of the pronephric marker pax2.1. Expression of a constitutively active receptor and transplantation experiments were used to confirm that BMP signaling in lateral mesoderm restricts subsequent hemato-vascular development. The results show that the BMP signaling pathway continues to function after cells are committed to a lateral mesoderm fate, and influences subsequent lineage decisions by restricting hemato-vascular fate in favor of pronephric development. -
DiBiase A, Harte RA, Zhou Y, Zon L, Kent WJ. 2006. Piloting the zebrafish genome browser. Developmental dynamics : an official publication of the American Association of Anatomists. 235(3):747-53. Pubmed: 16372332 DiBiase A, Harte RA, Zhou Y, Zon L, Kent WJ. 2006. Piloting the zebrafish genome browser. Developmental dynamics : an official publication of the American Association of Anatomists. 235(3):747-53. Pubmed: 16372332 This correspondence is a primer for the zebrafish research community on zebrafish tracks available in the UCSC Genome Browser at http://genome.ucsc.edu based on Sanger's Zv4 assembly. A primary capability of this facility is comparative informatics between humans (as well as many other model organisms) and zebrafish. The zebrafish genome sequencing project has played important roles in mutant mapping and cloning, and comparative genomic research projects. This easy-to-use genome browser aims to display and download useful genome sequence information for zebrafish mutant mapping and cloning projects. Its user-friendly interface expedites annotation of the zebrafish genome sequence. -
Burns CE, Zon LI. 2006. Homing sweet homing: odyssey of hematopoietic stem cells. Immunity. 25(6):859-62. Pubmed: 17174927 Burns CE, Zon LI. 2006. Homing sweet homing: odyssey of hematopoietic stem cells. Immunity. 25(6):859-62. Pubmed: 17174927 The lineage relationship between the blood cells found in the developmentally successive hematopoietic organs has remained elusive. In this issue of Immunity, Murayama et al. (2006) track the migration of nascent hematopoietic stem cells in zebrafish from their site of origin to a newly described intermediate location. -
Goishi K, Shimizu A, Najarro G, Watanabe S, Rogers R, Zon LI, Klagsbrun M. 2006. AlphaA-crystallin expression prevents gamma-crystallin insolubility and cataract formation in the zebrafish cloche mutant lens. Development (Cambridge, England). 133(13):2585-93. Pubmed: 16728471 Goishi K, Shimizu A, Najarro G, Watanabe S, Rogers R, Zon LI, Klagsbrun M. 2006. AlphaA-crystallin expression prevents gamma-crystallin insolubility and cataract formation in the zebrafish cloche mutant lens. Development (Cambridge, England). 133(13):2585-93. Pubmed: 16728471 Cataracts, the loss of lens transparency, are the leading cause of human blindness. The zebrafish embryo, with its transparency and relatively large eyes, is an excellent model for studying ocular disease in vivo. We found that the zebrafish cloche mutant, both the cloche(m39) and cloche(S5) alleles, which have defects in hematopoiesis and blood vessel development, also have lens cataracts. Quantitative examination of the living zebrafish lens by confocal microscopy showed significant increases in lens reflectance. Histological analysis revealed retention of lens fiber cell nuclei owing to impeded terminal differentiation. Proteomics identified gamma-crystallin as a protein that was substantially diminished in cloche mutants. Crystallins are the major structural proteins in mouse, human and zebrafish lens. Defects in crystallins have previously been shown in mice and humans to contribute to cataracts. The loss of gamma-crystallin protein in cloche was not due to lowered mRNA levels but rather to gamma-crystallin protein insolubility. AlphaA-crystallin is a chaperone that protects proteins from misfolding and becoming insoluble. The cloche lens is deficient in both alphaA-crystallin mRNA and protein during development from 2-5 dpf. Overexpression of exogenous alphaA-crystallin rescued the cloche lens phenotype, including solubilization of gamma-crystallin, increased lens transparency and induction of lens fiber cell differentiation. Taken together, these results indicate that alphaA-crystallin expression is required for normal lens development and demonstrate that cataract formation can be prevented in vivo. In addition, these results show that proteomics is a valuable tool for detecting protein alterations in zebrafish. -
Bansal D, Scholl C, Fröhling S, McDowell E, Lee BH, Döhner K, Ernst P, Davidson AJ, Daley GQ, Zon LI, Gilliland DG, Huntly BJ. 2006. Cdx4 dysregulates Hox gene expression and generates acute myeloid leukemia alone and in cooperation with Meis1a in a murine model. Proceedings of the National Academy of Sciences of the United States of America. 103(45):16924-9. Pubmed: 17068127 Bansal D, Scholl C, Fröhling S, McDowell E, Lee BH, Döhner K, Ernst P, Davidson AJ, Daley GQ, Zon LI, Gilliland DG, Huntly BJ. 2006. Cdx4 dysregulates Hox gene expression and generates acute myeloid leukemia alone and in cooperation with Meis1a in a murine model. Proceedings of the National Academy of Sciences of the United States of America. 103(45):16924-9. Pubmed: 17068127 HOX genes have emerged as critical effectors of leukemogenesis, but the mechanisms that regulate their expression in leukemia are not well understood. Recent data suggest that the caudal homeobox transcription factors CDX1, CDX2, and CDX4, developmental regulators of HOX gene expression, may contribute to HOX gene dysregulation in leukemia. We report here that CDX4 is expressed normally in early hematopoietic progenitors and is expressed aberrantly in approximately 25% of acute myeloid leukemia (AML) patient samples. Cdx4 regulates Hox gene expression in the adult murine hematopoietic system and dysregulates Hox genes that are implicated in leukemogenesis. Furthermore, bone marrow progenitors that are retrovirally engineered to express Cdx4 serially replate in methylcellulose cultures, grow in liquid culture, and generate a partially penetrant, long-latency AML in bone marrow transplant recipients. Coexpression of the Hox cofactor Meis1a accelerates the Cdx4 AML phenotype and renders it fully penetrant. Structure-function analysis demonstrates that leukemic transformation requires intact Cdx4 transactivation and DNA-binding domains but not the putative Pbx cofactor interaction motif. Together, these data indicate that Cdx4 regulates Hox gene expression in adult hematopoiesis and may serve as an upstream regulator of Hox gene expression in the induction of acute leukemia. Inasmuch as many human leukemias show dysregulated expression of a spectrum of HOX family members, these collective findings also suggest a central role for CDX4 expression in the genesis of acute leukemia. 2005
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Zhu H, Traver D, Davidson AJ, Dibiase A, Thisse C, Thisse B, Nimer S, Zon LI. 2005. Regulation of the lmo2 promoter during hematopoietic and vascular development in zebrafish. Developmental biology. 281(2):256-69. Pubmed: 15893977 Zhu H, Traver D, Davidson AJ, Dibiase A, Thisse C, Thisse B, Nimer S, Zon LI. 2005. Regulation of the lmo2 promoter during hematopoietic and vascular development in zebrafish. Developmental biology. 281(2):256-69. Pubmed: 15893977 The Lmo2 transcription factor, a T-cell oncoprotein, is required for both hematopoiesis and angiogenesis. To investigate the fate of lmo2-expressing cells and the transcriptional regulation of lmo2 in vivo, we generated stable transgenic zebrafish that express green fluorescent protein (EGFP) or DsRed under the control of an lmo2 promoter. A 2.5-kb fragment contains the cis-regulatory elements required to recapitulate endogenous lmo2 expression in embryonic hematopoietic and vascular tissues. We further characterized embryonic Lmo2+ cells through transplantation into vlad tepes (vlt), an erythropoietic mutant. These Lmo2+ primitive wave donor cells differentiated into circulating hematopoietic cells and extended the life span of vlt recipients, but did not demonstrate long-term repopulation of the erythroid lineage. Promoter analysis identified a 174-bp proximal promoter that was sufficient to recapitulate lmo2 expression. This element contains critical ETS-binding sites conserved between zebrafish and pufferfish. Furthermore, we show that ets1 is coexpressed with lmo2, and overexpression experiments indicate that ets1 can activate the lmo2 promoter through this element. Our studies elucidate the transcriptional regulation of this key transcription factor, and provide a transgenic system for the functional analysis of blood and blood vessels in zebrafish. -
Galloway JL, Wingert RA, Thisse C, Thisse B, Zon LI. 2005. Loss of gata1 but not gata2 converts erythropoiesis to myelopoiesis in zebrafish embryos. Developmental cell. 8(1):109-16. Pubmed: 15621534 Galloway JL, Wingert RA, Thisse C, Thisse B, Zon LI. 2005. Loss of gata1 but not gata2 converts erythropoiesis to myelopoiesis in zebrafish embryos. Developmental cell. 8(1):109-16. Pubmed: 15621534 The differentiation of hematopoietic progenitors into erythroid or myeloid cell lineages is thought to depend upon relative levels of the transcription factors gata1 and pu.1. While loss-of-function analysis shows that gata1 is necessary for terminal erythroid differentiation, no study has demonstrated that loss of gata1 alters myeloid differentiation during ontogeny. Here we provide in vivo evidence that loss of Gata1, but not Gata2, transforms primitive blood precursors into myeloid cells, resulting in a massive expansion of granulocytic neutrophils and macrophages at the expense of red blood cells. In addition to this fate change, expression of many erythroid genes was found to be differentially dependent on Gata1 alone, on both Gata1 and Gata2, or independent of both Gata factors, suggesting that multiple pathways regulate erythroid gene expression. Our studies establish a transcriptional hierarchy of Gata factor dependence during hematopoiesis and demonstrate that gata1 plays an integral role in directing myelo-erythroid lineage fate decisions during embryogenesis. -
de Jong JL, Zon LI. 2005. Use of the zebrafish system to study primitive and definitive hematopoiesis. Annual review of genetics. 39:481-501. Pubmed: 16285869 de Jong JL, Zon LI. 2005. Use of the zebrafish system to study primitive and definitive hematopoiesis. Annual review of genetics. 39:481-501. Pubmed: 16285869 The zebrafish (Danio rerio) has emerged as an ideal organism for the study of hematopoiesis, the process by which all the cellular elements of the blood are formed. These elements, including erythrocytes, granulocytes, monocytes, lymphocytes, and thrombocytes, are formed through complex genetic signaling pathways that are highly conserved throughout phylogeny. Large-scale forward genetic screens have identified numerous blood mutants in zebrafish, helping to elucidate specific signaling pathways important for hematopoietic stem cells (HSCs) and the various committed blood cell lineages. Here we review both primitive and definitive hematopoiesis in zebrafish, discuss various genetic methods available in the zebrafish model for studying hematopoiesis, and describe some of the zebrafish blood mutants identified to date, many of which have known human disease counterparts. -
Langenau DM, Zon LI. 2005. The zebrafish: a new model of T-cell and thymic development. Nature reviews. Immunology. 5(4):307-17. Pubmed: 15803150 Langenau DM, Zon LI. 2005. The zebrafish: a new model of T-cell and thymic development. Nature reviews. Immunology. 5(4):307-17. Pubmed: 15803150 T-cell and thymic development are processes that have been highly conserved throughout vertebrate evolution. Mammals, birds, reptiles and fish share common molecular signalling pathways that regulate the development of the adaptive immune system. This Review article focuses on defining the similarities and differences between zebrafish and mammalian T-cell immunobiology, and it highlights the advantages of using the zebrafish as a genetic model to uncover mutations that affect T-cell and thymic development. Finally, we summarize the use of the zebrafish as a new model for assessing stem-cell function and for drug discovery. -
Donovan A, Lima CA, Pinkus JL, Pinkus GS, Zon LI, Robine S, Andrews NC. 2005. The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis. Cell metabolism. 1(3):191-200. Pubmed: 16054062 Donovan A, Lima CA, Pinkus JL, Pinkus GS, Zon LI, Robine S, Andrews NC. 2005. The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis. Cell metabolism. 1(3):191-200. Pubmed: 16054062 Ferroportin (SLC40A1) is an iron transporter postulated to play roles in intestinal iron absorption and cellular iron release. Hepcidin, a regulatory peptide, binds to ferroportin and causes it to be internalized and degraded. If ferroportin is the major cellular iron exporter, ineffective hepcidin function could explain manifestations of human hemochromatosis disorders. To investigate this, we inactivated the murine ferroportin (Fpn) gene globally and selectively. Embryonic lethality of Fpn(null/null) animals indicated that ferroportin is essential early in development. Rescue of embryonic lethality through selective inactivation of ferroportin in the embryo proper suggested that ferroportin has an important function in the extraembryonic visceral endoderm. Ferroportin-deficient animals accumulated iron in enterocytes, macrophages, and hepatocytes, consistent with a key role for ferroportin in those cell types. Intestine-specific inactivation of ferroportin confirmed that it is critical for intestinal iron absorption. These observations define the major sites of ferroportin activity and give insight into hemochromatosis. -
Schönberger J, Wang L, Shin JT, Kim SD, Depreux FF, Zhu H, Zon L, Pizard A, Kim JB, Macrae CA, Mungall AJ, Seidman JG, Seidman CE. 2005. Mutation in the transcriptional coactivator EYA4 causes dilated cardiomyopathy and sensorineural hearing loss. Nature genetics. 37(4):418-22. Pubmed: 15735644 Schönberger J, Wang L, Shin JT, Kim SD, Depreux FF, Zhu H, Zon L, Pizard A, Kim JB, Macrae CA, Mungall AJ, Seidman JG, Seidman CE. 2005. Mutation in the transcriptional coactivator EYA4 causes dilated cardiomyopathy and sensorineural hearing loss. Nature genetics. 37(4):418-22. Pubmed: 15735644 We identified a human mutation that causes dilated cardiomyopathy and heart failure preceded by sensorineural hearing loss (SNHL). Unlike previously described mutations causing dilated cardiomyopathy that affect structural proteins, this mutation deletes 4,846 bp of the human transcriptional coactivator gene EYA4. To elucidate the roles of eya4 in heart function, we studied zebrafish embryos injected with antisense morpholino oligonucleotides. Attenuated eya4 transcript levels produced morphologic and hemodynamic features of heart failure. To determine why previously described mutated EYA4 alleles cause SNHL without heart disease, we examined biochemical interactions of mutant Eya4 peptides. Eya4 peptides associated with SNHL, but not the shortened 193-amino acid peptide associated with dilated cardiomyopathy and SNHL, bound wild-type Eya4 and associated with Six proteins. These data define unrecognized and crucial roles for Eya4-Six-mediated transcriptional regulation in normal heart function. -
Patton EE, Widlund HR, Kutok JL, Kopani KR, Amatruda JF, Murphey RD, Berghmans S, Mayhall EA, Traver D, Fletcher CD, Aster JC, Granter SR, Look AT, Lee C, Fisher DE, Zon LI. 2005. BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma. Current biology : CB. 15(3):249-54. Pubmed: 15694309 Patton EE, Widlund HR, Kutok JL, Kopani KR, Amatruda JF, Murphey RD, Berghmans S, Mayhall EA, Traver D, Fletcher CD, Aster JC, Granter SR, Look AT, Lee C, Fisher DE, Zon LI. 2005. BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma. Current biology : CB. 15(3):249-54. Pubmed: 15694309 Melanoma is the most lethal form of skin cancer, and the incidence and mortality rates are rapidly rising. Epidemiologically, high numbers of nevi (moles) are associated with higher risk of melanoma . The majority of melanomas exhibit activating mutations in the serine/threonine kinase BRAF . BRAF mutations may be critical for the initiation of melanoma ; however, the direct role of BRAF in nevi and melanoma has not been tested in an animal model. To directly test the role of activated BRAF in nevus and melanoma development, we have generated transgenic zebrafish expressing the most common BRAF mutant form (V600E) under the control of the melanocyte mitfa promoter. Expression of mutant, but not wild-type, BRAF led to dramatic patches of ectopic melanocytes, which we have termed fish (f)-nevi. Remarkably, in p53-deficient fish, activated BRAF induced formation of melanocyte lesions that rapidly developed into invasive melanomas, which resembled human melanomas and could be serially transplanted. These data provide direct evidence that BRAF activation is sufficient for f-nevus formation, that BRAF activation is among the primary events in melanoma development, and that the p53 and BRAF pathways interact genetically to produce melanoma. -
Dooley KA, Davidson AJ, Zon LI. 2005. Zebrafish scl functions independently in hematopoietic and endothelial development. Developmental biology. 277(2):522-36. Pubmed: 15617691 Dooley KA, Davidson AJ, Zon LI. 2005. Zebrafish scl functions independently in hematopoietic and endothelial development. Developmental biology. 277(2):522-36. Pubmed: 15617691 The SCL transcription factor is critically important for vertebrate hematopoiesis and angiogenesis, and has been postulated to induce hemangioblasts, bipotential precursors for blood and endothelial cells. To investigate the function of scl during zebrafish hematopoietic and endothelial development, we utilized site-directed, anti-sense morpholinos to inhibit scl mRNA. Knockdown of scl resulted in a loss of primitive and definitive hematopoietic cell lineages. However, the expression of early hematopoietic genes, gata2 and lmo2, was unaffected, suggesting that hematopoietic cells were present but unable to further differentiate. Using gene expression analysis and visualization of vessel formation in live animals harboring an lmo2 promoter-green fluorescent protein reporter transgene (Tg(lmo2:EGFP)), we show that angioblasts were specified normally in the absence of scl, but later defects in angiogenesis were evident. While scl was not required for angioblast specification, forced expression of exogenous scl caused an expansion of both hematopoietic and endothelial gene expression, and a loss of somitic tissue. In cloche and spadetail mutants, forced expression of scl resulted in an expansion of hematopoietic but not endothelial tissue. Surprisingly, in cloche, lmo2 was not induced in response to scl over-expression. Taken together, these findings support distinct roles for scl in hematopoietic and endothelial development, downstream of hemangioblast development. -
Berghmans S, Murphey RD, Wienholds E, Neuberg D, Kutok JL, Fletcher CD, Morris JP, Liu TX, Schulte-Merker S, Kanki JP, Plasterk R, Zon LI, Look AT. 2005. tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors. Proceedings of the National Academy of Sciences of the United States of America. 102(2):407-12. Pubmed: 15630097 Berghmans S, Murphey RD, Wienholds E, Neuberg D, Kutok JL, Fletcher CD, Morris JP, Liu TX, Schulte-Merker S, Kanki JP, Plasterk R, Zon LI, Look AT. 2005. tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors. Proceedings of the National Academy of Sciences of the United States of America. 102(2):407-12. Pubmed: 15630097 TP53 is the most frequently mutated tumor suppressor gene in human cancer, with nearly 50% of all tumors exhibiting a loss-of-function mutation. To further elucidate the genetic pathways involving TP53 and cancer, we have exploited the zebrafish, a powerful vertebrate model system that is amenable to whole-genome forward-genetic analysis and synthetic-lethal screens. Zebrafish lines harboring missense mutations in the tp53 DNA-binding domain were identified by using a target-selected mutagenesis strategy. Homozygous mutant fish from two of these lines were viable and exhibited mutations similar to those found in human cancers (tp53(N168K) and tp53(M214K)). Although homozygous tp53(N168K) mutants were temperature-sensitive and suppressed radiation-induced apoptosis only at 37 degrees C, cells in the tp53(M214K) embryos failed to undergo apoptosis in response to gamma radiation at both 28 and 37 degrees C. Unlike wild-type control embryos, irradiated tp53(M214K) embryos also failed to up-regulate p21 and did not arrest at the G(1)/S checkpoint. Beginning at 8.5 months of age, 28% of tp53(M214K) mutant fish developed malignant peripheral nerve sheath tumors. In addition to providing a model for studying the molecular pathogenic pathways of malignant peripheral nerve sheath tumors, these mutant zebrafish lines provide a unique platform for modifier screens to identify genetic mutations or small molecules that affect tp53-related pathways, including apoptosis, cell-cycle delay, and tumor suppression. -
Paffett-Lugassy NN, Zon LI. 2005. Analysis of hematopoietic development in the zebrafish. Methods in molecular medicine. 105:171-98. Pubmed: 15492396 Paffett-Lugassy NN, Zon LI. 2005. Analysis of hematopoietic development in the zebrafish. Methods in molecular medicine. 105:171-98. Pubmed: 15492396 The zebrafish (Danio rerio) has emerged as a powerful vertebrate genetic and developmental model that is particularly amenable to the study of hematopoiesis. The zebrafish embryo develops externally and its optical clarity allows the number and morphology of circulating blood cells to be visualized using a dissecting microscope. Both the morphology of the blood lineages and the expression of critical blood genes are highly conserved between zebrafish and mammals. The high fecundity and short generation time of zebrafish facilitate genetic analysis, and a number of large-scale mutagenesis screens have identified mutations in genes affecting blood development. The discovery of novel hematopoietic genes, as well as the cloning of zebrafish homologs of known hematopoietic genes, necessitates the use of efficacious and reliable methods for complete gene characterization. In this chapter, we illustrate frequently used techniques that are essential for evaluating hematopoiesis in the zebrafish, including whole-mount in situ hybridization, the detection of erythrocytes by o-dianisidine staining, and a description of the microinjection procedure, which has various applications, including overexpression of messenger ribonucleic acid, gene "knockdown" by antisense technology, and the creation of transgenic zebrafish. Also included is an explanation of the use of flow cytometry to separate hematopoietic lineages from the adult kidney and to isolate relatively pure populations of cell types from transgenic embryos based on the expression of fluorescent markers. -
Patton EE, Zon LI. 2005. Taking human cancer genes to the fish: a transgenic model of melanoma in zebrafish. Zebrafish. 1(4):363-8. Pubmed: 18248215 DOI:10.1089/zeb.2005.1.363 Patton EE, Zon LI. 2005. Taking human cancer genes to the fish: a transgenic model of melanoma in zebrafish. Zebrafish. 1(4):363-8. Pubmed: 18248215 DOI:10.1089/zeb.2005.1.363 -
Zon LI, Peterson RT. 2005. In vivo drug discovery in the zebrafish. Nature reviews. Drug discovery. 4(1):35-44. Pubmed: 15688071 Zon LI, Peterson RT. 2005. In vivo drug discovery in the zebrafish. Nature reviews. Drug discovery. 4(1):35-44. Pubmed: 15688071 The zebrafish has become a widely used model organism because of its fecundity, its morphological and physiological similarity to mammals, the existence of many genomic tools and the ease with which large, phenotype-based screens can be performed. Because of these attributes, the zebrafish might also provide opportunities to accelerate the process of drug discovery. By combining the scale and throughput of in vitro screens with the physiological complexity of animal studies, the zebrafish promises to contribute to several aspects of the drug development process, including target identification, disease modelling, lead discovery and toxicology. -
Hsia N, Zon LI. 2005. Transcriptional regulation of hematopoietic stem cell development in zebrafish. Experimental hematology. 33(9):1007-14. Pubmed: 16140148 Hsia N, Zon LI. 2005. Transcriptional regulation of hematopoietic stem cell development in zebrafish. Experimental hematology. 33(9):1007-14. Pubmed: 16140148 The zebrafish (Danio rerio) is a well-established vertebrate model for studying hematopoiesis. The major advantages of this system include robust experimental techniques in both genetics and embryology, which have been utilized to model many aspects of human development and disease. Although much is known about the transcription factors involved in the terminal differentiation of peripheral blood lineages, little is known about the development and maintenance of the hematopoietic stem cell (HSC). This review will focus on the current knowledge of the transcriptional regulation of the HSC in the context of the zebrafish. Future studies using new technologies in the zebrafish model will enhance our understanding of the molecular networks regulating HSC pluripotency and differentiation. -
Wingert RA, Galloway JL, Barut B, Foott H, Fraenkel P, Axe JL, Weber GJ, Dooley K, Davidson AJ, Schmid B, Paw BH, Shaw GC, Kingsley P, Palis J, Schubert H, Chen O, Kaplan J, Zon LI. 2005. Deficiency of glutaredoxin 5 reveals Fe-S clusters are required for vertebrate haem synthesis. Nature. 436(7053):1035-39. Pubmed: 16110529 Wingert RA, Galloway JL, Barut B, Foott H, Fraenkel P, Axe JL, Weber GJ, Dooley K, Davidson AJ, Schmid B, Paw BH, Shaw GC, Kingsley P, Palis J, Schubert H, Chen O, Kaplan J, Zon LI. 2005. Deficiency of glutaredoxin 5 reveals Fe-S clusters are required for vertebrate haem synthesis. Nature. 436(7053):1035-39. Pubmed: 16110529 Iron is required to produce haem and iron-sulphur (Fe-S) clusters, processes thought to occur independently. Here we show that the hypochromic anaemia in shiraz (sir) zebrafish mutants is caused by deficiency of glutaredoxin 5 (grx5), a gene required in yeast for Fe-S cluster assembly. We found that grx5 was expressed in erythroid cells of zebrafish and mice. Zebrafish grx5 rescued the assembly of grx5 yeast Fe-S, showing that the biochemical function of grx5 is evolutionarily conserved. In contrast to yeast, vertebrates use iron regulatory protein 1 (IRP1) to sense intracellular iron and regulate mRNA stability or the translation of iron metabolism genes. We found that loss of Fe-S cluster assembly in sir animals activated IRP1 and blocked haem biosynthesis catalysed by aminolaevulinate synthase 2 (ALAS2). Overexpression of ALAS2 RNA without the 5' iron response element that binds IRP1 rescued sir embryos, whereas overexpression of ALAS2 including the iron response element did not. Further, antisense knockdown of IRP1 restored sir embryo haemoglobin synthesis. These findings uncover a connection between haem biosynthesis and Fe-S clusters, indicating that haemoglobin production in the differentiating red cell is regulated through Fe-S cluster assembly. -
Shmukler BE, Kurschat CE, Ackermann GE, Jiang L, Zhou Y, Barut B, Stuart-Tilley AK, Zhao J, Zon LI, Drummond IA, Vandorpe DH, Paw BH, Alper SL. 2005. Zebrafish slc4a2/ae2 anion exchanger: cDNA cloning, mapping, functional characterization, and localization. American journal of physiology. Renal physiology. 289(4):F835-49. Pubmed: 15914778 Shmukler BE, Kurschat CE, Ackermann GE, Jiang L, Zhou Y, Barut B, Stuart-Tilley AK, Zhao J, Zon LI, Drummond IA, Vandorpe DH, Paw BH, Alper SL. 2005. Zebrafish slc4a2/ae2 anion exchanger: cDNA cloning, mapping, functional characterization, and localization. American journal of physiology. Renal physiology. 289(4):F835-49. Pubmed: 15914778 Although the zebrafish has been used increasingly for the study of pronephric kidney development, studies of renal ion transporters and channels of the zebrafish remain few. We report the cDNA cloning and characterization of the AE2 anion exchanger ortholog from zebrafish kidney, slc4a2/ae2. The ae2 gene in linkage group 2 encodes a polypeptide of 1,228 aa exhibiting 64% aa identity with mouse AE2a. The exon-intron boundaries of the zebrafish ae2 gene are nearly identical to those of the rodent and human genes. Whole-mount in situ hybridization detects ae2 mRNA in prospective midbrain as early as the five-somite stage, then later in the pronephric primordia and the forming pronephric duct, where it persists through 72 h postfertilization (hpf). Zebrafish Ae2 expressed in Xenopus laevis oocytes mediates Na(+)-independent, electroneutral (36)Cl(-)/Cl(-) exchange moderately sensitive to inhibition by DIDS, is inhibited by acidic intracellular pH and by acidic extracellular pH, but activated by (acidifying) ammonium and by hypertonicity. Zebrafish Ae2 also mediates Cl(-)/HCO(3)(-) exchange in X. laevis oocytes and accumulates in or near the plasma membrane in transfected HEK-293 cells. In 24-48 hpf zebrafish embryos, the predominant but not exclusive localization of Ae2 polypeptide is the apical membrane of pronephric duct epithelial cells. Thus Ae2 resembles its mammalian orthologs in function, mechanism, and acute regulation but differs in its preferentially apical expression in kidney. These results will inform tests of the role of Ae2 in zebrafish kidney development and function. -
Fraenkel PG, Traver D, Donovan A, Zahrieh D, Zon LI. 2005. Ferroportin1 is required for normal iron cycling in zebrafish. The Journal of clinical investigation. 115(6):1532-41. Pubmed: 15902304 Fraenkel PG, Traver D, Donovan A, Zahrieh D, Zon LI. 2005. Ferroportin1 is required for normal iron cycling in zebrafish. The Journal of clinical investigation. 115(6):1532-41. Pubmed: 15902304 Missense mutations in ferroportin1 (fpn1), an intestinal and macrophage iron exporter, have been identified between transmembrane helices 3 and 4 in the zebrafish anemia mutant weissherbst (weh(Tp85c-/-)) and in patients with type 4 hemochromatosis. To explore the effects of fpn1 mutation on blood development and iron homeostasis in the adult zebrafish, weh(Tp85c-/-) zebrafish were rescued by injection with iron dextran and studied in comparison with injected and uninjected WT zebrafish and heterozygotes. Although iron deposition was observed in all iron-injected fish, only weh(Tp85c-/-) zebrafish exhibited iron accumulation in the intestinal epithelium compatible with a block in iron export. Iron injections initially reversed the anemia. However, 8 months after iron injections were discontinued, weh(Tp85c-/-) zebrafish developed hypochromic anemia and impaired erythroid maturation despite the persistence of iron-loaded macrophages and elevated hepatic nonheme iron stores. Quantitative real-time RT-PCR revealed a significant decrease in mean hepatic transcript levels of the secreted iron-regulator hepcidin and increased intestinal expression of fpn1 in anemic weh(Tp85c-/-) adults. Injection of iron dextran into WT or mutant zebrafish embryos, however, resulted in significant increases in hepcidin expression 18 hours after injection, demonstrating that hepcidin expression in zebrafish is iron responsive and independent of fpn1's function as an iron exporter. -
Lin HF, Traver D, Zhu H, Dooley K, Paw BH, Zon LI, Handin RI. 2005. Analysis of thrombocyte development in CD41-GFP transgenic zebrafish. Blood. 106(12):3803-10. Pubmed: 16099879 Lin HF, Traver D, Zhu H, Dooley K, Paw BH, Zon LI, Handin RI. 2005. Analysis of thrombocyte development in CD41-GFP transgenic zebrafish. Blood. 106(12):3803-10. Pubmed: 16099879 Thrombocytes are the nucleated equivalent of platelets in nonmammalian vertebrates such as the zebrafish, Danio rerio. We have cloned zebrafish CD41 cDNA (alpha(IIb), glycoprotein IIb [GPIIb]) and its promoter and have generated transgenic zebrafish lines with green fluorescent protein (GFP)-tagged thrombocytes. CD41 mRNA transcripts appeared 42 hours after fertilization (hpf) by reverse-transcriptase-polymerase chain reaction (RT-PCR) and at 48 hpf in circulating hematopoietic cells. Flow sorting of thrombocytes from the mesonephros of adult CD41-GFP zebrafish showed a GFP(high) subset, which had the morphologic appearance of mature thrombocytes, and a GFP(low) subset with an immature appearance, suggesting that they may be thrombocyte precursors. Confocal laser microscopy of embryos 40 and 48 hpf also showed a nonmobile population of GFP+ cells in a discrete area between the dorsal aorta and caudal vein. Production of circulating thrombocytes was inhibited by the injection of antisense morpholinos for the stem-cell transcription factor scl and c-mpl, the receptor for thrombopoietin. The nonmobile pool of GFP+ cells was abolished by scl knockdown and partially inhibited by c-mpl knockdown. These studies have shown that it is possible to identify thrombocytes, thrombocyte precursors, and, possibly, early hematopoietic stem cells in zebrafish embryos and track their proliferation and maturation. -
Burns CE, Traver D, Mayhall E, Shepard JL, Zon LI. 2005. Hematopoietic stem cell fate is established by the Notch-Runx pathway. Genes & development. 19(19):2331-42. Pubmed: 16166372 Burns CE, Traver D, Mayhall E, Shepard JL, Zon LI. 2005. Hematopoietic stem cell fate is established by the Notch-Runx pathway. Genes & development. 19(19):2331-42. Pubmed: 16166372 Identifying the molecular pathways regulating hematopoietic stem cell (HSC) specification, self-renewal, and expansion remains a fundamental goal of both basic and clinical biology. Here, we analyzed the effects of Notch signaling on HSC number during zebrafish development and adulthood, defining a critical pathway for stem cell specification. The Notch signaling mutant mind bomb displays normal embryonic hematopoiesis but fails to specify adult HSCs. Surprisingly, transient Notch activation during embryogenesis via an inducible transgenic system led to a Runx1-dependent expansion of HSCs in the aorta-gonad-mesonephros (AGM) region. In irradiated adults, Notch activity induced runx1 gene expression and increased multilineage hematopoietic precursor cells approximately threefold in the marrow. This increase was followed by the accelerated recovery of all the mature blood cell lineages. These data define the Notch-Runx pathway as critical for the developmental specification of HSC fate and the subsequent homeostasis of HSC number, thus providing a mechanism for amplifying stem cells in vivo. -
Akashi K, Traver D, Zon LI. 2005. The complex cartography of stem cell commitment. Cell. 121(2):160-2. Pubmed: 15851023 Akashi K, Traver D, Zon LI. 2005. The complex cartography of stem cell commitment. Cell. 121(2):160-2. Pubmed: 15851023 In this issue of Cell, a study by Adolfsson and coworkers (Adolfsson et al., 2005) provides insight into the early lineage commitment events of multipotent hematopoietic stem cells (HSCs). These studies demonstrate the importance of the Flt3 receptor tyrosine kinase as the earliest marker of hematopoietic cell fate commitment in that erythrocyte and megakaryocyte potentials are lost first as HSCs differentiate to lymphocyte progenitors. -
Weber GJ, Choe SE, Dooley KA, Paffett-Lugassy NN, Zhou Y, Zon LI. 2005. Mutant-specific gene programs in the zebrafish. Blood. 106(2):521-30. Pubmed: 15827125 Weber GJ, Choe SE, Dooley KA, Paffett-Lugassy NN, Zhou Y, Zon LI. 2005. Mutant-specific gene programs in the zebrafish. Blood. 106(2):521-30. Pubmed: 15827125 Hematopoiesis involves the production of stem cells, followed by the orchestrated differentiation of the blood lineages. Genetic screens in zebrafish have identified mutants with defects that disrupt specific stages of hematopoiesis and vasculogenesis, including the cloche, spadetail (tbx16), moonshine (tif1g), bloodless, and vlad tepes (gata1) mutants. To better characterize the blood program, gene expression profiling was carried out in these mutants and in scl-morphants (scl(mo)). Distinct gene clusters were demarcated by stage-specific and mutant-specific gene regulation. These were found to correlate with the transcriptional program of hematopoietic progenitor cells, as well as of the erythroid, myeloid, and vascular lineages. Among these, several novel hematopoietic and vascular genes were detected, for instance, the erythroid transcription factors znfl2 and ncoa4. A specific regulation was found for myeloid genes, as they were more strongly expressed in vlt mutants compared with other erythroid mutants. A unique gene expression pattern of up-regulated isoprenoid synthesis genes was found in cloche and scl(mo), possibly in migrating cells. In conjunction with the high conservation of vertebrate hematopoiesis, the comparison of transcriptional profiles in zebrafish blood mutants represents a versatile and powerful tool to elucidate the genetic regulation of blood and blood vessel development. -
Gardiner MR, Daggett DF, Zon LI, Perkins AC. 2005. Zebrafish KLF4 is essential for anterior mesendoderm/pre-polster differentiation and hatching. Developmental dynamics : an official publication of the American Association of Anatomists. 234(4):992-6. Pubmed: 16222715 Gardiner MR, Daggett DF, Zon LI, Perkins AC. 2005. Zebrafish KLF4 is essential for anterior mesendoderm/pre-polster differentiation and hatching. Developmental dynamics : an official publication of the American Association of Anatomists. 234(4):992-6. Pubmed: 16222715 Gene knockout studies of Krüppel-like factors (KLFs) in mice have shown essential roles in organogenesis. A screen for KLF family members in zebrafish identified many KLFs. One of these, zebrafish KLF4 (zKLF4) is the homologue of neptune, a Xenopus laevis KLF. zKLF4 is expressed from approximately 80% epiboly a patch of dorsal/anterior mesendodermal cells called the pre-polster and, subsequently, in the polster and hatching gland. Here we investigate the function of zKLF4 using morpholino-based antisense oligonucleotides. Knockdown of zKLF4 resulted in complete absence of hatching gland formation and subsequent hatching in zebrafish. In addition, there was early knockdown of expression of the pre-polster/anterior mesendoderm markers CatL, cap1, and BMP4. These results indicate zKLF4 is expressed within the pre-polster, an early mesendodermal site, and that it plays a critical role in the differentiation of these cells into hatching gland cells.Copyright 2005 Wiley-Liss, Inc. -
Stern HM, Murphey RD, Shepard JL, Amatruda JF, Straub CT, Pfaff KL, Weber G, Tallarico JA, King RW, Zon LI. 2005. Small molecules that delay S phase suppress a zebrafish bmyb mutant. Nature chemical biology. 1(7):366-70. Pubmed: 16372403 Stern HM, Murphey RD, Shepard JL, Amatruda JF, Straub CT, Pfaff KL, Weber G, Tallarico JA, King RW, Zon LI. 2005. Small molecules that delay S phase suppress a zebrafish bmyb mutant. Nature chemical biology. 1(7):366-70. Pubmed: 16372403 Bmyb is a ubiquitously expressed transcription factor involved in cellular proliferation and cancer. Loss of bmyb function in the zebrafish mutant crash&burn (crb) results in decreased cyclin B1 expression, mitotic arrest and genome instability. These phenotypic observations in crb mutants could be attributed to the decreased expression of cyclin B1, a cell-cycle regulatory protein that is responsible for driving cell progression from G2 through mitosis. To identify small molecules that interact with the bmyb pathway, we developed an embryo-based suppressor screening strategy. In 16 weeks we screened a diverse approximately 16,000 compound library, and discovered one previously unknown compound, persynthamide (psy, 1), that suppressed bmyb-dependent mitotic defects. Psy-treated embryos showed an S-phase delay, and knockdown of the cell-cycle checkpoint regulator ataxia telangiectasia--and Rad-related kinase (ATR) abrogated the suppression of crb. The DNA synthesis inhibitors aphidicolin (2) and hydroxyurea (3) also suppressed crb. S-phase inhibition upregulated cyclin B1 mRNA, promoting the progression of cells through mitosis. Our study demonstrates that chemical suppressor screening in zebrafish can identify compounds with cell-cycle activity and can be used to identify pathways that interact with specific cell-cycle phenotypes. -
Shepard JL, Amatruda JF, Stern HM, Subramanian A, Finkelstein D, Ziai J, Finley KR, Pfaff KL, Hersey C, Zhou Y, Barut B, Freedman M, Lee C, Spitsbergen J, Neuberg D, Weber G, Golub TR, Glickman JN, Kutok JL, Aster JC, Zon LI. 2005. A zebrafish bmyb mutation causes genome instability and increased cancer susceptibility. Proceedings of the National Academy of Sciences of the United States of America. 102(37):13194-9. Pubmed: 16150706 Shepard JL, Amatruda JF, Stern HM, Subramanian A, Finkelstein D, Ziai J, Finley KR, Pfaff KL, Hersey C, Zhou Y, Barut B, Freedman M, Lee C, Spitsbergen J, Neuberg D, Weber G, Golub TR, Glickman JN, Kutok JL, Aster JC, Zon LI. 2005. A zebrafish bmyb mutation causes genome instability and increased cancer susceptibility. Proceedings of the National Academy of Sciences of the United States of America. 102(37):13194-9. Pubmed: 16150706 A major goal of cancer research has been to identify genes that contribute to cancer formation. The similar pathology between zebrafish and human tumors, as well as the past success of large-scale genetic screens in uncovering human disease genes, makes zebrafish an ideal system in which to find such new genes. Here, we show that a zebrafish forward genetic screen uncovered multiple cell proliferation mutants including one mutant, crash&burn (crb), that represents a loss-of-function mutation in bmyb, a transcriptional regulator and member of a putative proto-oncogene family. crb mutant embryos have defects in mitotic progression and spindle formation, and exhibit genome instability. Regulation of cyclin B levels by bmyb appears to be the mechanism of mitotic accumulation in crb. Carcinogenesis studies reveal increased cancer susceptibility in adult crb heterozygotes. Gene-expression signatures associated with loss of bmyb in zebrafish are also correlated with conserved signatures in human tumor samples, and down-regulation of the B-myb signature genes is associated with retention of p53 function. Our findings show that zebrafish screens can uncover cancer pathways, and demonstrate that loss of function of bmyb is associated with cancer. 2004
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Mayhall EA, Paffett-Lugassy N, Zon LI. 2004. The clinical potential of stem cells. Current opinion in cell biology. 16(6):713-20. Pubmed: 15530786 Mayhall EA, Paffett-Lugassy N, Zon LI. 2004. The clinical potential of stem cells. Current opinion in cell biology. 16(6):713-20. Pubmed: 15530786 Stem cells are defined by their capacity for self-renewal and multilineage differentiation, making them uniquely situated to treat a broad spectrum of human diseases. For example, because hematopoietic stem cells can reconstitute the entire blood system, bone marrow transplantation has long been used in the clinic to treat various diseases. Similarly, the transplantation of other tissue-specific stem cells, such as stem cells isolated from epithelial and neural tissues, can treat mouse disease models and human patients in which epithelial and neural cells are damaged. An alternative to tissue-specific stem cell therapy takes advantage of embryonic stem cells, which are capable of differentiating into any tissue type. Furthermore, nuclear transfer, the transfer of a post-mitotic somatic cell nucleus into an enucleated oocyte, creates a limitless source of autologous cells that, when combined with gene therapy, can serve as a powerful therapeutic tool. -
Bovenkamp DE, Goishi K, Bahary N, Davidson AJ, Zhou Y, Becker T, Becker CG, Zon LI, Klagsbrun M. 2004. Expression and mapping of duplicate neuropilin-1 and neuropilin-2 genes in developing zebrafish. Gene expression patterns : GEP. 4(4):361-70. Pubmed: 15183302 Bovenkamp DE, Goishi K, Bahary N, Davidson AJ, Zhou Y, Becker T, Becker CG, Zon LI, Klagsbrun M. 2004. Expression and mapping of duplicate neuropilin-1 and neuropilin-2 genes in developing zebrafish. Gene expression patterns : GEP. 4(4):361-70. Pubmed: 15183302 Previously, we described the isolation and characterization of the first zebrafish neuropilin gene, which we now call nrp1a, and found its protein to be a mediator of vascular endothelial growth factor (VEGF)-dependent angiogenesis [Proc. Natl Acad. Sci. USA 99 (2002) 10470]. Subsequently, we have isolated three other full-length neuropilin genes (nrp1b, nrp2a, and nrp2b) and find that they map to independent zebrafish linkage groups. The nrp1s and nrp2s had differential spatio-temporal gene expression profiles with nrp1a being most prominent in the gut, brain, retina, hypochord, motorneurons, fin bud and mandibular cartilage, nrp1b in the brain, dorsal aorta, melanophores, ventral fin, and heart, nrp2a in the brain, retina, heart, and caudal vessels, and nrp2b in the brain, retina, gut, fin bud, melanophores, heart, and caudal vessels. In addition, we have identified an alternatively-spliced transcript of the nrp1b gene (denoted as nrp1b(s)) which is predicted to encode a soluble form of Nrp1b, containing only the a, b, and c extracellular domains. Transcript expression of nrp1b(s) was different from full-length nrp1b transcript, with prominence in the brain, developing mouth, heart, and fin bud. The NRP1s were tested for VEGF-binding ability. Both 125 kDa Nrp1a and 145 kDa Nrp1b bound 125I-labelled VEGFA165. In summary, two nrp1 and two nrp2 genes, with expression patterns similar to higher vertebrates, have been isolated from zebrafish. -
Zon LI, Zoloth L, Kadereit S. 2004. Taking the stem cell debate to the public. PLoS biology. 2(6):e188. Pubmed: 15208732 DOI:e188 Zon LI, Zoloth L, Kadereit S. 2004. Taking the stem cell debate to the public. PLoS biology. 2(6):e188. Pubmed: 15208732 DOI:e188 In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholders -
Traver D, Winzeler A, Stern HM, Mayhall EA, Langenau DM, Kutok JL, Look AT, Zon LI. 2004. Effects of lethal irradiation in zebrafish and rescue by hematopoietic cell transplantation. Blood. 104(5):1298-305. Pubmed: 15142873 Traver D, Winzeler A, Stern HM, Mayhall EA, Langenau DM, Kutok JL, Look AT, Zon LI. 2004. Effects of lethal irradiation in zebrafish and rescue by hematopoietic cell transplantation. Blood. 104(5):1298-305. Pubmed: 15142873 The study of hematopoiesis has been greatly facilitated by transplantation of blood cell populations into recipient animals. Efficient engraftment of donor cells generally requires ablation of the host hematopoietic system. The zebrafish has recently emerged as a developmental and genetic system to study hematopoiesis. To enable the study of hematopoietic stem cell (HSC) biology, immune cell function, and leukemogenesis in zebrafish, we have developed hematopoietic cell transplantation (HCT) into adult recipient animals conditioned by gamma irradiation. Dose-response experiments showed that the minimum lethal dose (MLD) of 40 Gy led to the specific ablation of hematolymphoid cells and death by 14 days after irradiation. Sublethal irradiation doses of 20 Gy predominantly ablated lymphocytes and permitted transplantation of a lethal T-cell leukemia. Finally, transplantation of hematopoietic cells carrying transgenes yielding red fluorescent erythrocytes and green fluorescent leukocytes showed that HCT is sufficient to rescue the MLD, that recipient hematolymphoid tissues were repopulated by donor-derived cells, and that donor blood cell lineages can be independently visualized in living recipients. Together, these results establish transplantation assays to test for HSC function and oncogenic transformation in zebrafish. -
Langenau DM, Ferrando AA, Traver D, Kutok JL, Hezel JP, Kanki JP, Zon LI, Look AT, Trede NS. 2004. In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrafish. Proceedings of the National Academy of Sciences of the United States of America. 101(19):7369-74. Pubmed: 15123839 Langenau DM, Ferrando AA, Traver D, Kutok JL, Hezel JP, Kanki JP, Zon LI, Look AT, Trede NS. 2004. In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrafish. Proceedings of the National Academy of Sciences of the United States of America. 101(19):7369-74. Pubmed: 15123839 Transgenic zebrafish that express GFP under control of the T cell-specific tyrosine kinase (lck) promoter were used to analyze critical aspects of the immune system, including patterns of T cell development and T cell homing after transplant. GFP-labeled T cells could be ablated in larvae by either irradiation or dexamethasone added to the water, illustrating that T cells have evolutionarily conserved responses to chemical and radiation ablation. In transplant experiments, thymocytes from lck-GFP fish repopulated the thymus of irradiated wild-type fish only transiently, suggesting that the thymus contains only short-term thymic repopulating cells. By contrast, whole kidney marrow permanently reconstituted the T lymphoid compartment of irradiated wild-type fish, suggesting that long-term thymic repopulating cells reside in the kidney. -
Wingert RA, Brownlie A, Galloway JL, Dooley K, Fraenkel P, Axe JL, Davidson AJ, Barut B, Noriega L, Sheng X, Zhou Y, Zon LI. 2004. The chianti zebrafish mutant provides a model for erythroid-specific disruption of transferrin receptor 1. Development (Cambridge, England). 131(24):6225-35. Pubmed: 15563524 Wingert RA, Brownlie A, Galloway JL, Dooley K, Fraenkel P, Axe JL, Davidson AJ, Barut B, Noriega L, Sheng X, Zhou Y, Zon LI. 2004. The chianti zebrafish mutant provides a model for erythroid-specific disruption of transferrin receptor 1. Development (Cambridge, England). 131(24):6225-35. Pubmed: 15563524 Iron is a crucial metal for normal development, being required for the production of heme, which is incorporated into cytochromes and hemoglobin. The zebrafish chianti (cia) mutant manifests a hypochromic, microcytic anemia after the onset of embryonic circulation, indicative of a perturbation in red blood cell hemoglobin production. We show that cia encodes tfr1a, which is specifically expressed in the developing blood and requisite only for iron uptake in erythroid precursors. In the process of isolating zebrafish tfr1, we discovered two tfr1-like genes (tfr1a and tfr1b) and a single tfr2 ortholog. Abrogation of tfr1b function using antisense morpholinos revealed that this paralog was dispensable for hemoglobin production in red cells. tfr1b morphants exhibited growth retardation and brain necrosis, similar to the central nervous system defects observed in the Tfr1 null mouse, indicating that tfr1b is probably used by non-erythroid tissues for iron acquisition. Overexpression of mouse Tfr1, mouse Tfr2, and zebrafish tfr1b partially rescued hypochromia in cia embryos, establishing that each of these transferrin receptors are capable of supporting iron uptake for hemoglobin production in vivo. Taken together, these data show that zebrafish tfr1a and tfr1b share biochemical function but have restricted domains of tissue expression, and establish a genetic model to study the specific function of Tfr1 in erythroid cells. -
Bahary N, Davidson A, Ransom D, Shepard J, Stern H, Trede N, Zhou Y, Barut B, Zon LI. 2004. The Zon laboratory guide to positional cloning in zebrafish. Methods in cell biology. 77:305-29. Pubmed: 15602919 Bahary N, Davidson A, Ransom D, Shepard J, Stern H, Trede N, Zhou Y, Barut B, Zon LI. 2004. The Zon laboratory guide to positional cloning in zebrafish. Methods in cell biology. 77:305-29. Pubmed: 15602919 -
Hsu K, Traver D, Kutok JL, Hagen A, Liu TX, Paw BH, Rhodes J, Berman JN, Zon LI, Kanki JP, Look AT. 2004. The pu.1 promoter drives myeloid gene expression in zebrafish. Blood. 104(5):1291-7. Pubmed: 14996705 Hsu K, Traver D, Kutok JL, Hagen A, Liu TX, Paw BH, Rhodes J, Berman JN, Zon LI, Kanki JP, Look AT. 2004. The pu.1 promoter drives myeloid gene expression in zebrafish. Blood. 104(5):1291-7. Pubmed: 14996705 PU.1 is a member of the Ets family of transcription factors and plays an essential role in the development of both myeloid and lymphoid cells. To examine zebrafish pu.1 (zpu.1) expression in subpopulations of blood cells during zebrafish development, we linked a 9-kb zebrafish genomic fragment upstream of the zpu.1 initiator codon to green fluorescent protein (GFP) and microinjected this construct to generate stable transgenic lines. GFP-positive fluorescent myeloid precursors were observed migrating from the anterolateral mesoderm in living embryos from 16 to 28 hours after fertilization (hpf) in a pattern that overlaps the expression pattern of endogenous zpu.1 mRNA. Analysis of larval histologic sections revealed GFP-expressing hematopoietic cells in the developing zebrafish kidney. Flow cytometric analysis of cells from adult whole kidney marrow revealed 2 discrete subpopulations of GFP-positive cells, which after cell sorting exhibited either myeloid or early lymphoid morphology. Thus, the zebrafish zpu.1 promoter fragment used here is capable of driving reporter gene expression in subsets of embryonic and adult hematopoietic cells. These transgenic lines will be useful to dissect the cellular and molecular control of myeloid cell differentiation, and this promoter fragment may prove useful in the development of zebrafish models of acute myeloid leukemia. -
Caro J, Zon LI. 2004. Allan j. Erslev, m.d.: truly a great dane (1919-2003). Stem cells (Dayton, Ohio). 22(1):121-2. Pubmed: 14688399 Caro J, Zon LI. 2004. Allan j. Erslev, m.d.: truly a great dane (1919-2003). Stem cells (Dayton, Ohio). 22(1):121-2. Pubmed: 14688399 -
Zhu H, Zon LI. 2004. Use of the DsRed fluorescent reporter in zebrafish. Methods in cell biology. 76:3-12. Pubmed: 15602868 Zhu H, Zon LI. 2004. Use of the DsRed fluorescent reporter in zebrafish. Methods in cell biology. 76:3-12. Pubmed: 15602868 Green fluorescent protein (GFP) is firmly established as a fluorescent reporter for the imaging of specific tissues in zebrafish. The employment of other reporters such as DsRed in transgenic zebrafish has made multicolored labeling experiments possible. To date, several DsRed transgenic lines have been generated for lineage labeling, transplantation assays, and commercial applications. Advances in multicolored labeling experiments will depend on the implementation of newly engineered reporters and fusion proteins, as well as on innovative experiments that exploit the power of direct visualization. -
Ernst P, Mabon M, Davidson AJ, Zon LI, Korsmeyer SJ. 2004. An Mll-dependent Hox program drives hematopoietic progenitor expansion. Current biology : CB. 14(22):2063-9. Pubmed: 15556871 Ernst P, Mabon M, Davidson AJ, Zon LI, Korsmeyer SJ. 2004. An Mll-dependent Hox program drives hematopoietic progenitor expansion. Current biology : CB. 14(22):2063-9. Pubmed: 15556871 Chromosomal translocations disrupting the Mixed lineage leukemia (Mll) gene result in leukemia, with aberrant expression of some native Mll target genes (reviewed in). The Mll gene encodes a Trithorax-group chromatin regulator that is essential for the development of hematopoietic stem cells (HSCs) during embryogenesis. Like Trithorax, MLL positively regulates clustered homeodomain or Hox genes, yet the role of Hox genes collectively in the development of the mammalian hematopoietic system has been difficult to ascertain because of redundancy among Hox paralogs. Here, we show that in the absence of MLL, early hematopoietic progenitors develop despite reduced expression of HoxA, HoxB, and HoxC genes. However, these progenitors exhibit a marked reduction in their ability to generate hematopoietic colonies, a subsequent process requiring cell division and differentiation. Reactivation of a subset of Hox genes or, remarkably, reexpression of a single Hox gene in Mll-deficient progenitors rescued hematopoietic-colony frequency and growth. In contrast, expression of other MLL target genes such as Pitx2 or expression of anti-apoptotic BCL-2 failed to rescue hematopoietic-colony frequency. Furthermore, our results highlight a shared function of Hox proteins at this point in the development of the hematopoietic system. -
Trede NS, Langenau DM, Traver D, Look AT, Zon LI. 2004. The use of zebrafish to understand immunity. Immunity. 20(4):367-79. Pubmed: 15084267 Trede NS, Langenau DM, Traver D, Look AT, Zon LI. 2004. The use of zebrafish to understand immunity. Immunity. 20(4):367-79. Pubmed: 15084267 For decades immunologists have relied heavily on the mouse model for their experimental designs. With the realization of the important role innate immunity plays in orchestrating immune responses, invertebrates such as worms and flies have been added to the repertoire. Here, we discuss the advent of the zebrafish as a powerful vertebrate model organism that promises to positively impact immunologic research. -
Davidson AJ, Zon LI. 2004. The 'definitive' (and 'primitive') guide to zebrafish hematopoiesis. Oncogene. 23(43):7233-46. Pubmed: 15378083 Davidson AJ, Zon LI. 2004. The 'definitive' (and 'primitive') guide to zebrafish hematopoiesis. Oncogene. 23(43):7233-46. Pubmed: 15378083 Progressive advances using zebrafish as a model organism have provided hematologists with an additional genetic system to study blood cell formation and hematological malignancies. Despite extensive evolutionary divergence between bony fish (teleosts) and mammals, the molecular pathways governing hematopoiesis have been highly conserved. As a result, most (if not all) of the critical hematopoietic transcription factor genes identified in mammals have orthologues in zebrafish. As in other vertebrates, all of the teleost blood lineages are believed to originate from a pool of pluripotent, self-renewing hematopoietic stem cells. Here, we provide a detailed review of the timing, anatomical location, and transcriptional regulation of zebrafish 'primitive' and 'definitive' hematopoiesis as well as discuss a model of T-cell leukemia and recent advances in blood cell transplantation. Given that many of the regulatory genes that control embryonic hematopoiesis have been implicated in oncogenic pathways in adults, an understanding of blood cell ontogeny is likely to provide insights into the pathophysiology of human leukemias. -
Gong HY, Lin CJ, Chen MH, Hu MC, Lin GH, Zhou Y, Zon LI, Wu JL. 2004. Two distinct teleost hepatocyte nuclear factor 1 genes, hnf1alpha/tcf1 and hnf1beta/tcf2, abundantly expressed in liver, pancreas, gut and kidney of zebrafish. Gene. 338(1):35-46. Pubmed: 15302404 Gong HY, Lin CJ, Chen MH, Hu MC, Lin GH, Zhou Y, Zon LI, Wu JL. 2004. Two distinct teleost hepatocyte nuclear factor 1 genes, hnf1alpha/tcf1 and hnf1beta/tcf2, abundantly expressed in liver, pancreas, gut and kidney of zebrafish. Gene. 338(1):35-46. Pubmed: 15302404 Two distinct forms of zebrafish hepatocyte nuclear factor 1 (hnf1) were identified and referred to as hnf1alpha/tcf1 and hnf1beta/tcf2. Both hnf1 genes were shown to be expressed abundantly in liver, pancreas, gut and kidney. Zebrafish HNF1alpha and HNF1beta proteins contain all HNF1 signature domains including the dimerization domain, POU-like domain and atypical homeodomain. Sequence and phylogenetic analysis reveals that zebrafish hnf1alpha is closer to tetrapodian hnf1alpha than to tetrapodian hnf1beta and zebrafish hnf1beta is highly conserved with tetrapodian hnf1beta. Existences of hnf1alpha and hnf1beta in teleost zebrafish, tilapia and fugu suggest that hnf1 gene duplication might occur before the divergence of teleost and tetrapod ancestors. Zebrafish hnf1alpha and hnf1beta genes were mapped to linkage group LG8 and LG15 in T51 panel by RH mapping and are composed of 10 and 9 exons, respectively. Zebrafish hnf1beta gene with at least 11 genes in LG15 was identified to maintain the conserved synteny with those of human in chromosome 17 and those of mouse in chromosome 11. Our results indicate that distinct hnf1alpha and hnf1beta genes in teleosts had been evolved from the hnf1 ancestor gene of chordate. -
Ransom DG, Bahary N, Niss K, Traver D, Burns C, Trede NS, Paffett-Lugassy N, Saganic WJ, Lim CA, Hersey C, Zhou Y, Barut BA, Lin S, Kingsley PD, Palis J, Orkin SH, Zon LI. 2004. The zebrafish moonshine gene encodes transcriptional intermediary factor 1gamma, an essential regulator of hematopoiesis. PLoS biology. 2(8):E237. Pubmed: 15314655 DOI:e237 Ransom DG, Bahary N, Niss K, Traver D, Burns C, Trede NS, Paffett-Lugassy N, Saganic WJ, Lim CA, Hersey C, Zhou Y, Barut BA, Lin S, Kingsley PD, Palis J, Orkin SH, Zon LI. 2004. The zebrafish moonshine gene encodes transcriptional intermediary factor 1gamma, an essential regulator of hematopoiesis. PLoS biology. 2(8):E237. Pubmed: 15314655 DOI:e237 Hematopoiesis is precisely orchestrated by lineage-specific DNA-binding proteins that regulate transcription in concert with coactivators and corepressors. Mutations in the zebrafish moonshine (mon) gene specifically disrupt both embryonic and adult hematopoiesis, resulting in severe red blood cell aplasia. We report that mon encodes the zebrafish ortholog of mammalian transcriptional intermediary factor 1gamma (TIF1gamma) (or TRIM33), a member of the TIF1 family of coactivators and corepressors. During development, hematopoietic progenitor cells in mon mutants fail to express normal levels of hematopoietic transcription factors, including gata1, and undergo apoptosis. Three different mon mutant alleles each encode premature stop codons, and enforced expression of wild-type tif1gamma mRNA rescues embryonic hematopoiesis in homozygous mon mutants. Surprisingly, a high level of zygotic tif1gamma mRNA expression delineates ventral mesoderm during hematopoietic stem cell and progenitor formation prior to gata1 expression. Transplantation studies reveal that tif1gamma functions in a cell-autonomous manner during the differentiation of erythroid precursors. Studies in murine erythroid cell lines demonstrate that Tif1gamma protein is localized within novel nuclear foci, and expression decreases during erythroid cell maturation. Our results establish a major role for this transcriptional intermediary factor in the differentiation of hematopoietic cells in vertebrates. -
Song HD, Sun XJ, Deng M, Zhang GW, Zhou Y, Wu XY, Sheng Y, Chen Y, Ruan Z, Jiang CL, Fan HY, Zon LI, Kanki JP, Liu TX, Look AT, Chen Z. 2004. Hematopoietic gene expression profile in zebrafish kidney marrow. Proceedings of the National Academy of Sciences of the United States of America. 101(46):16240-5. Pubmed: 15520368 Song HD, Sun XJ, Deng M, Zhang GW, Zhou Y, Wu XY, Sheng Y, Chen Y, Ruan Z, Jiang CL, Fan HY, Zon LI, Kanki JP, Liu TX, Look AT, Chen Z. 2004. Hematopoietic gene expression profile in zebrafish kidney marrow. Proceedings of the National Academy of Sciences of the United States of America. 101(46):16240-5. Pubmed: 15520368 The zebrafish kidney marrow is considered to be the organ of definitive hematopoiesis, analogous to the mammalian bone marrow. We have sequenced 26,143 ESTs and isolated 304 cDNAs with putative full-length ORF from a zebrafish kidney marrow cDNA library. The ESTs formed 7,742 assemblies, representing both previously identified zebrafish ESTs (56%) and recently discovered zebrafish ESTs (44%). About 30% of these EST assemblies have orthologues in humans, including 1,282 disease-associated genes in the Online Mendelian Inheritance in Man (OMIM) database. Comparison of the effective and regulatory molecules related to erythroid functions across species suggests a good conservation from zebrafish to human. Interestingly, both embryonic and adult zebrafish globin genes showed higher homology to the human embryonic globin genes than to the human fetal/adult ones, consistent with evo-devo correlation hypothesis. In addition, conservation of a whole set of transcription factors involved in globin gene switch suggests the regulatory network for such remodeling mechanism existed before the divergence of the teleost and the ancestor of mammals. We also carried out whole-mount mRNA in situ hybridization assays for 493 cDNAs and identified 80 genes (16%) with tissue-specific expression during the first five days of zebrafish development. Twenty-six of these genes were specifically expressed in hematopoietic or vascular tissues, including three previously unidentified zebrafish genes: coro1a, nephrosin, and dab2. Our results indicate that conserved genetic programs regulate vertebrate hematopoiesis and vasculogenesis, and support the role of the zebrafish as an important animal model for studying both normal development and the molecular pathogenesis of human blood diseases. 2003
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Brownlie A, Hersey C, Oates AC, Paw BH, Falick AM, Witkowska HE, Flint J, Higgs D, Jessen J, Bahary N, Zhu H, Lin S, Zon L. 2003. Characterization of embryonic globin genes of the zebrafish. Developmental biology. 255(1):48-61. Pubmed: 12618133 Brownlie A, Hersey C, Oates AC, Paw BH, Falick AM, Witkowska HE, Flint J, Higgs D, Jessen J, Bahary N, Zhu H, Lin S, Zon L. 2003. Characterization of embryonic globin genes of the zebrafish. Developmental biology. 255(1):48-61. Pubmed: 12618133 Hemoglobin switching is a complex process by which distinct globin chains are produced during stages of development. In an effort to characterize the process of hemoglobin switching in the zebrafish model system, we have isolated and characterized several embryonic globin genes. The embryonic and adult globin genes are found in clusters in a head-to-head configuration. One cluster of embryonic and adult genes is localized to linkage group 3, whereas another embryonic cluster is localized on linkage group 12. Several embryonic globin genes demonstrate an erythroid-specific pattern of expression early during embryogenesis and later are downregulated as definitive hematopoiesis occurs. We utilized electrospray mass spectroscopy to correlate globin genes and protein expression in developing embryonic red cells. The mutation, zinfandel, has a hypochromic microcytic anemia as an embryo, but later recovers in adulthood. The zinfandel gene maps to linkage group 3 near the major globin gene locus, strongly suggesting that zinfandel represents an embryonic globin defect. Our studies are the first to systematically evaluate the embryonic globins in the zebrafish and will ultimately be useful in evaluating zebrafish mutants with defects in hemoglobin production and switching. -
Davidson AJ, Zon LI. 2003. Biomedicine. Love, honor, and protect (your liver). Science (New York, N.Y.). 299(5608):835-7. Pubmed: 12574609 Davidson AJ, Zon LI. 2003. Biomedicine. Love, honor, and protect (your liver). Science (New York, N.Y.). 299(5608):835-7. Pubmed: 12574609 -
Langenau DM, Traver D, Ferrando AA, Kutok JL, Aster JC, Kanki JP, Lin S, Prochownik E, Trede NS, Zon LI, Look AT. 2003. Myc-induced T cell leukemia in transgenic zebrafish. Science (New York, N.Y.). 299(5608):887-90. Pubmed: 12574629 Langenau DM, Traver D, Ferrando AA, Kutok JL, Aster JC, Kanki JP, Lin S, Prochownik E, Trede NS, Zon LI, Look AT. 2003. Myc-induced T cell leukemia in transgenic zebrafish. Science (New York, N.Y.). 299(5608):887-90. Pubmed: 12574629 The zebrafish is an attractive model organism for studying cancer development because of its genetic accessibility. Here we describe the induction of clonally derived T cell acute lymphoblastic leukemia in transgenic zebrafish expressing mouse c-myc under control of the zebrafish Rag2 promoter. Visualization of leukemic cells expressing a chimeric transgene encoding Myc fused to green fluorescent protein (GFP) revealed that leukemias arose in the thymus, spread locally into gill arches and retro-orbital soft tissue, and then disseminated into skeletal muscle and abdominal organs. Leukemic cells homed back to the thymus in irradiated fish transplanted with GFP-labeled leukemic lymphoblasts. This transgenic model provides a platform for drug screens and for genetic screens aimed at identifying mutations that suppress or enhance c-myc- induced carcinogenesis. -
Traver D, Herbomel P, Patton EE, Murphey RD, Yoder JA, Litman GW, Catic A, Amemiya CT, Zon LI, Trede NS. 2003. The zebrafish as a model organism to study development of the immune system. Advances in immunology. 81:253-330. Pubmed: 14711058 Traver D, Herbomel P, Patton EE, Murphey RD, Yoder JA, Litman GW, Catic A, Amemiya CT, Zon LI, Trede NS. 2003. The zebrafish as a model organism to study development of the immune system. Advances in immunology. 81:253-330. Pubmed: 14711058 -
Galloway JL, Zon LI. 2003. Ontogeny of hematopoiesis: examining the emergence of hematopoietic cells in the vertebrate embryo. Current topics in developmental biology. 53:139-58. Pubmed: 12510667 Galloway JL, Zon LI. 2003. Ontogeny of hematopoiesis: examining the emergence of hematopoietic cells in the vertebrate embryo. Current topics in developmental biology. 53:139-58. Pubmed: 12510667 Hematopoietic stem cells (HSCs) are responsible for generating all the lineages of the blood. During vertebrate development, waves of hematopoietic activity can be found in distinct anatomical sites, and they contribute to both embryonic and adult hematopoiesis. The origin of the HSCs that ultimately give rise to all the adult blood lineages has been a controversial issue in the field of hematopoiesis. Studies of amniotes have linked HSC activity to the aorta-gonad-mesonephros (AGM) region, whereas others suggest that the yolk sac is the true source of HSCs. This review describes both primitive and definitive hematopoiesis in mice, humans, chicks, frogs, and zebrafish and examines the current debate over the embryonic origins of HSCs. -
Traver D, Paw BH, Poss KD, Penberthy WT, Lin S, Zon LI. 2003. Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nature immunology. 4(12):1238-46. Pubmed: 14608381 Traver D, Paw BH, Poss KD, Penberthy WT, Lin S, Zon LI. 2003. Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nature immunology. 4(12):1238-46. Pubmed: 14608381 The zebrafish is firmly established as a genetic model for the study of vertebrate blood development. Here we have characterized the blood-forming system of adult zebrafish. Each major blood lineage can be isolated by flow cytometry, and with these lineal profiles, defects in zebrafish blood mutants can be quantified. We developed hematopoietic cell transplantation to study cell autonomy of mutant gene function and to establish a hematopoietic stem cell assay. Hematopoietic cell transplantation can rescue multilineage hematopoiesis in embryonic lethal gata1-/- mutants for over 6 months. Direct visualization of fluorescent donor cells in embryonic recipients allows engraftment and homing events to be imaged in real time. These results provide a cellular context in which to study the genetics of hematopoiesis. -
North TE, Zon LI. 2003. Modeling human hematopoietic and cardiovascular diseases in zebrafish. Developmental dynamics : an official publication of the American Association of Anatomists. 228(3):568-83. Pubmed: 14579393 North TE, Zon LI. 2003. Modeling human hematopoietic and cardiovascular diseases in zebrafish. Developmental dynamics : an official publication of the American Association of Anatomists. 228(3):568-83. Pubmed: 14579393 Zebrafish have emerged as a useful vertebrate model system in which unbiased large-scale screens have revealed hundreds of mutations affecting vertebrate development. Many zebrafish mutants closely resemble known human disorders, thus providing intriguing prospects for uncovering the genetic basis of human diseases and for the development of pharmacologic agents that inhibit or correct the progression of developmental disorders. The rapid pace of advances in genomic sequencing and map construction, in addition to morpholino targeting and transgenic techniques, have facilitated the identification and analysis of genes associated with zebrafish mutants, thus promoting the development of zebrafish as a model for human disorders. This review aims to illustrate how the zebrafish has been used to identify unknown genes, to assign function to known genes, and to delineate genetic pathways, all contributing valuable leads toward understanding human pathophysiology.Copyright 2003 Wiley-Liss, Inc. -
Davidson AJ, Ernst P, Wang Y, Dekens MP, Kingsley PD, Palis J, Korsmeyer SJ, Daley GQ, Zon LI. 2003. cdx4 mutants fail to specify blood progenitors and can be rescued by multiple hox genes. Nature. 425(6955):300-6. Pubmed: 13679919 Davidson AJ, Ernst P, Wang Y, Dekens MP, Kingsley PD, Palis J, Korsmeyer SJ, Daley GQ, Zon LI. 2003. cdx4 mutants fail to specify blood progenitors and can be rescued by multiple hox genes. Nature. 425(6955):300-6. Pubmed: 13679919 Organogenesis is dependent on the formation of distinct cell types within the embryo. Important to this process are the hox genes, which are believed to confer positional identities to cells along the anteroposterior axis. Here, we have identified the caudal-related gene cdx4 as the locus mutated in kugelig (kgg), a zebrafish mutant with an early defect in haematopoiesis that is associated with abnormal anteroposterior patterning and aberrant hox gene expression. The blood deficiency in kgg embryos can be rescued by overexpressing hoxb7a or hoxa9a but not hoxb8a, indicating that the haematopoietic defect results from perturbations in specific hox genes. Furthermore, the haematopoietic defect in kgg mutants is not rescued by scl overexpression, suggesting that cdx4 and hox genes act to make the posterior mesoderm competent for blood development. Overexpression of cdx4 during zebrafish development or in mouse embryonic stem cells induces blood formation and alters hox gene expression. Taken together, these findings demonstrate that cdx4 regulates hox genes and is necessary for the specification of haematopoietic cell fate during vertebrate embryogenesis. -
Ward AC, McPhee DO, Condron MM, Varma S, Cody SH, Onnebo SM, Paw BH, Zon LI, Lieschke GJ. 2003. The zebrafish spi1 promoter drives myeloid-specific expression in stable transgenic fish. Blood. 102(9):3238-40. Pubmed: 12869502 Ward AC, McPhee DO, Condron MM, Varma S, Cody SH, Onnebo SM, Paw BH, Zon LI, Lieschke GJ. 2003. The zebrafish spi1 promoter drives myeloid-specific expression in stable transgenic fish. Blood. 102(9):3238-40. Pubmed: 12869502 The spi1 (pu.1) gene has recently been identified as a useful marker of early myeloid cells in zebrafish. To enhance the versatility of this organism as a model for studying myeloid development, the promoter of this gene has been isolated and characterized. Transient transgenesis revealed that a 5.3 kilobase promoter fragment immediately upstream of the spi1 coding sequence was sufficient to drive expression of enhanced green fluorescent protein (EGFP) in injected embryos in a manner that largely recapitulated the native spi1 gene expression pattern. This fragment was successfully used to produce a germ line transgenic line of zebrafish with EGFP-expressing myeloid cells. These TG(spi1:EGFP)pA301 transgenic zebrafish represent a valuable tool for further studies of myeloid development and its perturbation. -
Goishi K, Lee P, Davidson AJ, Nishi E, Zon LI, Klagsbrun M. 2003. Inhibition of zebrafish epidermal growth factor receptor activity results in cardiovascular defects. Mechanisms of development. 120(7):811-22. Pubmed: 12915231 Goishi K, Lee P, Davidson AJ, Nishi E, Zon LI, Klagsbrun M. 2003. Inhibition of zebrafish epidermal growth factor receptor activity results in cardiovascular defects. Mechanisms of development. 120(7):811-22. Pubmed: 12915231 The physiological role of any of the epidermal growth factor (EGF) receptor tyrosine kinases has yet to be determined in zebrafish. We isolated a zebrafish homologue of EGFR (egfr) that shows a 63% amino acid overall identity to human EGFR but with 90% amino acid identity in the kinase domain. Whole mount in situ hybridization showed ubiquitous distribution of egfr transcripts during gastrulation, somitogenesis and later stages. When expressed in Chinese hamster ovary cells, zebrafish Egfr was a functional receptor that responded to EGF by receptor tyrosine phosphorylation and activation of MAP kinase. The function of zebrafish Egfr in vivo was determined by inhibiting its activity using EGFR kinase inhibitors and antisense morpholinos (MO), which inhibited Egfr kinase activity and translation of egfr messenger RNA into protein, respectively. The zebrafish is a particularly excellent model for studying cardiovascular development because zebrafish are transparent allowing direct visualization of the heart and circulation in the blood vessels. Inhibition of zebrafish Egfr activity in vivo impeded blood flow via the outflow tract into the aorta and impeded circulation in the axial and intersegmental vessels by 80 h post-fertilization. Analysis of the heart showed that the heart chambers and pericardial sacs were dilated and the outflow tracts were narrowed. Together these results suggested that zebrafish Egfr has a cardiovascular function in the developing zebrafish that is required for normal circulation. -
Stern HM, Zon LI. 2003. Cancer genetics and drug discovery in the zebrafish. Nature reviews. Cancer. 3(7):533-9. Pubmed: 12835673 Stern HM, Zon LI. 2003. Cancer genetics and drug discovery in the zebrafish. Nature reviews. Cancer. 3(7):533-9. Pubmed: 12835673 Fish have a long history of use in cancer toxicology studies, because they develop neoplasms that are histologically similar to human cancers. Because of considerable progress in zebrafish genetics and genomics over the past few years, the zebrafish system has provided many useful tools for studying basic biological processes. These tools include forward genetic screens, transgenic models, specific gene disruptions and small-molecule screens. By combining carcinogenesis assays, genetic analyses and small-molecule screening techniques, the zebrafish is emerging as a powerful system for identifying novel cancer genes and for cancer drug discovery. -
Piotrowski T, Ahn DG, Schilling TF, Nair S, Ruvinsky I, Geisler R, Rauch GJ, Haffter P, Zon LI, Zhou Y, Foott H, Dawid IB, Ho RK. 2003. The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans. Development (Cambridge, England). 130(20):5043-52. Pubmed: 12952905 Piotrowski T, Ahn DG, Schilling TF, Nair S, Ruvinsky I, Geisler R, Rauch GJ, Haffter P, Zon LI, Zhou Y, Foott H, Dawid IB, Ho RK. 2003. The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans. Development (Cambridge, England). 130(20):5043-52. Pubmed: 12952905 The van gogh (vgo) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1, a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2, genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS. -
Guyon JR, Mosley AN, Zhou Y, O'Brien KF, Sheng X, Chiang K, Davidson AJ, Volinski JM, Zon LI, Kunkel LM. 2003. The dystrophin associated protein complex in zebrafish. Human molecular genetics. 12(6):601-15. Pubmed: 12620966 Guyon JR, Mosley AN, Zhou Y, O'Brien KF, Sheng X, Chiang K, Davidson AJ, Volinski JM, Zon LI, Kunkel LM. 2003. The dystrophin associated protein complex in zebrafish. Human molecular genetics. 12(6):601-15. Pubmed: 12620966 Many cases of muscular dystrophy in humans are caused by mutations in members of the dystrophin associated protein complex (DAPC). Zebrafish are small vertebrates whose bodies are composed predominantly of skeletal muscle, making them attractive models for studying mammalian muscle disorders. Potential orthologs to most of the human DAPC proteins have been found in zebrafish by database screening. Expression of the sarcoglycans, dystroglycan and dystrophin has been confirmed by western blotting. Immunohistochemical and biochemical techniques localize these proteins to the muscle cell membrane in adult zebrafish. Morpholino (MO) experiments designed to inhibit the translation of dystrophin mRNA produce juvenile zebrafish that are less active than zebrafish injected with control morpholinos. Western blot analysis of the dystrophin morpholino-injected zebrafish shows concurrent reduction of dystrophin and the sarcoglycans, suggesting that these proteins, like those in mammals, are part of a complex whose integrity is dependent on dystrophin expression. These results indicate that the zebrafish is an excellent animal model in which to approach the study of dystrophin and its associated proteins. -
Paw BH, Davidson AJ, Zhou Y, Li R, Pratt SJ, Lee C, Trede NS, Brownlie A, Donovan A, Liao EC, Ziai JM, Drejer AH, Guo W, Kim CH, Gwynn B, Peters LL, Chernova MN, Alper SL, Zapata A, Wickramasinghe SN, Lee MJ, Lux SE, Fritz A, Postlethwait JH, Zon LI. 2003. Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency. Nature genetics. 34(1):59-64. Pubmed: 12669066 Paw BH, Davidson AJ, Zhou Y, Li R, Pratt SJ, Lee C, Trede NS, Brownlie A, Donovan A, Liao EC, Ziai JM, Drejer AH, Guo W, Kim CH, Gwynn B, Peters LL, Chernova MN, Alper SL, Zapata A, Wickramasinghe SN, Lee MJ, Lux SE, Fritz A, Postlethwait JH, Zon LI. 2003. Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency. Nature genetics. 34(1):59-64. Pubmed: 12669066 Most eukaryotic cell types use a common program to regulate the process of cell division. During mitosis, successful partitioning of the genetic material depends on spatially coordinated chromosome movement and cell cleavage. Here we characterize a zebrafish mutant, retsina (ret), that exhibits an erythroid-specific defect in cell division with marked dyserythropoiesis similar to human congenital dyserythropoietic anemia. Erythroblasts from ret fish show binuclearity and undergo apoptosis due to a failure in the completion of chromosome segregation and cytokinesis. Through positional cloning, we show that the ret mutation is in a gene (slc4a1) encoding the anion exchanger 1 (also called band 3 and AE1), an erythroid-specific cytoskeletal protein. We further show an association between deficiency in Slc4a1 and mitotic defects in the mouse. Rescue experiments in ret zebrafish embryos expressing transgenic slc4a1 with a variety of mutations show that the requirement for band 3 in normal erythroid mitosis is mediated through its protein 4.1R-binding domains. Our report establishes an evolutionarily conserved role for band 3 in erythroid-specific cell division and illustrates the concept of cell-specific adaptation for mitosis. 2002
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Best JL, Ganiatsas S, Agarwal S, Changou A, Salomoni P, Shirihai O, Meluh PB, Pandolfi PP, Zon LI. 2002. SUMO-1 protease-1 regulates gene transcription through PML. Molecular cell. 10(4):843-55. Pubmed: 12419228 Best JL, Ganiatsas S, Agarwal S, Changou A, Salomoni P, Shirihai O, Meluh PB, Pandolfi PP, Zon LI. 2002. SUMO-1 protease-1 regulates gene transcription through PML. Molecular cell. 10(4):843-55. Pubmed: 12419228 During a screen to identify c-Jun activators, we isolated a cysteine protease, SuPr-1, that induced c-Jun-dependent transcription independently of c-Jun phosphorylation. SuPr-1 is a member of a new family of proteases that hydrolyze the ubiquitin-like modifier, SUMO-1. SuPr-1 hydrolyzed SUMO-1-modified forms of the promyelocytic leukemia gene product, PML, and altered the subcellular distribution of PML in nuclear PODs (PML oncogenic domains). SuPr-1 also altered the distribution of other nuclear POD-associated proteins, such as CBP and Daxx, that act as transcriptional regulators. SuPr-1 action on transcription was enhanced by PML, and SuPr-1 failed to activate transcription in PML-deficient fibroblasts. Our studies establish an important role for SUMO proteases in transcription. -
Langenau DM, Palomero T, Kanki JP, Ferrando AA, Zhou Y, Zon LI, Look AT. 2002. Molecular cloning and developmental expression of Tlx (Hox11) genes in zebrafish (Danio rerio). Mechanisms of development. 117(1-2):243-8. Pubmed: 12204264 Langenau DM, Palomero T, Kanki JP, Ferrando AA, Zhou Y, Zon LI, Look AT. 2002. Molecular cloning and developmental expression of Tlx (Hox11) genes in zebrafish (Danio rerio). Mechanisms of development. 117(1-2):243-8. Pubmed: 12204264 Tlx (Hox11) genes are orphan homeobox genes that play critical roles in the regulation of early developmental processes in vertebrates. Here, we report the identification and expression patterns of three members of the zebrafish Tlx family. These genes share similar, but not identical, expression patterns with other vertebrate Tlx-1 and Tlx-3 genes. Tlx-1 is expressed early in the developing hindbrain and pharyngeal arches, and later in the putative splenic primordium. However, unlike its orthologues, zebrafish Tlx-1 is not expressed in the cranial sensory ganglia or spinal cord. Two homologues of Tlx-3 were identified: Tlx-3a and Tlx-3b, which are both expressed in discrete regions of the developing nervous system, including the cranial sensory ganglia and Rohon-Beard neurons. However, only Tlx-3a is expressed in the statoacoustic cranial ganglia, enteric neurons and non-neural tissues such as the fin bud and pharyngeal arches and Tlx-3b is only expressed in the dorsal root ganglia.Copyright 2002 Elsevier Science Ireland Ltd. -
Shafizadeh E, Paw BH, Foott H, Liao EC, Barut BA, Cope JJ, Zon LI, Lin S. 2002. Characterization of zebrafish merlot/chablis as non-mammalian vertebrate models for severe congenital anemia due to protein 4.1 deficiency. Development (Cambridge, England). 129(18):4359-70. Pubmed: 12183387 Shafizadeh E, Paw BH, Foott H, Liao EC, Barut BA, Cope JJ, Zon LI, Lin S. 2002. Characterization of zebrafish merlot/chablis as non-mammalian vertebrate models for severe congenital anemia due to protein 4.1 deficiency. Development (Cambridge, England). 129(18):4359-70. Pubmed: 12183387 The red blood cell membrane skeleton is an elaborate and organized network of structural proteins that interacts with the lipid bilayer and transmembrane proteins to maintain red blood cell morphology, membrane deformability and mechanical stability. A crucial component of red blood cell membrane skeleton is the erythroid specific protein 4.1R, which anchors the spectrin-actin based cytoskeleton to the plasma membrane. Qualitative and quantitative defects in protein 4.1R result in congenital red cell membrane disorders characterized by reduced cellular deformability and abnormal cell morphology. The zebrafish mutants merlot (mot) and chablis (cha) exhibit severe hemolytic anemia characterized by abnormal cell morphology and increased osmotic fragility. The phenotypic analysis of merlot indicates severe hemolysis of mutant red blood cells, consistent with the observed cardiomegaly, splenomegaly, elevated bilirubin levels and erythroid hyperplasia in the kidneys. The result of electron microscopic analysis demonstrates that mot red blood cells have membrane abnormalities and exhibit a severe loss of cortical membrane organization. Using positional cloning techniques and a candidate gene approach, we demonstrate that merlot and chablis are allelic and encode the zebrafish erythroid specific protein 4.1R. We show that mutant cDNAs from both alleles harbor nonsense point mutations, resulting in premature stop codons. This work presents merlot/chablis as the first characterized non-mammalian vertebrate models of hereditary anemia due to a defect in protein 4.1R integrity. -
Donovan A, Brownlie A, Dorschner MO, Zhou Y, Pratt SJ, Paw BH, Phillips RB, Thisse C, Thisse B, Zon LI. 2002. The zebrafish mutant gene chardonnay (cdy) encodes divalent metal transporter 1 (DMT1). Blood. 100(13):4655-9. Pubmed: 12393445 Donovan A, Brownlie A, Dorschner MO, Zhou Y, Pratt SJ, Paw BH, Phillips RB, Thisse C, Thisse B, Zon LI. 2002. The zebrafish mutant gene chardonnay (cdy) encodes divalent metal transporter 1 (DMT1). Blood. 100(13):4655-9. Pubmed: 12393445 Iron is an essential nutrient required for the function of all cells, most notably for the production of hemoglobin in red blood cells. Defects in the mechanisms of iron absorption, storage, or utilization can lead to disorders of iron-limited erythropoiesis or iron overload. In an effort to further understand these processes, we have used the zebrafish as a genetic system to study vertebrate iron metabolism. Here we characterized the phenotype of chardonnay (cdy), a zebrafish mutant with hypochromic, microcytic anemia, and positioned the mutant gene on linkage group 11. The cdy gene was isolated by a functional genomics approach in which we used a combination of expression studies, sequence analyses, and radiation hybrid panel mapping. We identified erythroid-specific genes using a whole embryo mRNA in situ hybridization screen and placed these genes on the zebrafish genomic map. One of these genes encoded the iron transporter divalent metal transporter 1 (DMT1) and colocalized with the cdy gene. We identified a nonsense mutation in the cdy allele and demonstrated that, whereas wild-type zebrafish DMT1 protein can transport iron, the truncated protein expressed in cdy mutants is not functional. Our studies further demonstrate the conservation of iron metabolism in vertebrates and suggest the existence of an alternative pathway of intestinal and red blood cell iron uptake. -
Murphey RD, Zon LI. 2002. Attack of the fish clones. Nature biotechnology. 20(8):785-6. Pubmed: 12148002 Murphey RD, Zon LI. 2002. Attack of the fish clones. Nature biotechnology. 20(8):785-6. Pubmed: 12148002 -
Lee P, Goishi K, Davidson AJ, Mannix R, Zon L, Klagsbrun M. 2002. Neuropilin-1 is required for vascular development and is a mediator of VEGF-dependent angiogenesis in zebrafish. Proceedings of the National Academy of Sciences of the United States of America. 99(16):10470-5. Pubmed: 12142468 Lee P, Goishi K, Davidson AJ, Mannix R, Zon L, Klagsbrun M. 2002. Neuropilin-1 is required for vascular development and is a mediator of VEGF-dependent angiogenesis in zebrafish. Proceedings of the National Academy of Sciences of the United States of America. 99(16):10470-5. Pubmed: 12142468 Neuropilin-1 (NRP1) is a cell-surface receptor for both vascular endothelial growth factor(165) (VEGF(165)) and class 3 semaphorins that is expressed by neurons and endothelial cells. NRP1 is required for normal developmental angiogenesis in mice. The zebrafish is an excellent system for analyzing vascular development. Zebrafish intersegmental vessels correspond to mammalian capillary sprouts, whereas the axial vessels correspond to larger blood vessels, such as arteries. The zebrafish NRP1 gene (znrp1) was isolated and when overexpressed in cells, zNRP1 protein was a functional receptor for human VEGF(165). Whole-mount in situ hybridization showed that transcripts for znrp1 during embryonic and early larval development were detected mainly in neuronal and vascular tissues. Morpholino-mediated knockdown of zNRP1 in embryos resulted in vascular defects, most notably impaired circulation in the intersegmental vessels. Circulation via trunk axial vessels was not affected. Embryos treated with VEGF receptor-2 kinase inhibitor had a similar intersegmental vessel defect suggesting that knockdown of zNRP1 reduces VEGF activity. To determine whether NRP1 and VEGF activities were interdependent in vivo, zNRP1 and VEGF morpholinos were coinjected into embryos at concentrations that individually did not significantly inhibit blood vessel development. The result was a potent inhibition of blood cell circulation via both intersegmental and axial vessels demonstrating that VEGF and NRP1 act synergistically to promote a functional circulatory system. These results provide the first physiological demonstration that NRP1 regulates angiogenesis through a VEGF-dependent pathway. -
Lieschke GJ, Oates AC, Paw BH, Thompson MA, Hall NE, Ward AC, Ho RK, Zon LI, Layton JE. 2002. Zebrafish SPI-1 (PU.1) marks a site of myeloid development independent of primitive erythropoiesis: implications for axial patterning. Developmental biology. 246(2):274-95. Pubmed: 12051816 Lieschke GJ, Oates AC, Paw BH, Thompson MA, Hall NE, Ward AC, Ho RK, Zon LI, Layton JE. 2002. Zebrafish SPI-1 (PU.1) marks a site of myeloid development independent of primitive erythropoiesis: implications for axial patterning. Developmental biology. 246(2):274-95. Pubmed: 12051816 The mammalian transcription factor SPI-1 (synonyms: SPI1, PU.1, or Sfpi1) plays a critical role in myeloid development. To examine early myeloid commitment in the zebrafish embryo, we isolated a gene from zebrafish that is a SPI-1 orthologue on the basis of homology and phylogenetic considerations. The zebrafish spi1 (pu1) gene was first expressed at 12 h postfertilization in rostral lateral plate mesoderm (LPM), anatomically isolated from erythroid development in caudal lateral plate mesoderm. Fate-mapping traced rostral LPM cells from the region of initial spi1 expression to a myeloid fate. spi1 expression was lost in the bloodless mutant cloche, but rostral spi1 expression and myeloid development were preserved in the mutant spadetail, despite its complete erythropoietic failure. This dissociation of myeloid and erythroid development was further explored in studies of embryos overexpressing BMP-4, or chordin, in bmp-deficient swirl and snailhouse mutants, and chordin-deficient chordino mutants. These studies demonstrate that, in zebrafish, spi1 marks a rostral population of LPM cells committed to a myeloid fate anatomically separated from and developmentally independent of erythroid commitment in the caudal LPM. Such complete anatomical and developmental dissociation of two hematopoietic lineages adds an interesting complexity to the understanding of vertebrate hematopoietic development and presents significant implications for the mechanisms regulating axial patterning. -
Ross S, Best JL, Zon LI, Gill G. 2002. SUMO-1 modification represses Sp3 transcriptional activation and modulates its subnuclear localization. Molecular cell. 10(4):831-42. Pubmed: 12419227 Ross S, Best JL, Zon LI, Gill G. 2002. SUMO-1 modification represses Sp3 transcriptional activation and modulates its subnuclear localization. Molecular cell. 10(4):831-42. Pubmed: 12419227 The GC box binding transcription factor Sp3 both activates and represses transcription. We have found that Sp3 activity is regulated by SUMO-1 modification. Endogenous Sp3 is sumoylated and localized to the nuclear periphery and in nuclear dots. Removal of SUMO-1 from Sp3 by mutation of the SUMO acceptor lysines or expression of the SUMO-1 protease SuPr-1 converted Sp3 to a strong activator with a diffuse nuclear localization. Covalent attachment of SUMO-1 to Sp3 by gene fusion was sufficient to repress Sp3-dependent transcription and relocalize Sp3 to the nuclear periphery and nuclear dots. These studies reveal a direct effect of SUMO-1 modification on activity of a dual function transcription factor and provide a mechanism for functional specificity within the Sp transcription factor family. -
Thisse C, Zon LI. 2002. Organogenesis--heart and blood formation from the zebrafish point of view. Science (New York, N.Y.). 295(5554):457-62. Pubmed: 11799232 Thisse C, Zon LI. 2002. Organogenesis--heart and blood formation from the zebrafish point of view. Science (New York, N.Y.). 295(5554):457-62. Pubmed: 11799232 Organs are specialized tissues used for enhanced physiology and environmental adaptation. The cells of the embryo are genetically programmed to establish organ form and function through conserved developmental modules. The zebrafish is a powerful model system that is poised to contribute to our basic understanding of vertebrate organogenesis. This review develops the theme of modules and illustrates how zebrafish have been particularly useful for understanding heart and blood formation. -
Liao EC, Trede NS, Ransom D, Zapata A, Kieran M, Zon LI. 2002. Non-cell autonomous requirement for the bloodless gene in primitive hematopoiesis of zebrafish. Development (Cambridge, England). 129(3):649-59. Pubmed: 11830566 Liao EC, Trede NS, Ransom D, Zapata A, Kieran M, Zon LI. 2002. Non-cell autonomous requirement for the bloodless gene in primitive hematopoiesis of zebrafish. Development (Cambridge, England). 129(3):649-59. Pubmed: 11830566 Vertebrate hematopoiesis occurs in two distinct phases, primitive (embryonic) and definitive (adult). Genes that are required specifically for the definitive program, or for both phases of hematopoiesis, have been described. However, a specific regulator of primitive hematopoiesis has yet to be reported. The zebrafish bloodless (bls) mutation causes absence of embryonic erythrocytes in a dominant but incompletely penetrant manner. Primitive macrophages appear to develop normally in bls mutants. Although the thymic epithelium forms normally in bls mutants, lymphoid precursors are absent. Nonetheless, the bloodless mutants can progress through embryogenesis, where red cells begin to accumulate after 5 days post-fertilization (dpf). Lymphocytes also begin to populate the thymic organs by 7.5 dpf. Expression analysis of hematopoietic genes suggests that formation of primitive hematopoietic precursors is deficient in bls mutants and those few blood precursors that are specified fail to differentiate and undergo apoptosis. Overexpression of scl, but not bmp4 or gata1, can lead to partial rescue of embryonic blood cells in bls. Cell transplantation experiments show that cells derived from bls mutant donors can differentiate into blood cells in a wild-type host, but wild-type donor cells fail to form blood in the mutant host. These observations demonstrate that the bls gene product is uniquely required in a non-cell autonomous manner for primitive hematopoiesis, potentially acting via regulation of scl. -
Burns CE, DeBlasio T, Zhou Y, Zhang J, Zon L, Nimer SD. 2002. Isolation and characterization of runxa and runxb, zebrafish members of the runt family of transcriptional regulators. Experimental hematology. 30(12):1381-9. Pubmed: 12482499 Burns CE, DeBlasio T, Zhou Y, Zhang J, Zon L, Nimer SD. 2002. Isolation and characterization of runxa and runxb, zebrafish members of the runt family of transcriptional regulators. Experimental hematology. 30(12):1381-9. Pubmed: 12482499 Array -
Burns CE, Zon LI. 2002. Portrait of a stem cell. Developmental cell. 3(5):612-3. Pubmed: 12431368 Burns CE, Zon LI. 2002. Portrait of a stem cell. Developmental cell. 3(5):612-3. Pubmed: 12431368 There is great enthusiasm for the potential use of stem cells in treating tissue degenerative disorders, but little is known about the intrinsic molecular programs defining self-renewal and differentiation. New data sets produced by transcriptional profiling of purified stem cell populations begin to establish the nature of "stemness." -
Zhu H, Zon LI. 2002. Use of zebrafish models for the analysis of human disease. Current protocols in human genetics. Chapter 15:Unit 15.3. Pubmed: 18428328 DOI:10.1002/0471142905.hg1503s34 Zhu H, Zon LI. 2002. Use of zebrafish models for the analysis of human disease. Current protocols in human genetics. Chapter 15:Unit 15.3. Pubmed: 18428328 DOI:10.1002/0471142905.hg1503s34 The zebrafish has emerged as a powerful animal model for human diseases. While it has long informed us about the biology of early development, it has recently come into favor for the investigation of clinically relevant problems. Genes conserved from fish to humans can be rapidly analyzed using the zebrafish embryo in what is essentially a transparent in vivo assay. This unit describes methodologies including genetic screening, targeted knockdowns, ectopic overexpression, and transgenesis. -
Pratt SJ, Drejer A, Foott H, Barut B, Brownlie A, Postlethwait J, Kato Y, Yamamoto M, Zon LI. 2002. Isolation and characterization of zebrafish NFE2. Physiological genomics. 11(2):91-8. Pubmed: 12388799 Pratt SJ, Drejer A, Foott H, Barut B, Brownlie A, Postlethwait J, Kato Y, Yamamoto M, Zon LI. 2002. Isolation and characterization of zebrafish NFE2. Physiological genomics. 11(2):91-8. Pubmed: 12388799 Vertebrate hematopoiesis is regulated by distinct cell-specific transcription factors such as GATA-1 and SCL. Mammalian p45-NFE2 was characterized for its ability to bind the hypersensitive sites of the globin locus control region. NFE2 is a member of a cap'n'collar (CNC) and basic zipper (BZIP) superfamily that regulates gene transcription. It has been implicated in diverse processes such as globin gene expression, oxidative stress, and platelet lineage differentiation. Here, we have isolated the zebrafish ortholog of NFE2. The gene is highly homologous, particularly in the DNA-binding domain. Mapping the zebrafish NFE2 to linkage group 23 establishes a region of chromosomal synteny with human chromosome 12, further suggesting evolutionary conservation. During embryogenesis, the zebrafish gene is expressed specifically in erythroid cells and also in the developing ear. NFE2 expression is lacking in zebrafish mutants that have no hematopoietic cells. An analysis of the sauternes mutant, which carries a mutation in the ALAS-2 gene and thus has defective heme synthesis, demonstrates higher levels of NFE2 expression than normal. This further establishes the block to erythroid differentiation in the sauternes mutant. Our studies demonstrate conservation of the vertebrate genetic program for the erythroid lineage. -
Orkin SH, Zon LI. 2002. Hematopoiesis and stem cells: plasticity versus developmental heterogeneity. Nature immunology. 3(4):323-8. Pubmed: 11919568 Orkin SH, Zon LI. 2002. Hematopoiesis and stem cells: plasticity versus developmental heterogeneity. Nature immunology. 3(4):323-8. Pubmed: 11919568 Hematopoietic stem cells (HSCs) provide for blood formation throughout the life of the individual. Studies of HSCs form a conceptual framework for the analysis of stem cells of other organ systems. We review here the origin of HSCs during embryological development, the relationship between hematopoiesis and vascular development and the potential plasticity of HSCs and other tissue stem cells. Recent experiments in the mouse have been widely interpreted as evidence for unprecedented transdifferentiation of tissue stem cells. The use of enriched, but impure, cell populations allows for alternative interpretation. In considering these findings, we draw a distinction here between the plasticity of adult stem cells and the heterogeneity of stem cell types that pre-exist within tissues. -
Amatruda JF, Shepard JL, Stern HM, Zon LI. 2002. Zebrafish as a cancer model system. Cancer cell. 1(3):229-31. Pubmed: 12086858 Amatruda JF, Shepard JL, Stern HM, Zon LI. 2002. Zebrafish as a cancer model system. Cancer cell. 1(3):229-31. Pubmed: 12086858 The zebrafish, with its combination of forward genetics and vertebrate biology, has great potential as a cancer model system. -
Liu TX, Zhou Y, Kanki JP, Deng M, Rhodes J, Yang HW, Sheng XM, Zon LI, Look AT. 2002. Evolutionary conservation of zebrafish linkage group 14 with frequently deleted regions of human chromosome 5 in myeloid malignancies. Proceedings of the National Academy of Sciences of the United States of America. 99(9):6136-41. Pubmed: 11983906 Liu TX, Zhou Y, Kanki JP, Deng M, Rhodes J, Yang HW, Sheng XM, Zon LI, Look AT. 2002. Evolutionary conservation of zebrafish linkage group 14 with frequently deleted regions of human chromosome 5 in myeloid malignancies. Proceedings of the National Academy of Sciences of the United States of America. 99(9):6136-41. Pubmed: 11983906 Recurring interstitial loss of all or part of the long arm of chromosome 5, del(5q), is a hallmark of myelodysplastic syndrome and acute myeloid leukemia. Although the genes affected by these changes have not been identified, two critically deleted regions (CDRs) are well established. We have identified 76 zebrafish cDNAs orthologous to genes located in these 5q CDRs. Radiation hybrid mapping revealed that 33 of the 76 zebrafish orthologs are clustered in a genomic region on linkage group 14 (LG14). Fifteen others are located on LG21, and two on LG10. Although there are large blocks of conserved syntenies, the gene order between human and zebrafish is extensively inverted and transposed. Thus, intrachromosomal rearrangements and inversions appear to have occurred more frequently than translocations during evolution from a common chordate ancestor. Interestingly, of the 33 orthologs located on LG14, three have duplicates on LG21, suggesting that the duplication event occurred early in the evolution of teleosts. Murine orthologs of human 5q CDR genes are distributed among three chromosomes, 18, 11, and 13. The order of genes within the three syntenic mouse chromosomes appears to be more colinear with the human order, suggesting that translocations occurred more frequently than inversions during mammalian evolution. Our comparative map should enhance understanding of the evolution of the del(5q) chromosomal region. Mutant fish harboring deletions affecting the 5q CDR syntenic region may provide useful animal models for investigating the pathogenesis of myelodysplastic syndrome and acute myeloid leukemia. -
Traver D, Zon LI. 2002. Walking the walk: migration and other common themes in blood and vascular development. Cell. 108(6):731-4. Pubmed: 11955426 Traver D, Zon LI. 2002. Walking the walk: migration and other common themes in blood and vascular development. Cell. 108(6):731-4. Pubmed: 11955426 In this issue of Cell, a study by N. Cho and coworkers provides insight into the role of vascular endothelial growth factor (VEGF) signaling in Drosophila hematopoiesis. Their work suggests that an ancestral function of VEGF was to guide blood cell migration and highlights the conservation of at least one aspect of VEGF signaling during evolution. 2001
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Lyons SE, Shue BC, Lei L, Oates AC, Zon LI, Liu PP. 2001. Molecular cloning, genetic mapping, and expression analysis of four zebrafish c/ebp genes. Gene. 281(1-2):43-51. Pubmed: 11750126 Lyons SE, Shue BC, Lei L, Oates AC, Zon LI, Liu PP. 2001. Molecular cloning, genetic mapping, and expression analysis of four zebrafish c/ebp genes. Gene. 281(1-2):43-51. Pubmed: 11750126 The CCAAT/enhancer binding protein family (C/EBP) are transcription factors that play integral roles in the development and function of many organ systems, including hematopoietic cells, adipose tissues, and liver. We have identified and characterized putative zebrafish orthologs of mammalian C/EBP alpha, beta, gamma, and delta using low-stringency hybridization screening and computer searches of the GenBank EST database. c/ebpa and g were mapped within 1 cM of each other on linkage group (LG) 7, syntenic with human CEBPA and G genes on chromosome 19. c/ebpb was mapped to LG8, and c/ebpd was mapped to LG24, on the same LG as a recently identified unique c/ebp in zebrafish, c/ebp1. The mapping of these genes established new syntenic relationships between LG8 and human chromosome 20, extended existing synteny between LG7 and human chromosome 19, and confirmed the synteny between LG24 and human chromosome 8. In addition, these syntenies between zebrafish and human chromosomes are also conserved in the mouse genome. To characterize the expression of these genes, RNA in situ hybridization in embryos of wild type and a hematopoietic mutant, cloche, was performed. The results showed that zebrafish c/ebpa, b, g, and d were expressed in many embryonic tissues. c/ebpa and b were expressed in a subset of hematopoietic cells in a region consistent with myeloid expression. In addition, there was expression of c/ebpa and b in the liver and c/ebpa, b, and d in regions of the gastrointestinal tract. The expression of the c/ebps may serve as important markers for analysis of myelopoiesis, hepatic development, and other developmental processes in the future. -
Hukriede N, Fisher D, Epstein J, Joly L, Tellis P, Zhou Y, Barbazuk B, Cox K, Fenton-Noriega L, Hersey C, Miles J, Sheng X, Song A, Waterman R, Johnson SL, Dawid IB, Chevrette M, Zon LI, McPherson J, Ekker M. 2001. The LN54 radiation hybrid map of zebrafish expressed sequences. Genome research. 11(12):2127-32. Pubmed: 11731504 Hukriede N, Fisher D, Epstein J, Joly L, Tellis P, Zhou Y, Barbazuk B, Cox K, Fenton-Noriega L, Hersey C, Miles J, Sheng X, Song A, Waterman R, Johnson SL, Dawid IB, Chevrette M, Zon LI, McPherson J, Ekker M. 2001. The LN54 radiation hybrid map of zebrafish expressed sequences. Genome research. 11(12):2127-32. Pubmed: 11731504 To increase the density of a gene map of the zebrafish, Danio rerio, we have placed 3119 expressed sequence tags (ESTs) and cDNA sequences on the LN54 radiation hybrid (RH) panel. The ESTs and genes mapped here join 748 SSLp markers and 459 previously mapped genes and ESTs, bringing the total number of markers on the LN54 RH panel to 4226. Addition of these new markers brings the total LN54 map size to 14,372 cR, with 118 kb/cR. The distribution of ESTs according to linkage groups shows relatively little variation (minimum, 73; maximum, 201). This observation, combined with a relatively uniform size for zebrafish chromosomes, as previously indicated by karyotyping, indicates that there are no especially gene-rich or gene-poor chromosomes in this species. We developed an algorithm to provide a semiautomatic method for the selection of additional framework markers for the LN54 map. This algorithm increased the total number of framework markers to 1150 and permitted the mapping of a high percentage of sequences that could not be placed on a previous version of the LN54 map. The increased concentration of expressed sequences on the LN54 map of the zebrafish genome will facilitate the molecular characterization of mutations in this species. -
Lyons SE, Shue BC, Oates AC, Zon LI, Liu PP. 2001. A novel myeloid-restricted zebrafish CCAAT/enhancer-binding protein with a potent transcriptional activation domain. Blood. 97(9):2611-7. Pubmed: 11313249 Lyons SE, Shue BC, Oates AC, Zon LI, Liu PP. 2001. A novel myeloid-restricted zebrafish CCAAT/enhancer-binding protein with a potent transcriptional activation domain. Blood. 97(9):2611-7. Pubmed: 11313249 The CCAAT/enhancer-binding protein (C/EBP) family consists of transcription factors essential for hematopoiesis. The defining feature of the C/EBPs is a highly conserved carboxy-terminal bZIP domain that is necessary and sufficient for dimerization and DNA binding, whereas their amino-terminal domains are unique. This study reports a novel c/ebp gene (c/ebp1) from zebrafish that encodes a protein homologous to mammalian C/EBPs within the bZIP domain, but with an amino terminus lacking homology to any C/EBP or to any known sequence. In zebrafish embryos, c/ebp1 expression was initially observed in cells within the yolk sac circulation valley at approximately the 16-to 18-somite stage, and at 24 hours postfertilization (hpf), also in circulating cells. Most c/ebp1(+) cells also expressed a known early macrophage marker, leukocyte-specific plastin (l-plastin). Expression of both markers was lost in cloche, a mutant affecting hematopoiesis at the level of the hemangioblast. Expression of both markers was retained in m683 and spadetail, mutants affecting erythropoiesis, but not myelopoiesis. Further, c/ebp1 expression was lost in a mutant with defective myelopoiesis, but intact erythropoiesis. These data suggest that c/ebp1 is expressed exclusively in myeloid cells. In electrophoretic mobility shift assays, c/ebp1 was able to bind a C/EBP consensus DNA site. Further, a chimeric protein containing the amino-terminal domain of c/ebp1 fused to the DNA-binding domain of GAL4 induced a GAL4 reporter 4000-fold in NIH3T3 cells. These results suggest that c/ebp1 is a novel member of the C/EBP family that may function as a potent transcriptional activator in myeloid cells. -
Patton EE, Zon LI. 2001. The art and design of genetic screens: zebrafish. Nature reviews. Genetics. 2(12):956-66. Pubmed: 11733748 Patton EE, Zon LI. 2001. The art and design of genetic screens: zebrafish. Nature reviews. Genetics. 2(12):956-66. Pubmed: 11733748 Inventive genetic screens in zebrafish are revealing new genetic pathways that control vertebrate development, disease and behaviour. By exploiting the versatility of zebrafish, biological processes that had been previously obscured can be visualized and many of the responsible genes can be isolated. Coupled with gene knockdown and overexpression technologies, and small-molecule-induced phenotypes, genetic screens in zebrafish provide a powerful system by which to dissect vertebrate gene function and gene networks. -
Bennett CM, Kanki JP, Rhodes J, Liu TX, Paw BH, Kieran MW, Langenau DM, Delahaye-Brown A, Zon LI, Fleming MD, Look AT. 2001. Myelopoiesis in the zebrafish, Danio rerio. Blood. 98(3):643-51. Pubmed: 11468162 Bennett CM, Kanki JP, Rhodes J, Liu TX, Paw BH, Kieran MW, Langenau DM, Delahaye-Brown A, Zon LI, Fleming MD, Look AT. 2001. Myelopoiesis in the zebrafish, Danio rerio. Blood. 98(3):643-51. Pubmed: 11468162 Genome-wide chemical mutagenesis screens in the zebrafish (Danio rerio) have led to the identification of novel genes affecting vertebrate erythropoiesis. In determining if this approach could also be used to clarify the molecular genetics of myelopoiesis, it was found that the developmental hierarchy of myeloid precursors in the zebrafish kidney is similar to that in human bone marrow. Zebrafish neutrophils resembled human neutrophils, possessing segmented nuclei and myeloperoxidase-positive cytoplasmic granules. The zebrafish homologue of the human myeloperoxidase (MPO) gene, which is specific to cells of the neutrophil lineage, was cloned and used to synthesize antisense RNA probes for in situ hybridization analyses of zebrafish embryos. Granulocytic cells expressing zebrafish mpo were first evident at 18 hours after fertilization (hpf) in the posterior intermediate cell mass (ICM) and on the anterior yolk sac by 20 hpf. By 24 hpf, mpo-expressing cells were observed along the ICM and within the developing vascular system. Thus, the mpo gene should provide a useful molecular probe for identifying zebrafish mutants with defects in granulopoiesis. The expression of zebrafish homologues was also examined in 2 other mammalian hematopoietic genes, Pu.1, which appears to initiate a commitment step in normal mammalian myeloid development, and L-Plastin, a gene expressed by human monocytes and macrophages. The results demonstrate a high level of conservation of the spatio-temporal expression patterns of these genes between zebrafish and mammals. The morphologic and molecular genetic evidence presented here supports the zebrafish as an informative model system for the study of normal and aberrant human myelopoiesis. (Blood. 2001;98:643-651) -
Mead PE, Deconinck AE, Huber TL, Orkin SH, Zon LI. 2001. Primitive erythropoiesis in the Xenopus embryo: the synergistic role of LMO-2, SCL and GATA-binding proteins. Development (Cambridge, England). 128(12):2301-8. Pubmed: 11493549 Mead PE, Deconinck AE, Huber TL, Orkin SH, Zon LI. 2001. Primitive erythropoiesis in the Xenopus embryo: the synergistic role of LMO-2, SCL and GATA-binding proteins. Development (Cambridge, England). 128(12):2301-8. Pubmed: 11493549 Hematopoietic stem cells are derived from ventral mesoderm during vertebrate development. Gene targeting experiments in the mouse have demonstrated key roles for the basic helix-loop-helix transcription factor SCL and the GATA-binding protein GATA-1 in hematopoiesis. When overexpressed in Xenopus animal cap explants, SCL and GATA-1 are each capable of specifying mesoderm to become blood. Forced expression of either factor in whole embryos, however, does not lead to ectopic blood formation. This apparent paradox between animal cap assays and whole embryo phenotype has led to the hypothesis that additional factors are involved in specifying hematopoietic mesoderm. SCL and GATA-1 interact in a transcriptional complex with the LIM domain protein LMO-2. We have cloned the Xenopus homolog of LMO-2 and show that it is expressed in a similar pattern to SCL during development. LMO-2 can specify hematopoietic mesoderm in animal cap assays. SCL and LMO-2 act synergistically to expand the blood island when overexpressed in whole embryos. Furthermore, co-expression of GATA-1 with SCL and LMO-2 leads to embryos that are ventralized and have blood throughout the dorsal-ventral axis. The synergistic effect of SCL, LMO-2 and GATA-1, taken together with the findings that these factors can form a complex in vitro, suggests that this complex specifies mesoderm to become blood during embryogenesis. -
Chayama K, Papst PJ, Garrington TP, Pratt JC, Ishizuka T, Webb S, Ganiatsas S, Zon LI, Sun W, Johnson GL, Gelfand EW. 2001. Role of MEKK2-MEK5 in the regulation of TNF-alpha gene expression and MEKK2-MKK7 in the activation of c-Jun N-terminal kinase in mast cells. Proceedings of the National Academy of Sciences of the United States of America. 98(8):4599-604. Pubmed: 11274363 Chayama K, Papst PJ, Garrington TP, Pratt JC, Ishizuka T, Webb S, Ganiatsas S, Zon LI, Sun W, Johnson GL, Gelfand EW. 2001. Role of MEKK2-MEK5 in the regulation of TNF-alpha gene expression and MEKK2-MKK7 in the activation of c-Jun N-terminal kinase in mast cells. Proceedings of the National Academy of Sciences of the United States of America. 98(8):4599-604. Pubmed: 11274363 Cross-linking of the high-affinity IgE receptor (FcepsilonRI) on mast cells with IgE and multivalent antigen triggers mitogen-activated protein (MAP) kinase activation and cytokine gene expression. We report here that MAP kinase kinase 4 (MKK4) gene disruption does not affect either MAP kinase activation or cytokine gene expression in response to cross-linking of FcepsilonRI in embryonic stem cell-derived mast cells. MKK7 is activated in response to cross-linking of FcepsilonRI, and this activation is inhibited by MAP/ERK kinase (MEK) kinase 2 (MEKK2) gene disruption. In addition, expression of kinase-inactive MKK7 in the murine mast cell line MC/9 inhibits c-Jun NH(2)-terminal kinase (JNK) activation in response to cross-linking of FcepsilonRI, whereas expression of kinase-inactive MKK4 does not affect JNK activation by this stimulus. However, FcepsilonRI-induced activation of the tumor necrosis factor-alpha (TNF-alpha) gene promoter is not affected by expression of kinase-inactive MKK7. We describe an alternative pathway by which MEKK2 activates MEK5 and big MAP kinase1/extracellular signal-regulated kinase 5 in addition to MKK7 and JNK, and interruption of this pathway inhibits TNF-alpha promoter activation. These findings suggest that JNK activation by antigen cross-linking is dependent on the MEKK2-MKK7 pathway, and cytokine production in mast cells is regulated in part by the signaling complex MEKK2-MEK5-ERK5. -
Zon LI. 2001. Self-renewal versus differentiation, a job for the mighty morphogens. Nature immunology. 2(2):142-3. Pubmed: 11175810 Zon LI. 2001. Self-renewal versus differentiation, a job for the mighty morphogens. Nature immunology. 2(2):142-3. Pubmed: 11175810 -
Miyoshi K, Cui Y, Riedlinger G, Robinson P, Lehoczky J, Zon L, Oka T, Dewar K, Hennighausen L. 2001. Structure of the mouse Stat 3/5 locus: evolution from Drosophila to zebrafish to mouse. Genomics. 71(2):150-5. Pubmed: 11161808 Miyoshi K, Cui Y, Riedlinger G, Robinson P, Lehoczky J, Zon L, Oka T, Dewar K, Hennighausen L. 2001. Structure of the mouse Stat 3/5 locus: evolution from Drosophila to zebrafish to mouse. Genomics. 71(2):150-5. Pubmed: 11161808 Signal transducers and activators of transcription (Stat) are transcription factors that can be activated by many cytokines. While Drosophila contains only one Stat (d-Stat), mammals contain seven, with STATs 3, 5a, and 5b being the closest functional relatives. To understand the evolutionary relationship between d-Stat and vertebrate STATs 3 and 5, we isolated, sequenced, and analyzed the zebrafish Stat3 (z-Stat3) gene and a 500-kb region spanning mouse chromosome 11, 60.5 cM containing three Stat genes (m-Stats). Within this region we identified the genes encoding m-Stats 3, 5a, and 5b, Cnp1, Hcrt/Orexin, Ptrf, GCN5, mDj11, and four new genes. The 5' ends of the m-Stat5a and m-Stat5b genes are juxtaposed to each other, and the 3' ends of the m-Stat3 and Stat5a genes face each other. While the m-Stat5a and m-Stat3 genes have one promoter each, which are active in many tissues, the m-Stat5b gene acquired two distinct promoters. The distal promoter is expressed ubiquitously, and transcription from the proximal promoter is restricted to liver, muscle, and mammary tissue. Through a comparison of exon-intron boundaries from the m-Stat3, m-Stat5a, and m-Stat5b, z-Stat3, and d-Stat genes, we deduced their evolutionary relationship. We propose that the Stat3 and Stat5 lineages are derived from the duplication of a common primordial gene and that d-Stat is a part of the Stat5 lineage.Copyright 2001 Academic Press. -
Bahary N, Zon LI. 2001. Development. Endothelium--chicken soup for the endoderm. Science (New York, N.Y.). 294(5542):530-1. Pubmed: 11577202 Bahary N, Zon LI. 2001. Development. Endothelium--chicken soup for the endoderm. Science (New York, N.Y.). 294(5542):530-1. Pubmed: 11577202 Endothelial cells in blood vessels are known to be important during the later stages of organ development in the embryo. However, their involvement at the induction stage of organ formation has not been previously documented. As Bahary and Zon explain in their Perspective, new work demonstrates that endothelial cells secrete factors early in development that induce embryonic endoderm to become liver or pancreas (Matsumoto et al., Lammert et al.). -
Huber TL, Perkins AC, Deconinck AE, Chan FY, Mead PE, Zon LI. 2001. neptune, a Krüppel-like transcription factor that participates in primitive erythropoiesis in Xenopus. Current biology : CB. 11(18):1456-61. Pubmed: 11566106 Huber TL, Perkins AC, Deconinck AE, Chan FY, Mead PE, Zon LI. 2001. neptune, a Krüppel-like transcription factor that participates in primitive erythropoiesis in Xenopus. Current biology : CB. 11(18):1456-61. Pubmed: 11566106 The specification of the erythroid lineage from hematopoietic stem cells requires the expression and activity of lineage-specific transcription factors. One transcription factor family that has several members involved in hematopoiesis is the Krüppel-like factor (KLF) family [1]. For example, erythroid KLF (EKLF) regulates beta-globin expression during erythroid differentiation [2-6]. KLFs share a highly conserved zinc finger-based DNA binding domain (DBD) that mediates binding to CACCC-box and GC-rich sites, both of which are frequently found in the promoters of hematopoietic genes. Here, we identified a novel Xenopus KLF gene, neptune, which is highly expressed in the ventral blood island (VBI), cranial ganglia, and hatching and cement glands. neptune expression is induced in response to components of the BMP-4 signaling pathway in injected animal cap explants. Similar to its family member, EKLF, Neptune can bind CACCC-box and GC-rich DNA elements. We show that Neptune cooperates with the hematopoietic transcription factor XGATA-1 to enhance globin induction in animal cap explants. A fusion protein comprised of Neptune's DBD and the Drosophila engrailed repressor domain suppresses the induction of globin in ventral marginal zones and in animal caps. These studies demonstrate that Neptune is a positive regulator of primitive erythropoiesis in Xenopus. -
Oates AC, Pratt SJ, Vail B, Yan Yl, Ho RK, Johnson SL, Postlethwait JH, Zon LI. 2001. The zebrafish klf gene family. Blood. 98(6):1792-801. Pubmed: 11535513 Oates AC, Pratt SJ, Vail B, Yan Yl, Ho RK, Johnson SL, Postlethwait JH, Zon LI. 2001. The zebrafish klf gene family. Blood. 98(6):1792-801. Pubmed: 11535513 The Krüppel-like factor (KLF) family of genes encodes transcriptional regulatory proteins that play roles in differentiation of a diverse set of cells in mammals. For instance, the founding member KLF1 (also known as EKLF) is required for normal globin production in mammals. Five new KLF genes have been isolated from the zebrafish, Danio rerio, and the structure of their products, their genetic map positions, and their expression during development of the zebrafish have been characterized. Three genes closely related to mammalian KLF2 and KLF4 were found, as was an ortholog of mammalian KLF12. A fifth gene, apparently missing from the genome of mammals and closely related to KLF1 and KLF2, was also identified. Analysis demonstrated the existence of novel conserved domains in the N-termini of these proteins. Developmental expression patterns suggest potential roles for these zebrafish genes in diverse processes, including hematopoiesis, blood vessel function, and fin and epidermal development. The studies imply a high degree of functional conservation of the zebrafish genes with their mammalian homologs. These findings further the understanding of the KLF genes in vertebrate development and indicate an ancient role in hematopoiesis for the Krüppel-like factor gene family. -
Moretti PA, Davidson AJ, Baker E, Lilley B, Zon LI, D'Andrea RJ. 2001. Molecular cloning of a human Vent-like homeobox gene. Genomics. 76(1-3):21-9. Pubmed: 11549314 Moretti PA, Davidson AJ, Baker E, Lilley B, Zon LI, D'Andrea RJ. 2001. Molecular cloning of a human Vent-like homeobox gene. Genomics. 76(1-3):21-9. Pubmed: 11549314 We have isolated a previously unknown human homeobox-containing cDNA, VENT-like homeobox-2 (VENTX2), using PCR with a bone marrow cDNA library and primers designed from the VENTX1 (alias HPX42) homeobox sequence. Here we describe the molecular cloning, chromosomal localization to 10q26.3, and functional analysis of this gene. The 2.4-kb human VENTX2 cDNA encoded a protein with a predicted molecular weight of 28 kDa containing a homeodomain with 65% identity to the Xenopus laevis ventralizing gene Xvent2B. VENTX2 antisera detected a 28-kDa protein in cells transfected with a VENTX2 expression construct, in a human erythroleukemic cell line and in bone marrow samples obtained from patients in recovery phase after chemotherapy. The similarity of the homeodomains from VENTX2 and the X. laevis Vent gene family places them in the same homeodomain class. Consistent with this structural classification, overexpression of VENTX2 in zebrafish embryos led to anterior truncations and failure to form a notochord, which are characteristics of ventralization. -
Hamazaki T, Iiboshi Y, Oka M, Papst PJ, Meacham AM, Zon LI, Terada N. 2001. Hepatic maturation in differentiating embryonic stem cells in vitro. FEBS letters. 497(1):15-9. Pubmed: 11376655 Hamazaki T, Iiboshi Y, Oka M, Papst PJ, Meacham AM, Zon LI, Terada N. 2001. Hepatic maturation in differentiating embryonic stem cells in vitro. FEBS letters. 497(1):15-9. Pubmed: 11376655 We investigated the potential of mouse embryonic stem (ES) cells to differentiate into hepatocytes in vitro. Differentiating ES cells expressed endodermal-specific genes, such as alpha-fetoprotein, transthyretin, alpha 1-anti-trypsin and albumin, when cultured without additional growth factors and late differential markers of hepatic development, such as tyrosine aminotransferase (TAT) and glucose-6-phosphatase (G6P), when cultured in the presence of growth factors critical for late embryonic liver development. Further, induction of TAT and G6P expression was induced regardless of expression of the functional SEK1 gene, which is thought to provide a survival signal for hepatocytes during an early stage of liver morphogenesis. The data indicate that the in vitro ES differentiation system has a potential to generate mature hepatocytes. The system has also been found useful in analyzing the role of growth factors and intracellular signaling molecules in hepatic development. -
Trede NS, Zapata A, Zon LI. 2001. Fishing for lymphoid genes. Trends in immunology. 22(6):302-7. Pubmed: 11377288 Trede NS, Zapata A, Zon LI. 2001. Fishing for lymphoid genes. Trends in immunology. 22(6):302-7. Pubmed: 11377288 Thymic organogenesis and T-cell lymphopoiesis are crucial interdependent processes that establish a functional vertebrate immune system. The current understanding of vertebrate thymic development during embryogenesis remains incomplete and would benefit from novel approaches. The zebrafish Danio rerio is a powerful developmental and genetic system for the dissection of early events in the ontogeny of the immune system. Forward genetic screens have uncovered genes involved in hematopoiesis, and specific screens are being designed to examine the genes that regulate T-cell development and the origin of the thymus. Studies of the zebrafish should improve our understanding of lymphoid development in vertebrates. 2000
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Dooley K, Zon LI. 2000. Zebrafish: a model system for the study of human disease. Current opinion in genetics & development. 10(3):252-6. Pubmed: 10826982 Dooley K, Zon LI. 2000. Zebrafish: a model system for the study of human disease. Current opinion in genetics & development. 10(3):252-6. Pubmed: 10826982 The zebrafish (Danio rerio) is a powerful model organism for the study of vertebrate biology, being well suited to both developmental and genetic analysis. Large-scale genetic screens have identified hundreds of mutant phenotypes, many of which resemble human clinical disorders. The creation of critical genetic reagents, coupled with the rapid progress of the zebrafish genome initiative directed by the National Institutes of Health, are bringing this model system to its full potential for the study of vertebrate biology, physiology and human disease. -
Shepard JL, Zon LI. 2000. Developmental derivation of embryonic and adult macrophages. Current opinion in hematology. 7(1):3-8. Pubmed: 10608497 Shepard JL, Zon LI. 2000. Developmental derivation of embryonic and adult macrophages. Current opinion in hematology. 7(1):3-8. Pubmed: 10608497 The macrophage cell lineage continually arises from hematopoietic stem cells during embryonic, fetal, and adult life. Previous theories proposed that macrophages are the recent progeny of bone marrow-derived monocytes and that they function primarily in phagocytosis. More recently, however, observations have shown that the ontogeny of macrophages in early mouse and human embryos is different from that occurring during adult development, and that the embryonic macrophages do not follow the monocyte pathway. Fetal macrophages are thought to differentiate from yolk sac-derived primitive macrophages before the development of adult monocytes. Further support for a separate lineage of fetal macrophages has come from studies of several species, including chicken, zebrafish, Xenopus, Drosophila, and C. elegans. The presence of fetal macrophages in PU.1-null mice indicates their independence from monocyte precursors and their existence as an alternative macrophage lineage. -
Deconinck AE, Mead PE, Tevosian SG, Crispino JD, Katz SG, Zon LI, Orkin SH. 2000. FOG acts as a repressor of red blood cell development in Xenopus. Development (Cambridge, England). 127(10):2031-40. Pubmed: 10769228 Deconinck AE, Mead PE, Tevosian SG, Crispino JD, Katz SG, Zon LI, Orkin SH. 2000. FOG acts as a repressor of red blood cell development in Xenopus. Development (Cambridge, England). 127(10):2031-40. Pubmed: 10769228 Members of the GATA family of zinc-finger transcription factors have critical roles in a variety of cell types. GATA-1, GATA-2 and GATA-3 are required for proliferation and differentiation of several hematopoietic lineages, whereas GATA-4, GATA-5 and GATA-6 activate cardiac and endoderm gene expression programs. Two GATA cofactors have recently been identified. Friend of GATA-1 (FOG-1) interacts with GATA-1 and is expressed principally in hematopoietic lineages, whereas FOG-2 is expressed predominantly in heart and brain. Although gene targeting experiments are consistent with an essential role for FOG-1 as an activator of GATA-1 function, reporter assays in transfected cells indicate that FOG-1 and FOG-2 can act as repressors. We have cloned a Xenopus laevis homologue of FOG that is structurally most similar to FOG-1, but is expressed predominantly in heart and brain, as well as the ventral blood island and adult spleen. Ectopic expression and explant assays demonstrate that FOG proteins can act as repressors in vivo, in part through interaction with the transcriptional co-repressor, C-terminal Binding Protein (CtBP). FOG may regulate the differentiation of red blood cells by modulating expression and activity of GATA-1 and GATA-2. We propose that the FOG proteins participate in the switch from progenitor proliferation to red blood cell maturation and differentiation. -
Barut BA, Zon LI. 2000. Realizing the potential of zebrafish as a model for human disease. Physiological genomics. 2(2):49-51. Pubmed: 11015581 Barut BA, Zon LI. 2000. Realizing the potential of zebrafish as a model for human disease. Physiological genomics. 2(2):49-51. Pubmed: 11015581 The value of the zebrafish (Danio rerio) as a model for human disease has been substantiated by a number of recently published papers. Several zebrafish mutants with "human" diseases have been found, spanning a variety of human pathologies. These successful studies utilizing the zebrafish have been made possible by the development of key reagents such as YAC, PAC, and BAC libraries, as well as radiation hybrid panels. With the further establishment of new tools and access to the newly generated resources, the zebrafish is poised to serve as a novel model for human disease. -
Paw BH, Zon LI. 2000. Zebrafish: a genetic approach in studying hematopoiesis. Current opinion in hematology. 7(2):79-84. Pubmed: 10698293 Paw BH, Zon LI. 2000. Zebrafish: a genetic approach in studying hematopoiesis. Current opinion in hematology. 7(2):79-84. Pubmed: 10698293 The zebrafish (Danio rerio) has emerged in recent years as an exciting animal model system for studying vertebrate organ development and, in particular, the development of the hematopoietic system. The combined advantages of developmental biology and genetic screens for mutations in zebrafish have provided insights into early events in hematopoiesis and identified several genes required for normal blood development in vertebrates. As a result of the large-scale mutagenesis screens for developmental mutants, several zebrafish mutants with defects in blood development have been recovered. This review discusses how these blood mutations in zebrafish have given new perspectives on hematopoietic development. -
Donovan A, Brownlie A, Zhou Y, Shepard J, Pratt SJ, Moynihan J, Paw BH, Drejer A, Barut B, Zapata A, Law TC, Brugnara C, Lux SE, Pinkus GS, Pinkus JL, Kingsley PD, Palis J, Fleming MD, Andrews NC, Zon LI. 2000. Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter. Nature. 403(6771):776-81. Pubmed: 10693807 Donovan A, Brownlie A, Zhou Y, Shepard J, Pratt SJ, Moynihan J, Paw BH, Drejer A, Barut B, Zapata A, Law TC, Brugnara C, Lux SE, Pinkus GS, Pinkus JL, Kingsley PD, Palis J, Fleming MD, Andrews NC, Zon LI. 2000. Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter. Nature. 403(6771):776-81. Pubmed: 10693807 Defects in iron absorption and utilization lead to iron deficiency and overload disorders. Adult mammals absorb iron through the duodenum, whereas embryos obtain iron through placental transport. Iron uptake from the intestinal lumen through the apical surface of polarized duodenal enterocytes is mediated by the divalent metal transporter, DMTi. A second transporter has been postulated to export iron across the basolateral surface to the circulation. Here we have used positional cloning to identify the gene responsible for the hypochromic anaemia of the zebrafish mutant weissherbst. The gene, ferroportin1, encodes a multiple-transmembrane domain protein, expressed in the yolk sac, that is a candidate for the elusive iron exporter. Zebrafish ferroportin1 is required for the transport of iron from maternally derived yolk stores to the circulation and functions as an iron exporter when expressed in Xenopus oocytes. Human Ferroportin1 is found at the basal surface of placental syncytiotrophoblasts, suggesting that it also transports iron from mother to embryo. Mammalian Ferroportin1 is expressed at the basolateral surface of duodenal enterocytes and could export cellular iron into the circulation. We propose that Ferroportin1 function may be perturbed in mammalian disorders of iron deficiency or overload. -
White RA, Pasztor LM, Richardson PM, Zon LI. 2000. The gene encoding TBC1D1 with homology to the tre-2/USP6 oncogene, BUB2, and cdc16 maps to mouse chromosome 5 and human chromosome 4. Cytogenetics and cell genetics. 89(3-4):272-5. Pubmed: 10965142 White RA, Pasztor LM, Richardson PM, Zon LI. 2000. The gene encoding TBC1D1 with homology to the tre-2/USP6 oncogene, BUB2, and cdc16 maps to mouse chromosome 5 and human chromosome 4. Cytogenetics and cell genetics. 89(3-4):272-5. Pubmed: 10965142 TBC1D1 is the founding member of a family of related proteins with homology to tre-2/UPS6, BUB2, and cdc16 and containing the tbc box motif of 180-220 amino acids. This protein family is thought to have a role in differentiation and in regulating cell growth. We set out to map the TBC1D1 gene in mouse and human. Segregation analysis of a TBC1D1 RFLP in two independent mouse RI (recombinant inbred) lines reveals that mouse Tbc1d1 is closely linked to Pgm1 on chromosome 5. The human TBC1D1 gene was assigned to human chromosome 4p15.1-->4q21 using Southern blot analyses of genomic DNAs from rodent-human somatic cell lines. A human-specific genomic fragment was observed in the somatic cell lines containing human chromosome 4 or the 4p15.1-->4q21 region of the chromosome. TBC1D1 maps to the region containing the ortholog of mouse Pgm1 adding another locus to this long region of conserved synteny between mouse and man.Copyright 2000 S. Karger AG, Basel. -
Davidson AJ, Zon LI. 2000. Turning mesoderm into blood: the formation of hematopoietic stem cells during embryogenesis. Current topics in developmental biology. 50:45-60. Pubmed: 10948449 Davidson AJ, Zon LI. 2000. Turning mesoderm into blood: the formation of hematopoietic stem cells during embryogenesis. Current topics in developmental biology. 50:45-60. Pubmed: 10948449 The formation of hematopoietic stem cells during development occurs by a multistep process that begins with the induction of ventral mesoderm. This mesoderm is patterned during gastrulation by a bone morphogenetic protein (BMP) signaling pathway that is mediated, at least in part, by members of the Mix and Vent families of homeobox transcription factors. Following gastrulation, a subset of ventral mesoderm is specified to become hematopoietic stem cells. Key determinants of hematopoietic fate include the product of the zebrafish cloche gene and the basic helix-loop-helix transcription factor SCL. Future studies in Xenopus and zebrafish should reveal other critical factors in this developmental pathway. -
Blake T, Adya N, Kim CH, Oates AC, Zon L, Chitnis A, Weinstein BM, Liu PP. 2000. Zebrafish homolog of the leukemia gene CBFB: its expression during embryogenesis and its relationship to scl and gata-1 in hematopoiesis. Blood. 96(13):4178-84. Pubmed: 11110689 Blake T, Adya N, Kim CH, Oates AC, Zon L, Chitnis A, Weinstein BM, Liu PP. 2000. Zebrafish homolog of the leukemia gene CBFB: its expression during embryogenesis and its relationship to scl and gata-1 in hematopoiesis. Blood. 96(13):4178-84. Pubmed: 11110689 Mammalian CBFB encodes a transcription factor (CBF beta) that in combination with CBF alpha 2 binds to specific DNA sequences and regulates expression of a number of hematopoietic genes. CBFB is associated with human leukemias through a chromosome 16 inversion and is essential for definitive hematopoiesis during mouse embryo development. We have isolated a zebrafish cbfb complementary DNA (cDNA) clone from a zebrafish kidney cDNA library. This cbfb is highly homologous to human and mouse CBFB/Cbfb genes at both the DNA and protein level. In biochemical analyses, cbfbeta binds to human CBF alpha 2 and enhances its DNA binding. During zebrafish development, cbfb is expressed in the lateral plate mesoderm at tail bud stage and in the intermediate cell mass (ICM, the location of embryonic hematopoiesis) between the 21- to 26-somite stages. The cbfb is also expressed in Rohon-Beard cells, cranial nerve ganglia, hindbrain, retina, branchial arches, jaw, and fin buds. Expression of cbfb is decreased or absent in the ICM and Rohon-Beard cells in some hematopoietic mutants and is unaffected in others. We have also analyzed the expression of scl and gata-1 in the same hematopoietic mutants to ascertain the relative order of these transcription factors to cbfb in zebrafish hematopoiesis. Our results indicate that cbfb is expressed in early hematopoietic progenitors and that its expression pattern in the hematopoietic mutants is similar to that of scl. (Blood. 2000;96:4178-4184) -
MacLean JR, Zon L. 2000. A healthy community through health system partnerships: the approach of Markham Stouffville Hospital. HealthcarePapers. 1(2):96-103; discussion 104-7. Pubmed: 12811070 MacLean JR, Zon L. 2000. A healthy community through health system partnerships: the approach of Markham Stouffville Hospital. HealthcarePapers. 1(2):96-103; discussion 104-7. Pubmed: 12811070 The structure of the Canadian healthcare system, particularly in Ontario, has remained remarkably stable over the past 25 years. No other private sector industry employing hundreds of thousands of people, spending tens of billions of dollars annually and serving millions of consumers every day has survived for 30 years without the need to reinvent itself in quite fundamental ways. How then has the healthcare sector in Canada avoided the pressure to "reinvent itself?" -
Liao EC, Paw BH, Peters LL, Zapata A, Pratt SJ, Do CP, Lieschke G, Zon LI. 2000. Hereditary spherocytosis in zebrafish riesling illustrates evolution of erythroid beta-spectrin structure, and function in red cell morphogenesis and membrane stability. Development (Cambridge, England). 127(23):5123-32. Pubmed: 11060238 Liao EC, Paw BH, Peters LL, Zapata A, Pratt SJ, Do CP, Lieschke G, Zon LI. 2000. Hereditary spherocytosis in zebrafish riesling illustrates evolution of erythroid beta-spectrin structure, and function in red cell morphogenesis and membrane stability. Development (Cambridge, England). 127(23):5123-32. Pubmed: 11060238 Spectrins are key cytoskeleton proteins with roles in membrane integrity, cell morphology, organelle transport and cell polarity of varied cell types during development. Defects in erythroid spectrins in humans result in congenital hemolytic anemias with altered red cell morphology. Although well characterized in mammals and invertebrates, analysis of the structure and function of non-mammalian vertebrate spectrins has been lacking. The zebrafish riesling (ris) suffers from profound anemia, where the developing red cells fail to assume terminally differentiated erythroid morphology. Using comparative genomics, erythroid beta-spectrin (sptb) was identified as the gene mutated in ris. Zebrafish Sptb shares 62.3% overall identity with the human ortholog and phylogenetic comparisons suggest intragenic duplication and divergence during evolution. Unlike the human and murine orthologs, the pleckstrin homology domain of zebrafish Sptb is not removed in red cells by alternative splicing. In addition, apoptosis and abnormal microtubule marginal band aggregation contribute to hemolysis of mutant erythrocytes, which are features not present in mammalian red cells with sptb defects. This study presents the first genetic characterization of a non-mammalian vertebrate sptb and demonstrates novel features of red cell hemolysis in non-mammalian red cells. Further, we propose that the distinct mammalian erythroid morphology may have evolved from specific modifications of Sptb structure and function. -
LaPatra SE, Barone L, Jones GR, Zon LI. 2000. Effects of infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus infection on hematopoietic precursors of the zebrafish. Blood cells, molecules & diseases. 26(5):445-52. Pubmed: 11112382 LaPatra SE, Barone L, Jones GR, Zon LI. 2000. Effects of infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus infection on hematopoietic precursors of the zebrafish. Blood cells, molecules & diseases. 26(5):445-52. Pubmed: 11112382 The zebrafish Danio rerio is a new model system for studying the genetics of hematopoiesis. To define naturally occurring viruses which could infect and replicate within hematopoietic precursors of the zebrafish, infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) were studied. Infection of whole fish with viral supernatants demonstrated infectious replicants for both viruses, indicating that the virus host range includes the zebrafish. In other species, infection with these viruses leads to prominent hematopoietic necrosis of the head kidney, the major site of adult hematopoiesis. We detected a transient toxicity of the virus to hematopoietic precursors and terminally differentiated red cells after viral infections. The kinetics of hematopoietic defects between IHNV and IPNV infection differed; fish infected with either virus, however, recovered by 6 days postinfection. In contrast to other fish infected with the virus, hematocrit did not change appreciably during this time. These studies are the first to demonstrate IHNV and IPNV infection of the zebrafish and reveal the potential for use of such viruses for gene transfer experiments to infect zebrafish hematopoietic cells.Copyright 2000 Academic Press. -
Jagadeeswaran P, Gregory M, Zhou Y, Zon L, Padmanabhan K, Hanumanthaiah R. 2000. Characterization of zebrafish full-length prothrombin cDNA and linkage group mapping. Blood cells, molecules & diseases. 26(5):479-89. Pubmed: 11112385 Jagadeeswaran P, Gregory M, Zhou Y, Zon L, Padmanabhan K, Hanumanthaiah R. 2000. Characterization of zebrafish full-length prothrombin cDNA and linkage group mapping. Blood cells, molecules & diseases. 26(5):479-89. Pubmed: 11112385 In this paper, we report the complete cDNA sequence of zebrafish prothrombin. The cDNA sequence predicts that zebrafish prothrombin is synthesized as a pre-proprotein consisting of a Gla domain, two kringle domains, and a two-chain protease domain. Zebrafish prothrombin is structurally very similar to human and other vertebrate prothrombins. Zebrafish and human prothrombin share 53% amino acid identity whereas zebrafish and hagfish prothrombin share 51% identity. Amino acid alignments of various prothrombins identified conservation of many of the functional/structural motifs suggesting that the vertebrate prothrombins may have similar functions. The three-dimensional structure of prothrombin predicted by homology modeling also revealed that the prothrombin fragment 1 and the catalytic domain structures are well conserved except for the insertion of an extra 7-amino-acid loop in the connecting region (CR) between the Gla and kringle I domain of fragment 1. Linkage analysis revealed that the prothrombin gene locus on linkage group 7 in zebrafish is syntenic to the human chromosome 11-prothrombin region suggesting its preservation through evolution. The availability of this cDNA sequence in zebrafish adds to our knowledge of the zebrafish hemostatic system and provides support for the view that similarities between zebrafish and mammalian coagulation exist, thus underscoring the relevance of the zebrafish model for studying human hemostasis.Copyright 2000 Academic Press. -
Tsai M, Wedemeyer J, Ganiatsas S, Tam SY, Zon LI, Galli SJ. 2000. In vivo immunological function of mast cells derived from embryonic stem cells: an approach for the rapid analysis of even embryonic lethal mutations in adult mice in vivo. Proceedings of the National Academy of Sciences of the United States of America. 97(16):9186-90. Pubmed: 10908668 Tsai M, Wedemeyer J, Ganiatsas S, Tam SY, Zon LI, Galli SJ. 2000. In vivo immunological function of mast cells derived from embryonic stem cells: an approach for the rapid analysis of even embryonic lethal mutations in adult mice in vivo. Proceedings of the National Academy of Sciences of the United States of America. 97(16):9186-90. Pubmed: 10908668 An important goal of tissue engineering is to achieve reconstitution of specific functionally active cell types by transplantation of differentiated cell populations derived from normal or genetically altered embryonic stem cells in vitro. We find that mast cells derived in vitro from wild-type or genetically manipulated embryonic stem cells can survive and orchestrate immunologically specific IgE-dependent reactions after transplantation into mast cell-deficient Kit(W)/Kit(W-v) mice. These findings define a unique approach for analyzing the effects of mutations of any genes that are expressed in mast cells, including embryonic lethal mutations, in vitro or in vivo. -
Parichy DM, Ransom DG, Paw B, Zon LI, Johnson SL. 2000. An orthologue of the kit-related gene fms is required for development of neural crest-derived xanthophores and a subpopulation of adult melanocytes in the zebrafish, Danio rerio. Development (Cambridge, England). 127(14):3031-44. Pubmed: 10862741 Parichy DM, Ransom DG, Paw B, Zon LI, Johnson SL. 2000. An orthologue of the kit-related gene fms is required for development of neural crest-derived xanthophores and a subpopulation of adult melanocytes in the zebrafish, Danio rerio. Development (Cambridge, England). 127(14):3031-44. Pubmed: 10862741 Developmental mechanisms underlying traits expressed in larval and adult vertebrates remain largely unknown. Pigment patterns of fishes provide an opportunity to identify genes and cell behaviors required for postembryonic morphogenesis and differentiation. In the zebrafish, Danio rerio, pigment patterns reflect the spatial arrangements of three classes of neural crest-derived pigment cells: black melanocytes, yellow xanthophores and silver iridophores. We show that the D. rerio pigment pattern mutant panther ablates xanthophores in embryos and adults and has defects in the development of the adult pattern of melanocyte stripes. We find that panther corresponds to an orthologue of the c-fms gene, which encodes a type III receptor tyrosine kinase and is the closest known homologue of the previously identified pigment pattern gene, kit. In mouse, fms is essential for the development of macrophage and osteoclast lineages and has not been implicated in neural crest or pigment cell development. In contrast, our analyses demonstrate that fms is expressed and required by D. rerio xanthophore precursors and that fms promotes the normal patterning of melanocyte death and migration during adult stripe formation. Finally, we show that fms is required for the appearance of a late developing, kit-independent subpopulation of adult melanocytes. These findings reveal an unexpected role for fms in pigment pattern development and demonstrate that parallel neural crest-derived pigment cell populations depend on the activities of two essentially paralogous genes, kit and fms. 1999
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Ransom DG, Zon LI. 1999. Collection, storage, and use of zebrafish sperm. Methods in cell biology. 60:365-72. Pubmed: 9891349 Ransom DG, Zon LI. 1999. Collection, storage, and use of zebrafish sperm. Methods in cell biology. 60:365-72. Pubmed: 9891349 -
Paw BH, Zon LI. 1999. Primary fibroblast cell culture. Methods in cell biology. 59:39-43. Pubmed: 9891354 Paw BH, Zon LI. 1999. Primary fibroblast cell culture. Methods in cell biology. 59:39-43. Pubmed: 9891354 -
Johnson SL, Zon LI. 1999. Genetic backgrounds and some standard stocks and strains used in zebrafish developmental biology and genetics. Methods in cell biology. 60:357-9. Pubmed: 9891347 Johnson SL, Zon LI. 1999. Genetic backgrounds and some standard stocks and strains used in zebrafish developmental biology and genetics. Methods in cell biology. 60:357-9. Pubmed: 9891347 -
Parker LH, Zon LI, Stainier DY. 1999. Vascular and blood gene expression. Methods in cell biology. 59:313-36. Pubmed: 9891367 Parker LH, Zon LI, Stainier DY. 1999. Vascular and blood gene expression. Methods in cell biology. 59:313-36. Pubmed: 9891367 -
Ransom DG, Zon LI. 1999. Mapping zebrafish mutations by AFLP. Methods in cell biology. 60:195-211. Pubmed: 9891339 Ransom DG, Zon LI. 1999. Mapping zebrafish mutations by AFLP. Methods in cell biology. 60:195-211. Pubmed: 9891339 -
Liao EC, Zon LI. 1999. Simple sequence-length polymorphism analysis. Methods in cell biology. 60:181-3. Pubmed: 9891337 Liao EC, Zon LI. 1999. Simple sequence-length polymorphism analysis. Methods in cell biology. 60:181-3. Pubmed: 9891337 -
Amatruda JF, Zon LI. 1999. Dissecting hematopoiesis and disease using the zebrafish. Developmental biology. 216(1):1-15. Pubmed: 10588859 Amatruda JF, Zon LI. 1999. Dissecting hematopoiesis and disease using the zebrafish. Developmental biology. 216(1):1-15. Pubmed: 10588859 The study of blood has often defined paradigms that are relevant to the biology of other vertebrate organ systems. As examples, stem cell physiology and the structure of the membrane cytoskeleton were first described in hematopoietic cells. Much of the reason for these successes resides in the ease with which blood cells can be isolated and manipulated in vitro. The cell biology of hematopoiesis can also be illuminated by the study of human disease states such as anemia, immunodeficiency, and leukemia. The sequential development of the blood system in vertebrates is characterized by ventral mesoderm induction, hematopoietic stem cell specification, and subsequent cell lineage differentiation. Some of the key regulatory steps in this process have been uncovered by studies in mouse, chicken, and Xenopus. More recently, the genetics of the zebrafish (Danio rerio) have been employed to define novel points of regulation of the hematopoietic program. In this review, we describe the advantages of the zebrafish system for the study of blood cell development and the initial success of the system in this pursuit. The striking similarity of zebrafish mutant phenotypes and human diseases emphasizes the utility of this model system for elucidating pathophysiologic mechanisms. New screens for lineage-specific mutations are beginning, and the availability of transgenics promises a better understanding of lineage-specific gene expression. The infrastructure of the zebrafish system is growing with an NIH-directed genome initiative, providing a detailed map of the zebrafish genome and an increasing number of candidate genes for the mutations. The zebrafish is poised to contribute greatly to our understanding of normal and disease-related hematopoiesis.Copyright 1999 Academic Press. -
Zon LI. 1999. Zebrafish: a new model for human disease. Genome research. 9(2):99-100. Pubmed: 10022974 Zon LI. 1999. Zebrafish: a new model for human disease. Genome research. 9(2):99-100. Pubmed: 10022974 -
Kane DA, Zon LI, Detrich HW. 1999. Centromeric markers in the zebrafish. Methods in cell biology. 60:361-3. Pubmed: 9891348 Kane DA, Zon LI, Detrich HW. 1999. Centromeric markers in the zebrafish. Methods in cell biology. 60:361-3. Pubmed: 9891348 -
Kieran MW, Katz S, Vail B, Zon LI, Mayer BJ. 1999. Concentration-dependent positive and negative regulation of a MAP kinase by a MAP kinase kinase. Oncogene. 18(48):6647-57. Pubmed: 10597270 Kieran MW, Katz S, Vail B, Zon LI, Mayer BJ. 1999. Concentration-dependent positive and negative regulation of a MAP kinase by a MAP kinase kinase. Oncogene. 18(48):6647-57. Pubmed: 10597270 There are at least three distinct MAP kinase signaling modules in mammalian cells, distinguished by the family of kinases (Erk, SAPK/JNK, or p38) that is ultimately activated. Many input signals activate multiple MAP kinase cascades, and the mechanisms that control the specificity of signal output are not well understood. We show that SEK1/MKK4, a MAP kinase kinase proposed to activate SAPK/JNK, is a very potent inhibitor of p54 SAPK beta/JNK3 both in vitro and in vivo if present at equimolar or higher ratios. In contrast SEK can activate SAPK when present in substoichiometric amounts, but this activation is slow, consistent with the rate-limiting step in activation being the dissociation of an inactive SEK:SAPK complex. The N-terminal unique region of SEK is both necessary and partially sufficient for inhibition of SAPK, and is also necessary for activation of SAPK by SEK in vitro. We have also used the p38 MAP kinase and its activator MKK6 to examine the regulatory relationships among different kinases involved in stress responses. We show using purified kinases that inhibitory activity is specific for the combination of SEK and SAPK: SEK can activate but not inhibit p38, and MKK6 can activate but not inhibit SAPK beta and p38. These results reveal a potential mechanism for regulating stress-activated kinases, adding to a growing body of evidence suggesting that MAP kinases are controlled by relatively stable interactions with their activators. -
Hukriede NA, Joly L, Tsang M, Miles J, Tellis P, Epstein JA, Barbazuk WB, Li FN, Paw B, Postlethwait JH, Hudson TJ, Zon LI, McPherson JD, Chevrette M, Dawid IB, Johnson SL, Ekker M. 1999. Radiation hybrid mapping of the zebrafish genome. Proceedings of the National Academy of Sciences of the United States of America. 96(17):9745-50. Pubmed: 10449765 Hukriede NA, Joly L, Tsang M, Miles J, Tellis P, Epstein JA, Barbazuk WB, Li FN, Paw B, Postlethwait JH, Hudson TJ, Zon LI, McPherson JD, Chevrette M, Dawid IB, Johnson SL, Ekker M. 1999. Radiation hybrid mapping of the zebrafish genome. Proceedings of the National Academy of Sciences of the United States of America. 96(17):9745-50. Pubmed: 10449765 The zebrafish is an excellent genetic system for the study of vertebrate development and disease. In an effort to provide a rapid and robust tool for zebrafish gene mapping, a panel of radiation hybrids (RH) was produced by fusion of irradiated zebrafish AB9 cells with mouse B78 cells. The overall retention of zebrafish sequences in the 93 RH cell lines that constitute the LN54 panel is 22%. Characterization of the LN54 panel with 849 simple sequence length polymorphism markers, 84 cloned genes and 122 expressed sequence tags allowed the production of an RH map whose total size was 11,501 centiRays. From this value, we estimated the average breakpoint frequency of the LN54 RH panel to correspond to 1 centiRay = 148 kilobase. Placement of a group of 235 unbiased markers on the RH map suggests that the map generated for the LN54 panel, at present, covers 88% of the zebrafish genome. Comparison of marker positions in RH and meiotic maps indicated a 96% concordance. Mapping expressed sequence tags and cloned genes by using the LN54 panel should prove to be a valuable method for the identification of candidate genes for specific mutations in zebrafish. -
Porcher C, Liao EC, Fujiwara Y, Zon LI, Orkin SH. 1999. Specification of hematopoietic and vascular development by the bHLH transcription factor SCL without direct DNA binding. Development (Cambridge, England). 126(20):4603-15. Pubmed: 10498694 Porcher C, Liao EC, Fujiwara Y, Zon LI, Orkin SH. 1999. Specification of hematopoietic and vascular development by the bHLH transcription factor SCL without direct DNA binding. Development (Cambridge, England). 126(20):4603-15. Pubmed: 10498694 Transcription factors, such as those of the basic-helix-loop-helix (bHLH) and homeodomain classes, are primary regulators of cell fate decisions and differentiation. It is considered axiomatic that they control their respective developmental programs via direct binding to cognate DNA sequences in critical targets genes. Here we test this widely held paradigm by in vivo functional assay of the leukemia oncoprotein SCL, a bHLH factor that resembles myogenic and neurogenic proteins and is essential for both hematopoietic and vascular development in vertebrates. Contrary to all expectation, we find that SCL variants unable to bind DNA rescue hematopoiesis from gene-targeted SCL(-)(/)(-) embryonic stem cells and complement hematopoietic and vascular deficits in the zebrafish mutant cloche. Our findings establish DNA-binding-independent functions of SCL critical for transcriptional specification, and should encourage reassessment of presumed requirements for direct DNA binding by other transcription factors during initiation of developmental programs. -
Oates AC, Brownlie A, Pratt SJ, Irvine DV, Liao EC, Paw BH, Dorian KJ, Johnson SL, Postlethwait JH, Zon LI, Wilks AF. 1999. Gene duplication of zebrafish JAK2 homologs is accompanied by divergent embryonic expression patterns: only jak2a is expressed during erythropoiesis. Blood. 94(8):2622-36. Pubmed: 10515866 Oates AC, Brownlie A, Pratt SJ, Irvine DV, Liao EC, Paw BH, Dorian KJ, Johnson SL, Postlethwait JH, Zon LI, Wilks AF. 1999. Gene duplication of zebrafish JAK2 homologs is accompanied by divergent embryonic expression patterns: only jak2a is expressed during erythropoiesis. Blood. 94(8):2622-36. Pubmed: 10515866 Members of the JAK family of protein tyrosine kinase (PTK) proteins are required for the transmission of signals from a variety of cell surface receptors, particularly those of the cytokine receptor family. JAK function has been implicated in hematopoiesis and regulation of the immune system, and recent data suggest that the vertebrate JAK2 gene may play a role in leukemia. We have isolated and characterized jak cDNAs from the zebrafish Danio rerio. The zebrafish genome possesses 2 jak2 genes that occupy paralogous chromosome segments in the zebrafish genome, and these segments conserve syntenic relationships with orthologous genes in mammalian genomes, suggesting an ancient duplication in the zebrafish lineage. The jak2a gene is expressed at high levels in erythroid precursors of primitive and definitive waves and at a lower level in early central nervous system and developing fin buds. jak2b is expressed in the developing lens and nephritic ducts, but not in hematopoietic tissue. The expression of jak2a was examined in hematopoietic mutants and found to be disrupted in cloche and spadetail, suggesting an early role in hematopoiesis. Taken together with recent gene knockout data in the mouse, we suggest that jak2a may be functionally equivalent to mammalian Jak2, with a role in early erythropoiesis. -
Amemiya CT, Zhong TP, Silverman GA, Fishman MC, Zon LI. 1999. Zebrafish YAC, BAC, and PAC genomic libraries. Methods in cell biology. 60:235-58. Pubmed: 9891341 Amemiya CT, Zhong TP, Silverman GA, Fishman MC, Zon LI. 1999. Zebrafish YAC, BAC, and PAC genomic libraries. Methods in cell biology. 60:235-58. Pubmed: 9891341 Numerous positional cloning projects directed at isolating genes responsible for the myriads of observed developmental defects in the zebrafish are anticipated in the very near future. In this chapter, we have reviewed the YAC, BAC, and PAC large-insert genomic resources available to the zebrafish community. We have discussed how these resources are screened and used in a positional cloning scheme and have pointed out frequently formidable logistical considerations in the approach. Despite being extremely tedious, positional cloning projects in the zebrafish will be comparatively easier to accomplish than in human and mouse, because of unique biological advantages of the zebrafish system. Moreover, the ease and speed at which genes are identified and cloned should rapidly increase as more mapping reagents and information become available, thereby paving the way for meaningful biological studies. -
Meriin AB, Yaglom JA, Gabai VL, Zon L, Ganiatsas S, Mosser DD, Zon L, Sherman MY. 1999. Protein-damaging stresses activate c-Jun N-terminal kinase via inhibition of its dephosphorylation: a novel pathway controlled by HSP72. Molecular and cellular biology. 19(4):2547-55. Pubmed: 10082520 Meriin AB, Yaglom JA, Gabai VL, Zon L, Ganiatsas S, Mosser DD, Zon L, Sherman MY. 1999. Protein-damaging stresses activate c-Jun N-terminal kinase via inhibition of its dephosphorylation: a novel pathway controlled by HSP72. Molecular and cellular biology. 19(4):2547-55. Pubmed: 10082520 Various stresses activate the c-Jun N-terminal kinase (JNK), which is involved in the regulation of many aspects of cellular physiology, including apoptosis. Here we demonstrate that in contrast to UV irradiation, heat shock causes little or no stimulation of the JNK-activating kinase SEK1, while knocking out the SEK1 gene completely blocks heat-induced JNK activation. Therefore, we tested whether heat shock activates JNK via inhibition of JNK dephosphorylation. The rate of JNK dephosphorylation in unstimulated cells was high, and exposure to UV irradiation, osmotic shock, interleukin-1, or anisomycin did not affect this process. Conversely, exposure of cells to heat shock and other protein-damaging conditions, including ethanol, arsenite, and oxidative stress, strongly reduced the rate of JNK dephosphorylation. Under these conditions, we did not observe any effects on dephosphorylation of the homologous p38 kinase, suggesting that suppression of dephosphorylation is specific to JNK. Together, these data indicate that activation of JNK by protein-damaging treatments is mediated primarily by inhibition of a JNK phosphatase(s). Elevation of cellular levels of the major heat shock protein Hsp72 inhibited a repression of JNK dephosphorylation by these stressful treatments, which explains recent reports of the suppression of JNK activation by Hsp72. -
Amemiya CT, Zon LI. 1999. Generation of a zebrafish P1 artificial chromosome library. Genomics. 58(2):211-3. Pubmed: 10366454 Amemiya CT, Zon LI. 1999. Generation of a zebrafish P1 artificial chromosome library. Genomics. 58(2):211-3. Pubmed: 10366454 We have constructed a genomic P1 artificial chromosome library from the zebrafish. The library has been arrayed and archived in two hundred seventy-one 384-well microtiter dishes. It encompasses four to five genome equivalents with an average insert size of approximately 115 kb and is readily accessible to the scientific community. The library has been used by numerous investigators in the community and shown to be a useful reagent for chromosomal walking and positional cloning.Copyright 1999 Academic Press. -
Detrich HW, Westerfield M, Zon LI. 1999. Overview of the Zebrafish system. Methods in cell biology. 59:3-10. Pubmed: 9891351 Detrich HW, Westerfield M, Zon LI. 1999. Overview of the Zebrafish system. Methods in cell biology. 59:3-10. Pubmed: 9891351 -
Oates AC, Wollberg P, Pratt SJ, Paw BH, Johnson SL, Ho RK, Postlethwait JH, Zon LI, Wilks AF. 1999. Zebrafish stat3 is expressed in restricted tissues during embryogenesis and stat1 rescues cytokine signaling in a STAT1-deficient human cell line. Developmental dynamics : an official publication of the American Association of Anatomists. 215(4):352-70. Pubmed: 10417824 Oates AC, Wollberg P, Pratt SJ, Paw BH, Johnson SL, Ho RK, Postlethwait JH, Zon LI, Wilks AF. 1999. Zebrafish stat3 is expressed in restricted tissues during embryogenesis and stat1 rescues cytokine signaling in a STAT1-deficient human cell line. Developmental dynamics : an official publication of the American Association of Anatomists. 215(4):352-70. Pubmed: 10417824 Transcription factors of the STAT family are required for cellular responses to multiple signaling molecules. After ligand binding-induced activation of cognate receptors, STAT proteins are phosphorylated, hetero- or homodimerize, and translocate to the nucleus. Subsequent STAT binding to specific DNA elements in the promoters of signal-responsive genes alters the transcriptional activity of these loci. STAT function has been implicated in the transduction of signals for growth, reproduction, viral defense, and immune regulation. We have isolated and characterized two STAT homologs from the zebrafish Danio rerio. The stat3 gene is expressed in a tissue-restricted manner during embryogenesis, and larval development with highest levels of transcript are detected in the anterior hypoblast, eyes, cranial sensory ganglia, gut, pharyngeal arches, cranial motor nuclei, and lateral line system. In contrast, the stat1 gene is not expressed during early development. The stat3 gene maps to a chromosomal position syntenic with the mouse and human STAT3 homologs, whereas the stat1 gene does not. Despite a higher rate of evolutionary change in stat1 relative to stat3, the stat1 protein rescues interferon-signaling functions in a STAT1-deficient human cell line, indicating that cytokine-signaling mechanisms are likely to be conserved between fish and tetrapods. Dev Dyn 1999;215:352-370.Copyright 1999 Wiley-Liss, Inc. -
Rubin DA, Hellman P, Zon LI, Lobb CJ, Bergwitz C, Jüppner H. 1999. A G protein-coupled receptor from zebrafish is activated by human parathyroid hormone and not by human or teleost parathyroid hormone-related peptide. Implications for the evolutionary conservation of calcium-regulating peptide hormones. The Journal of biological chemistry. 274(33):23035-42. Pubmed: 10438471 Rubin DA, Hellman P, Zon LI, Lobb CJ, Bergwitz C, Jüppner H. 1999. A G protein-coupled receptor from zebrafish is activated by human parathyroid hormone and not by human or teleost parathyroid hormone-related peptide. Implications for the evolutionary conservation of calcium-regulating peptide hormones. The Journal of biological chemistry. 274(33):23035-42. Pubmed: 10438471 Genomic and cDNA clones encoding portions of a putative catfish parathyroid hormone (PTH) 2 receptor (PTH2R) led to the isolation of a cDNA encoding a full-length zebrafish PTH2R (zPTH2R). The zPTH2R shared 63 and 60% amino acid sequence identity with human and rat PTH2Rs, respectively, 47-52% identity with mammalian and frog PTH/PTHrP receptors (PTH1R), and less than 37% with other members of this family of G protein-coupled receptors. COS-7 cells expressing zPTH2R(43), a 5' splice variant that lacked 17 amino acids in the amino-terminal extracellular domain, showed cAMP accumulation when challenged with [Tyr(34)]hPTH(1-34)-amide (hPTH) (EC(50), 1.64 +/- 0. 95 nM) and [Ile(5),Trp(23),Tyr(36)]hPTHrP-(1-36)-amide ([Ile(5), Trp(23)]hPTHrP) (EC(50), 46.8 +/- 12.1 nM) but not when stimulated with [Tyr(36)]hPTHrP-(1-36)-amide (hPTHrP), [Trp(23), Tyr(36)]hPTHrP-(1-36)-amide ([Trp(23)]hPTHrP), or [Ala(29),Glu(30), Ala(34),Glu(35),Tyr(36)]fugufish PTHrP-(1-36)amide (fuguPTHrP). FuguPTHrP also failed to activate the human PTH2R but had similar efficiency and efficacy as hPTH and hPTHrP when tested with cells expressing the human PTH1R. Agonist-dependent activation of zPTH2R was less efficient than that of zPTH2R(43), and both receptor variants showed no cAMP accumulation when stimulated with either secretin, growth hormone-releasing hormone, or calcitonin. The zPTH2R thus has ligand specificity similar to that of the human homolog, which raises the possibility that a PTH-like molecule exists in zebrafish, species which lack parathyroid glands. 1998
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Liao EC, Paw BH, Oates AC, Pratt SJ, Postlethwait JH, Zon LI. 1998. SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish. Genes & development. 12(5):621-6. Pubmed: 9499398 Liao EC, Paw BH, Oates AC, Pratt SJ, Postlethwait JH, Zon LI. 1998. SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish. Genes & development. 12(5):621-6. Pubmed: 9499398 SCL/Tal-1 is a transcription factor necessary for hematopoietic stem cell differentiation. Although SCL is also expressed in endothelial and neural progenitors, SCL function in these cells remains unknown. In the zebrafish mutant cloche (clo), SCL expression is nearly abolished in hematopoietic and vascular tissues. Correspondingly, it was shown previously that clo fails to differentiate blood and angioblasts. Genetic analysis demonstrates that the clo mutation is not linked to the SCL locus. Forced expression of SCL in clo embryos rescues the blood and vascular defects, suggesting that SCL acts downstream of clo to specify hematopoietic and vascular differentiation. -
Swat W, Fujikawa K, Ganiatsas S, Yang D, Xavier RJ, Harris NL, Davidson L, Ferrini R, Davis RJ, Labow MA, Flavell RA, Zon LI, Alt FW. 1998. SEK1/MKK4 is required for maintenance of a normal peripheral lymphoid compartment but not for lymphocyte development. Immunity. 8(5):625-34. Pubmed: 9620683 Swat W, Fujikawa K, Ganiatsas S, Yang D, Xavier RJ, Harris NL, Davidson L, Ferrini R, Davis RJ, Labow MA, Flavell RA, Zon LI, Alt FW. 1998. SEK1/MKK4 is required for maintenance of a normal peripheral lymphoid compartment but not for lymphocyte development. Immunity. 8(5):625-34. Pubmed: 9620683 SAPK is a member of the group of evolutionary conserved stress-activated kinases that mediate control of cellular death and proliferation. In lymphocytes, the SAPK pathway has been implicated in signaling from antigen, costimulatory, and death receptors; SEK1, which directly activates SAPK, is required for early embryonic development and has also been reported to be essential for normal lymphocyte development. In contrast to the latter findings, we have used RAG-2-deficient blastocyst complementation to show that SEK1-deficient embryonic stem cells support unimpaired T and B lymphocyte development. Moreover, mature SEK1-deficient lymphocytes are capable of SAPK activation. Surprisingly, however, aging SEK1-deficient chimeric mice frequently develop lymphadenopathy and polyclonal B and T cell expansions. Thus, SEK1 is not required for lymphocyte development, but is required for maintaining peripheral lymphoid homeostasis. -
Trede NS, Zon LI. 1998. Development of T-cells during fish embryogenesis. Developmental and comparative immunology. 22(3):253-63. Pubmed: 9700456 Trede NS, Zon LI. 1998. Development of T-cells during fish embryogenesis. Developmental and comparative immunology. 22(3):253-63. Pubmed: 9700456 -
Jessen JR, Meng A, McFarlane RJ, Paw BH, Zon LI, Smith GR, Lin S. 1998. Modification of bacterial artificial chromosomes through chi-stimulated homologous recombination and its application in zebrafish transgenesis. Proceedings of the National Academy of Sciences of the United States of America. 95(9):5121-6. Pubmed: 9560239 Jessen JR, Meng A, McFarlane RJ, Paw BH, Zon LI, Smith GR, Lin S. 1998. Modification of bacterial artificial chromosomes through chi-stimulated homologous recombination and its application in zebrafish transgenesis. Proceedings of the National Academy of Sciences of the United States of America. 95(9):5121-6. Pubmed: 9560239 The modification of yeast artificial chromosomes through homologous recombination has become a useful genetic tool for studying gene function and enhancer/promoter activity. However, it is difficult to purify intact yeast artificial chromosome DNA at a concentration sufficient for many applications. Bacterial artificial chromosomes (BACs) are vectors that can accommodate large DNA fragments and can easily be purified as plasmid DNA. We report herein a simple procedure for modifying BACs through homologous recombination using a targeting construct containing properly situated Chi sites. To demonstrate a usage for this technique, we modified BAC clones containing the zebrafish GATA-2 genomic locus by replacing the first coding exon with the green fluorescent protein (GFP) reporter gene. Molecular analyses confirmed that the modification occurred without additional deletions or rearrangements of the BACs. Microinjection demonstrated that GATA-2 expression patterns can be recapitulated in living zebrafish embryos by using these GFP-modified GATA-2 BACs. Embryos microinjected with the modified BAC clones were less mosaic and had improved GFP expression in hematopoietic progenitor cells compared with smaller plasmid constructs. The precise modification of BACs through Chi-stimulated homologous recombination should be useful for studying gene function and regulation in cultured cells or organisms where gene transfer is applicable. -
Postlethwait JH, Yan YL, Gates MA, Horne S, Amores A, Brownlie A, Donovan A, Egan ES, Force A, Gong Z, Goutel C, Fritz A, Kelsh R, Knapik E, Liao E, Paw B, Ransom D, Singer A, Thomson M, Abduljabbar TS, Yelick P, Beier D, Joly JS, Larhammar D, Rosa F, Westerfield M, Zon LI, Johnson SL, Talbot WS. 1998. Vertebrate genome evolution and the zebrafish gene map. Nature genetics. 18(4):345-9. Pubmed: 9537416 Postlethwait JH, Yan YL, Gates MA, Horne S, Amores A, Brownlie A, Donovan A, Egan ES, Force A, Gong Z, Goutel C, Fritz A, Kelsh R, Knapik E, Liao E, Paw B, Ransom D, Singer A, Thomson M, Abduljabbar TS, Yelick P, Beier D, Joly JS, Larhammar D, Rosa F, Westerfield M, Zon LI, Johnson SL, Talbot WS. 1998. Vertebrate genome evolution and the zebrafish gene map. Nature genetics. 18(4):345-9. Pubmed: 9537416 In chordate phylogeny, changes in the nervous system, jaws, and appendages transformed meek filter feeders into fearsome predators. Gene duplication is thought to promote such innovation. Vertebrate ancestors probably had single copies of genes now found in multiple copies in vertebrates and gene maps suggest that this occurred by polyploidization. It has been suggested that one genome duplication event occurred before, and one after the divergence of ray-finned and lobe-finned fishes. Holland et al., however, have argued that because various vertebrates have several HOX clusters, two rounds of duplication occurred before the origin of jawed fishes. Such gene-number data, however, do not distinguish between tandem duplications and polyploidization events, nor whether independent duplications occurred in different lineages. To investigate these matters, we mapped 144 zebrafish genes and compared the resulting map with mammalian maps. Comparison revealed large conserved chromosome segments. Because duplicated chromosome segments in zebrafish often correspond with specific chromosome segments in mammals, it is likely that two polyploidization events occurred prior to the divergence of fish and mammal lineages. This zebrafish gene map will facilitate molecular identification of mutated zebrafish genes, which can suggest functions for human genes known only by sequence. -
Huber TL, Zon LI. 1998. Transcriptional regulation of blood formation during Xenopus development. Seminars in immunology. 10(2):103-9. Pubmed: 9618755 Huber TL, Zon LI. 1998. Transcriptional regulation of blood formation during Xenopus development. Seminars in immunology. 10(2):103-9. Pubmed: 9618755 Hematopoiesis is the generation, proliferation and differentiation of hematopoietic stem cells (HSCs) into the erythroid, myeloid and lymphoid lineages. The HSC is a ventral mesodermal derivative that arises due to inductive and patterning processes triggered by bone morphogenetic protein 4 (BMP-4). Recent studies in Xenopus laevis have identified families of transcription factors that participate in hematopoietic development. Transcription factor families, such as the SMADs, Mixs and Vents pattern mesoderm to a ventral fate which ultimately results in the expression of hematopoietic-specific transcription factors such as GATA-1 and SCL. This transcriptional regulatory network enforces the correct temporal and spatial expression of the blood program. -
Mead PE, Zon LI. 1998. Molecular insights into early hematopoiesis. Current opinion in hematology. 5(2):156-60. Pubmed: 9570710 Mead PE, Zon LI. 1998. Molecular insights into early hematopoiesis. Current opinion in hematology. 5(2):156-60. Pubmed: 9570710 During embryogenesis, a pool of pluripotential, self-renewing hematopoietic stem cells is specified from undifferentiated ventral mesoderm. These cells proliferate and differentiate to yield all the peripheral blood lineages. Although the mechanisms involved in the commitment and differentiation of multipotential progenitors have been well documented, little focus has been directed to the molecular events underlying the formation of hematopoietic stem cells. In this review we discuss the recent advances made in defining a bone morphogenetic protein-responsive signal cascade that regulates the earliest phase of blood formation during embryogenesis. -
Bahary N, Zon LI. 1998. Use of the zebrafish (Danio rerio) to define hematopoiesis. Stem cells (Dayton, Ohio). 16 Suppl 2:67-78. Pubmed: 11012178 Bahary N, Zon LI. 1998. Use of the zebrafish (Danio rerio) to define hematopoiesis. Stem cells (Dayton, Ohio). 16 Suppl 2:67-78. Pubmed: 11012178 Hematopoiesis in the vertebrate is characterized by the induction of ventral mesoderm to form hematopoietic stem cells and the eventual differentiation of these progenitors to form the peripheral blood lineages. Several genes have been implicated in the differentiation and development of hematopoietic and vascular progenitor cells, yet our understanding of the discrete steps involved in the induction of these cells from the ventral mesoderm is still incomplete. One method of delineating these processes is based on the use of lower vertebrates. The zebrafish (Danio rerio) is an especially robust vertebrate system for both isolating and characterizing genes involved in these processes. Hematopoietic mutants have been generated with defects in many of the steps of both the primitive and definitive hematopoietic programs. Cloning of the genes that underlie these mutations should yield valuable details of hematopoiesis and may have therapeutic implications for bone marrow transplantation and stem cell gene therapy. -
Thompson MA, Ransom DG, Pratt SJ, MacLennan H, Kieran MW, Detrich HW, Vail B, Huber TL, Paw B, Brownlie AJ, Oates AC, Fritz A, Gates MA, Amores A, Bahary N, Talbot WS, Her H, Beier DR, Postlethwait JH, Zon LI. 1998. The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis. Developmental biology. 197(2):248-69. Pubmed: 9630750 Thompson MA, Ransom DG, Pratt SJ, MacLennan H, Kieran MW, Detrich HW, Vail B, Huber TL, Paw B, Brownlie AJ, Oates AC, Fritz A, Gates MA, Amores A, Bahary N, Talbot WS, Her H, Beier DR, Postlethwait JH, Zon LI. 1998. The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis. Developmental biology. 197(2):248-69. Pubmed: 9630750 In vertebrates, hematopoietic and vascular progenitors develop from ventral mesoderm. The first primitive wave of hematopoiesis yields embryonic red blood cells, whereas progenitor cells of subsequent definitive waves form all hematopoietic cell lineages. In this report we examine the development of hematopoietic and vasculogenic cells in normal zebrafish and characterize defects in cloche and spadetail mutant embryos. The zebrafish homologs of lmo2, c-myb, fli1, flk1, and flt4 have been cloned and characterized in this study. Expression of these genes identifies embryonic regions that contain hematopoietic and vascular progenitor cells. The expression of c-myb also identifies definitive hematopoietic cells in the ventral wall of the dorsal aorta. Analysis of b316 mutant embryos that carry a deletion of the c-myb gene demonstrates that c-myb is not required for primitive erythropoiesis in zebrafish even though it is expressed in these cells. Both cloche and spadetail mutant embryos have defects in primitive hematopoiesis and definitive hematopoiesis. The cloche mutants also have significant decreases in vascular gene expression, whereas spadetail mutants expressed normal levels of these genes. These studies demonstrate that the molecular mechanisms that regulate hematopoiesis and vasculogenesis have been conserved throughout vertebrate evolution and the clo and spt genes are key regulators of these programs. -
Yamaguchi Y, Zon LI, Ackerman SJ, Yamamoto M, Suda T. 1998. Forced GATA-1 expression in the murine myeloid cell line M1: induction of c-Mpl expression and megakaryocytic/erythroid differentiation. Blood. 91(2):450-7. Pubmed: 9427697 Yamaguchi Y, Zon LI, Ackerman SJ, Yamamoto M, Suda T. 1998. Forced GATA-1 expression in the murine myeloid cell line M1: induction of c-Mpl expression and megakaryocytic/erythroid differentiation. Blood. 91(2):450-7. Pubmed: 9427697 The "zinc-finger" transcription factor GATA-1 was first shown in cells of erythroid lineage. It is also expressed in cells of other hematopoietic lineages including megakaryocytes, mast cells, and eosinophils. GATA-1 is now considered to be one of the central regulators in hematopoietic cell differentiation. To further analyze the role of GATA-1 in controlling differentiation from hematopoietic stem cells, we investigated the phenotypic changes induced by the overexpression of murine GATA-1 in the murine myeloid leukemic cell line, M1. Forced expression of GATA-1 induced the appearance of erythroid cells and megakaryocytes as assessed by cellular morphology, acetylcholinesterase activity, and expression of platelet factor 4 and beta-globin mRNA synthesis. Because the c-mpl ligand, thrombopoietin, plays an important role in megakaryopoiesis, the expression of c-mpl and c-mpl ligand (thrombopoietin) mRNA was analyzed by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in M1 cells overexpressing GATA-1. The c-mpl ligand mRNA was equally expressed both in parental M1 cells and in those transfected with the GATA-1 expression vector. In contrast, the mRNA expression of c-mpl was increased only in GATA-1 expressing M1 cells differentiated towards erythroid and megakaryocyte lineages. The increased expression of c-mpl mRNA induced by GATA-1 raised the question as to whether or not GATA-1 transactivated the c-mpl promoter. The activity of the c-mpl promoter in the presence of cotransfected GATA-1 was significantly increased compared with that of the control. A plasmid with the mutated GATA-binding site did not show transactivation ability in the cotransfection with a GATA expression vector. These findings suggest that the upregulation of c-mpl induced by GATA-1 expression in M1 cells is closely associated with erythroid and megakaryocytic differentiation. -
Brownlie A, Donovan A, Pratt SJ, Paw BH, Oates AC, Brugnara C, Witkowska HE, Sassa S, Zon LI. 1998. Positional cloning of the zebrafish sauternes gene: a model for congenital sideroblastic anaemia. Nature genetics. 20(3):244-50. Pubmed: 9806542 Brownlie A, Donovan A, Pratt SJ, Paw BH, Oates AC, Brugnara C, Witkowska HE, Sassa S, Zon LI. 1998. Positional cloning of the zebrafish sauternes gene: a model for congenital sideroblastic anaemia. Nature genetics. 20(3):244-50. Pubmed: 9806542 Many human anaemias are caused by defects in haemoglobin synthesis. The zebrafish mutant sauternes (sau) has a microcytic, hypochromic anaemia, suggesting that haemoglobin production is perturbed. During embryogenesis, sau mutants have delayed erythroid maturation and abnormal globin gene expression. Using positional cloning techniques, we show that sau encodes the erythroid-specific isoform of delta-aminolevulinate synthase (ALAS2; also known as ALAS-E), the enzyme required for the first step in haem biosynthesis. As mutations in ALAS2 cause congenital sideroblastic anaemia (CSA) in humans, sau represents the first animal model of this disease. -
Bahary N, Zon LI. 1998. Use of the zebrafish (Danio rerio) to define hematopoiesis. Stem cells (Dayton, Ohio). 16(2):89-98. Pubmed: 9554032 Bahary N, Zon LI. 1998. Use of the zebrafish (Danio rerio) to define hematopoiesis. Stem cells (Dayton, Ohio). 16(2):89-98. Pubmed: 9554032 Hematopoiesis in the vertebrate is characterized by the induction of ventral mesoderm to form hematopoietic stem cells and the eventual differentiation of these progenitors to form the peripheral blood lineages. Several genes have been implicated in the differentiation and development of hematopoietic and vascular progenitor cells, yet our understanding of the discrete steps involved in the induction of these cells from the ventral mesoderm is still incomplete. One method of delineating these processes is based on the use of lower vertebrates. The zebrafish (Danio rerio) is an especially robust vertebrate system for both isolating and characterizing genes involved in these processes. Hematopoietic mutants have been generated with defects in many of the steps of both the primitive and definitive hematopoietic programs. Cloning of the genes that underlie these mutations should yield valuable details of hematopoiesis and may have therapeutic implications for bone marrow transplantation and stem cell gene therapy. -
Huber TL, Zhou Y, Mead PE, Zon LI. 1998. Cooperative effects of growth factors involved in the induction of hematopoietic mesoderm. Blood. 92(11):4128-37. Pubmed: 9834218 Huber TL, Zhou Y, Mead PE, Zon LI. 1998. Cooperative effects of growth factors involved in the induction of hematopoietic mesoderm. Blood. 92(11):4128-37. Pubmed: 9834218 Hematopoietic induction occurs on the ventral side of Xenopus gastrulae and is thought to be triggered by the growth factor bone morphogenetic protein 4 (BMP-4). To characterize this process, we developed a quantitative and sensitive assay for the induction of erythroid cells from totipotent ectoderm of the embryo. When high doses of BMP-4 were used in this explant assay, few erythroid cells were detected. In contrast, large numbers of differentiated erythroid cells were induced when ectoderm was treated with BMP-4 and the mesoderm inducers, activin, or fibroblast growth factor (FGF). Ectopic expression of GATA-1 also induced abundant erythroid cells in ectoderm treated with bFGF. This induction of erythroid cells by GATA-1 was blocked by coexpression with a dominant negative BMP-4 receptor, showing that GATA-1 requires the BMP signaling cascade to function. These results suggest that BMP-4 requires mesoderm induction to generate a program of gene expression, which regulates the specification of hematopoietic mesoderm by GATA factors. -
Ganiatsas S, Kwee L, Fujiwara Y, Perkins A, Ikeda T, Labow MA, Zon LI. 1998. SEK1 deficiency reveals mitogen-activated protein kinase cascade crossregulation and leads to abnormal hepatogenesis. Proceedings of the National Academy of Sciences of the United States of America. 95(12):6881-6. Pubmed: 9618507 Ganiatsas S, Kwee L, Fujiwara Y, Perkins A, Ikeda T, Labow MA, Zon LI. 1998. SEK1 deficiency reveals mitogen-activated protein kinase cascade crossregulation and leads to abnormal hepatogenesis. Proceedings of the National Academy of Sciences of the United States of America. 95(12):6881-6. Pubmed: 9618507 SEK1 (MKK4/JNKK) is a mitogen-activated protein kinase activator that has been shown to participate in vitro in two stress-activated cascades terminating with the SAPK and p38 kinases. To define the role of SEK1 in vivo, we studied stress-induced signaling in SEK1(-/-) embryonic stem and fibroblast cells and evaluated the phenotype of SEK1(-/-) mouse embryos during development. Studies of SEK1(-/-) embryonic stem cells demonstrated defects in stimulated SAPK phosphorylation but not in the phosphorylation of p38 kinase. In contrast, SEK1(-/-) fibroblasts exhibited defects in both SAPK and p38 phosphorylation, demonstrating that crosstalk exists between the stress-activated cascades. Tumor necrosis factor alpha and interleukin 1 stimulation of both stress-activated cascades are severely affected in the SEK1(-/-) fibroblast cells. SEK1 deficiency leads to embryonic lethality after embryonic day 12.5 and is associated with abnormal liver development. This phenotype is similar to c-jun null mouse embryos and suggests that SEK1 is required for phosphorylation and activation of c-jun during the organo-genesis of the liver. -
Mead PE, Kelley CM, Hahn PS, Piedad O, Zon LI. 1998. SCL specifies hematopoietic mesoderm in Xenopus embryos. Development (Cambridge, England). 125(14):2611-20. Pubmed: 9636076 Mead PE, Kelley CM, Hahn PS, Piedad O, Zon LI. 1998. SCL specifies hematopoietic mesoderm in Xenopus embryos. Development (Cambridge, England). 125(14):2611-20. Pubmed: 9636076 Targeted gene disruption experiments in the mouse have demonstrated an absolute requirement for several transcription factors for the development of hematopoietic progenitors during embryogenesis. Disruption of the basic helix-loop-helix gene SCL (stem cell leukemia) causes a block early in the hematopoietic program with defects in all hematopoietic lineages. To understand how SCL participates in the organogenesis of blood, we have isolated cDNAs encoding Xenopus SCL and characterized the function of SCL during embryogenesis. We demonstrate that SCL is expressed in ventral mesoderm early in embryogenesis. SCL expression is induced by BMP-4, and a dominant negative BMP-4 receptor inhibits SCL expression in the ventral region of the embryo. Expression of SCL in either bFGF-treated animal pole explants or dorsal marginal zone explants leads to the expression of globin protein. Furthermore, over-expression of SCL does not alter normal dorsal-ventral patterning in the embryo, indicating that SCL acts to specify mesoderm to a hematopoietic fate after inductive and patterning events have occurred. We propose that SCL is both necessary and sufficient to specify hematopoietic mesoderm, and that it has a similar role in specifying hematopoietic cell fate as MyoD has in specifying muscle cell fate. -
Lee KH, Marden JJ, Thompson MS, MacLennan H, Kishimoto Y, Pratt SJ, Schulte-Merker S, Hammerschmidt M, Johnson SL, Postlethwaite JH, Beier DC, Zon LI. 1998. Cloning and genetic mapping of zebrafish BMP-2. Developmental genetics. 23(2):97-103. Pubmed: 9770266 Lee KH, Marden JJ, Thompson MS, MacLennan H, Kishimoto Y, Pratt SJ, Schulte-Merker S, Hammerschmidt M, Johnson SL, Postlethwaite JH, Beier DC, Zon LI. 1998. Cloning and genetic mapping of zebrafish BMP-2. Developmental genetics. 23(2):97-103. Pubmed: 9770266 The BMP family of polypeptide growth factors has been shown to play diverse roles in establishing embryonic patterning and tissue fates. We report the cloning of the zebrafish homologue of BMP-2, examine its expression during embryogenesis, and find that it is localized to the distal end of the long arm of zebrafish chromosome 20. A missense mutation of the bmp2 gene has recently been shown to be responsible for the early dorsalized phenotype of the zebrafish swirl mutant [Kishimoto et al., 1997]. Given the dynamic expression of bmp2 in the developing embryo and the complex interactions of BMP signaling response in vertebrates, it is possible that other mutant phenotypes, due to altered bmp2 gene expression, will eventually map to or interact with this genetic locus. -
Alberola-Ila J, Levin SD, Barton G, Forbush K, Zon LI, Perlmutter RM. 1998. Analysis of the role of MKK-4/Sek-1 in T cell development and apoptosis. International immunology. 10(8):1077-82. Pubmed: 9723693 Alberola-Ila J, Levin SD, Barton G, Forbush K, Zon LI, Perlmutter RM. 1998. Analysis of the role of MKK-4/Sek-1 in T cell development and apoptosis. International immunology. 10(8):1077-82. Pubmed: 9723693 The stress-activated protein kinases (SAPK) are a group of dual-specificity kinases with potential roles in the control of apoptosis and proliferation. In most cells they are regulated through phosphorylation by MKK-4. We have investigated the role of MKK-4 in T cell development and function by generating transgenic animals expressing catalytically inactive MKK-4 (dMKK-4) in the thymus. Our results show that overexpression of dMKK-4 does not interfere with normal T cell development. Furthermore, expression of dMKK-4 inhibits Fas- but not phorbol ester plus ionomycin-induced activation of SAPK, suggesting that a SAPK kinase different from MKK-4 is responsible for the regulation of SAPK activation after stimulation of T cells with phorbol ester plus ionomycin. We then analyzed the effect of dMKK-4 on Fas-induced apoptosis of thymocytes. Our results show that activation of SAPK is not a necessary event in Fas-induced apoptosis of thymocytes. -
Kwok C, Korn RM, Davis ME, Burt DW, Critcher R, McCarthy L, Paw BH, Zon LI, Goodfellow PN, Schmitt K. 1998. Characterization of whole genome radiation hybrid mapping resources for non-mammalian vertebrates. Nucleic acids research. 26(15):3562-6. Pubmed: 9671819 Kwok C, Korn RM, Davis ME, Burt DW, Critcher R, McCarthy L, Paw BH, Zon LI, Goodfellow PN, Schmitt K. 1998. Characterization of whole genome radiation hybrid mapping resources for non-mammalian vertebrates. Nucleic acids research. 26(15):3562-6. Pubmed: 9671819 Radiation hybrid panels are already available for genome mapping in human and mouse. In this study we have used two model organisms (chicken and zebrafish) to show that hybrid panels that contain a full complement of the donor genome can be generated by fusion to hamster cells. The quality of the resulting hybrids has been assessed using PCR and FISH. We confirmed the utility of our panels by establishing the percentage of donor DNA present in the hybrids. Our hybrid resources will allow inexpensive gene mapping and we expect that this technology can be transferred to many other species. Such successes are providing the basis for a new era of mapping tools, in the form of whole genome radiation hybrid panels, and are opening new possibilities for systematic genome analysis in the animal genetics community. -
Mead PE, Zhou Y, Lustig KD, Huber TL, Kirschner MW, Zon LI. 1998. Cloning of Mix-related homeodomain proteins using fast retrieval of gel shift activities, (FROGS), a technique for the isolation of DNA-binding proteins. Proceedings of the National Academy of Sciences of the United States of America. 95(19):11251-6. Pubmed: 9736722 Mead PE, Zhou Y, Lustig KD, Huber TL, Kirschner MW, Zon LI. 1998. Cloning of Mix-related homeodomain proteins using fast retrieval of gel shift activities, (FROGS), a technique for the isolation of DNA-binding proteins. Proceedings of the National Academy of Sciences of the United States of America. 95(19):11251-6. Pubmed: 9736722 We have developed a technique, fast retrieval of gel shift activities (FROGS), that allows for the rapid isolation of proteins that interact with DNA. Using this technique, we have isolated two proteins that are structurally similar to Mix.1, a PAX class homeodomain protein with ventralizing activity in Xenopus. The Mix family of proteins are expressed during late blastula and gastrula stages of Xenopus development. During gastrulation, these genes are expressed at high levels in distinct, yet overlapping regions in mesoderm and endoderm. The members of the Mix family heterodimerize with each other and overexpression of each results in severe axial abnormalities. Mix.3 and Mix.4 can directly induce primitive ectoderm to become endoderm whereas Mix.1 cannot. Injection of Mix.3 or Mix.4 RNA in the whole embryo results in extensive ectopic endodermin mRNA expression. The expression of the Mix family homeoproteins is differentially regulated by activin, Vg1, BMP-4, and fibroblast growth factor, supporting a model in which the Mix homeoproteins are downstream effectors of growth factor signaling during endoderm and ventral mesoderm formation. -
Förnzler D, Her H, Knapik EW, Clark M, Lehrach H, Postlethwait JH, Zon LI, Beier DR. 1998. Gene mapping in zebrafish using single-strand conformation polymorphism analysis. Genomics. 51(2):216-22. Pubmed: 9722944 Förnzler D, Her H, Knapik EW, Clark M, Lehrach H, Postlethwait JH, Zon LI, Beier DR. 1998. Gene mapping in zebrafish using single-strand conformation polymorphism analysis. Genomics. 51(2):216-22. Pubmed: 9722944 To exploit fully the power of the zebrafish system as a model for vertebrate development, it will be necessary to develop efficient tools for genomic analysis. In this report we have tested whether single-strand conformation polymorphism analysis (SSCP) can be utilized for gene mapping in zebrafish. Over 100 primer pairs derived from noncoding regions of known genes and partially characterized cDNAs were analyzed, and a polymorphism frequency of approximately 50% was detected in zebrafish strains used for genetic mapping studies. A subset of these polymorphic cDNAs was localized on the zebrafish map. SSCP thus represents an efficient strategy for mapping transcribed sequences with a high resolution in the zebrafish genome, which will facilitate the integration of existing zebrafish framework maps, the generation of a zebrafish EST map, and the application of alternative gene localization strategies such as comparative mapping. 1997
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Molnár A, Theodoras AM, Zon LI, Kyriakis JM. 1997. Cdc42Hs, but not Rac1, inhibits serum-stimulated cell cycle progression at G1/S through a mechanism requiring p38/RK. The Journal of biological chemistry. 272(20):13229-35. Pubmed: 9148940 Molnár A, Theodoras AM, Zon LI, Kyriakis JM. 1997. Cdc42Hs, but not Rac1, inhibits serum-stimulated cell cycle progression at G1/S through a mechanism requiring p38/RK. The Journal of biological chemistry. 272(20):13229-35. Pubmed: 9148940 Antimitogenic stimuli such as environmental or genotoxic stress, transforming growth factor-beta, and the inflammatory cytokines tumor necrosis factor and interleukin-1 activate two extracellular signal-regulated kinase (ERK)-based signaling pathways: the stress-activated protein kinase (SAPK/JNK) pathway and the p38 pathway. Activated p38 phosphorylates transcription factors important in the regulation of cell growth and apoptosis, including activating transcription factor 2 (ATF2), Max, cAMP response element-binding protein-homologous protein/growth arrest DNA damage 153 (CHDP/GADD153). In turn, p38 lies downstream of the Rho family GTPases Cdc42Hs and Rac1, as well as at least three mitogen-activated protein kinase (MAPK)/ERK-kinases (MEKs): MAPK kinases-3, -6, and SAPK/ERK-kinase-1. Although many of the stimuli that activate p38 can also inhibit cell cycle progression, a clear-cut role for the p38 pathway in cell cycle regulation has not been established. Using a quantitative microinjection approach, we show here that Cdc42Hs, but not Rac1 or RhoA, can inhibit cell cycle progression at G1/S through a mechanism requiring activation of p38. These results suggest a novel role for Cdc42Hs in cell cycle inhibition. Furthermore, these results suggest that although both Cdc42Hs and Rac1 can activate p38 in situ, the effects of Cdc42Hs and Rac1 on cell cycle progression are, in fact, quite distinct. -
Lustig KD, Stukenberg PT, McGarry TJ, King RW, Cryns VL, Mead PE, Zon LI, Yuan J, Kirschner MW. 1997. Small pool expression screening: identification of genes involved in cell cycle control, apoptosis, and early development. Methods in enzymology. 283:83-99. Pubmed: 9251013 Lustig KD, Stukenberg PT, McGarry TJ, King RW, Cryns VL, Mead PE, Zon LI, Yuan J, Kirschner MW. 1997. Small pool expression screening: identification of genes involved in cell cycle control, apoptosis, and early development. Methods in enzymology. 283:83-99. Pubmed: 9251013 -
Orkin SH, Zon LI. 1997. Genetics of erythropoiesis: induced mutations in mice and zebrafish. Annual review of genetics. 31:33-60. Pubmed: 9442889 Orkin SH, Zon LI. 1997. Genetics of erythropoiesis: induced mutations in mice and zebrafish. Annual review of genetics. 31:33-60. Pubmed: 9442889 Production of red blood cells (erythropoiesis) in the vertebrate embryo is critical to its survival and subsequent development. As red cells are the first blood cells to appear in embryogenesis, their origin reflects commitment of mesoderm to an hematopoietic fate and provides an avenue by which to examine the development of the hematopoietic system, including the hematopoietic stem cell (HSC). We discuss the genetics of erythropoiesis as studied in two systems: the mouse and zebrafish (Danio rerio). In the mouse, targeted disruption has established several genes as essential at different stages of hematopoiesis or erythroid precursor cell maturation. In the zebrafish, numerous mutants displaying a wide range of phenotypes have been isolated, although the affected genes are unknown. In comparing mouse knockout and zebrafish mutant phenotypes, we propose a pathway for erythropoiesis that emphasizes the apparent similarity of the mutants and the complementary nature of investigation in the two species. We speculate that further genetic studies in mouse and zebrafish will identify the majority of essential genes and define a regulatory network for hematopoiesis in vertebrates. -
Hirai Si, Katoh M, Terada M, Kyriakis JM, Zon LI, Rana A, Avruch J, Ohno S. 1997. MST/MLK2, a member of the mixed lineage kinase family, directly phosphorylates and activates SEK1, an activator of c-Jun N-terminal kinase/stress-activated protein kinase. The Journal of biological chemistry. 272(24):15167-73. Pubmed: 9182538 Hirai Si, Katoh M, Terada M, Kyriakis JM, Zon LI, Rana A, Avruch J, Ohno S. 1997. MST/MLK2, a member of the mixed lineage kinase family, directly phosphorylates and activates SEK1, an activator of c-Jun N-terminal kinase/stress-activated protein kinase. The Journal of biological chemistry. 272(24):15167-73. Pubmed: 9182538 c-Jun N-terminal kinases/stress-activated protein kinases (JNKs/SAPKs) are mitogen-activated protein kinase (MAPK)-related protein kinases that are involved in several cellular events, including growth, differentiation, and apoptosis. Mixed lineage kinases (MLKs) form a family of protein kinases sharing two leucine zipper-like motifs and a kinase domain whose primary structure is similar to both the tyrosine-specific and the serine/threonine-specific kinase classes. We have reported that a member of the MLK family, MUK/DLK/ZPK, can activate JNK/SAPK in vivo, and here we show that another member of the MLK family, MST/MLK2, activates JNK/SAPK. Both MUK/DLK/ZPK and MST/MLK2 cause a slight activation of p38/Mpk2 when overexpressed in COS-1 cells, whereas MST/MLK2, but not MUK/DLK/ZPK, activates extracellular response kinase (ERK) to a certain degree. The activity of SEK1/MKK4/JNKK, a MAPK kinase class protein kinase designated as a direct activator of JNK/SAPK, is also induced by MUK/DLK/ZPK or MST/MLK2 overexpression. Furthermore, recombinant MST/MLK2 produced in bacteria directly phosphorylates and activates SEK1/MKK4/JNKK in vitro, showing that MST/MLK2 acts like a MAPK kinase kinase. Taken together, these results suggest that MLK family members are MAPK kinase kinases preferentially acting on the JNK/SAPK pathway. -
Turpen JB, Kelley CM, Mead PE, Zon LI. 1997. Bipotential primitive-definitive hematopoietic progenitors in the vertebrate embryo. Immunity. 7(3):325-34. Pubmed: 9324353 Turpen JB, Kelley CM, Mead PE, Zon LI. 1997. Bipotential primitive-definitive hematopoietic progenitors in the vertebrate embryo. Immunity. 7(3):325-34. Pubmed: 9324353 Two regions of the vertebrate embryo, the blood islands and the dorsal lateral plate (DLP), participate in early hematopoietic development. In Xenopus, primitive erythrocytes are derived solely from the ventral blood islands (VBI), while definitive hematopoietic cells such as lymphocytes are derived from both VBI and DLP. We have utilized a transplantation technique to demonstrate in vivo that all hematopoietic cells (embryonic, fetal, or adult) originate from ventral mesoderm. Reciprocal grafts between VBI and DLP demonstrated that both regions are bipotential with respect to primitive and definitive hematopoiesis. Commitment of the VBI to primitive erythropoiesis and restriction of the DLP to definitive hematopoiesis occurs during neurula stages. Thus, hematopoietic development involves the induction of the blood program on the ventral axis of the embryo followed by environmentally regulated specification to the primitive or definitive lineages. -
Kishimoto Y, Lee KH, Zon L, Hammerschmidt M, Schulte-Merker S. 1997. The molecular nature of zebrafish swirl: BMP2 function is essential during early dorsoventral patterning. Development (Cambridge, England). 124(22):4457-66. Pubmed: 9409664 Kishimoto Y, Lee KH, Zon L, Hammerschmidt M, Schulte-Merker S. 1997. The molecular nature of zebrafish swirl: BMP2 function is essential during early dorsoventral patterning. Development (Cambridge, England). 124(22):4457-66. Pubmed: 9409664 Early dorsoventral pattern formation in vertebrate embryos is regulated by opposing activities of ventralizing bone morphogenetic proteins (BMPs) and dorsal-specific BMP antagonists such as Chordin, Noggin and Follistatin. Specific defects in early dorsoventral patterning have been recently found in a number of zebrafish mutants, which exhibit either a ventralized or dorsalized phenotype. One of these, the ventralized mutant chordino (originally called dino) is caused by a mutation in the zebrafish chordin homologue and interacts genetically with the dorsalized mutant swirl. In swirl mutant embryos, dorsal structures such as notochord and somites are expanded while ventral structures such as blood and nephros are missing. Here we demonstrate that the swirl phenotype is caused by mutations in the zebrafish bmp2 gene (zbmp2). While injection of mRNAs encoded by the mutant alleles has no ventralizing effect, injection of wild-type zbmp2 mRNA leads to a complete rescue of the swirl mutant phenotype. Fertile adult mutant fish were obtained, showing that development after gastrulation is not dependent on zbmp2 function. In addition zBMP2 has no maternal role in mesoderm induction. Our analysis shows that swirl/BMP2, unlike mouse BMP2 but like mouse BMP4, is required for early dorsoventral patterning of the zebrafish embryo. -
Chan FY, Robinson J, Brownlie A, Shivdasani RA, Donovan A, Brugnara C, Kim J, Lau BC, Witkowska HE, Zon LI. 1997. Characterization of adult alpha- and beta-globin genes in the zebrafish. Blood. 89(2):688-700. Pubmed: 9002973 Chan FY, Robinson J, Brownlie A, Shivdasani RA, Donovan A, Brugnara C, Kim J, Lau BC, Witkowska HE, Zon LI. 1997. Characterization of adult alpha- and beta-globin genes in the zebrafish. Blood. 89(2):688-700. Pubmed: 9002973 Developmental switching of hemoglobins (Hbs) occurs in most vertebrates, yet the cellular and molecular basis for this process remains elusive. The zebrafish is a new genetic and developmental system that can be used to study embryogenesis, and mutants with a variety of defects in hematopoiesis have recently been derived. To initiate our studies on Hb switching in this organism, we have characterized the globins expressed in the adult. Reversed-phase high performance liquid chromatography and mass spectrometric analyses of adult peripheral blood hemolysates showed that there are three major alpha globins and two beta globins in circulating erythroid cells. In addition, we have isolated and characterized zebrafish adult alpha- and beta-globin cDNA clones that encode some of these globins. High levels of alpha- and beta-globin gene expression were detected in adult erythroid cells, whereas embryonic erythroid cells expressed little, if any, of these RNAs. We have also shown that the alpha- and beta-globin genes are tightly linked on the same chromosome and are arrayed in a 3'-5' to 5'-3' configuration, respectively. The characterization of these genes and regulatory elements in this globin locus will provide insight into the process of globin gene transcription. With these reagents, future studies of Hb switching in zebrafish mutants with defective hematopoiesis will be possible. 1996
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Kieran MW, Zon LI. 1996. Stress- and mitogen-activated signal transduction in hematopoietic cells. Current opinion in hematology. 3(1):27-34. Pubmed: 9372048 Kieran MW, Zon LI. 1996. Stress- and mitogen-activated signal transduction in hematopoietic cells. Current opinion in hematology. 3(1):27-34. Pubmed: 9372048 The ability of an organism to respond to its environment is critical to survival. The mechanisms by which cells recognize and interpret different stimuli vary enormously and can be manifested by proliferation, differentiation, apoptosis, or altered metabolic activity. Recently, a series of signaling cascades was identified that links actions on the cell surface with activation or suppression of transcription. These signals are transmitted via phosphorylation and include the stress-activated protein kinases and the mitogen-activated protein kinases. This review describes these cascades in reference to the hematopoietic cell lineages. -
Haire RN, Ota T, Rast JP, Litman RT, Chan FY, Zon LI, Litman GW. 1996. A third Ig light chain gene isotype in Xenopus laevis consists of six distinct VL families and is related to mammalian lambda genes. Journal of immunology (Baltimore, Md. : 1950). 157(4):1544-50. Pubmed: 8759737 Haire RN, Ota T, Rast JP, Litman RT, Chan FY, Zon LI, Litman GW. 1996. A third Ig light chain gene isotype in Xenopus laevis consists of six distinct VL families and is related to mammalian lambda genes. Journal of immunology (Baltimore, Md. : 1950). 157(4):1544-50. Pubmed: 8759737 Xenopus laevis is a unique model for studying the ontogenetic development of immune functions. A short primer PCR amplification method was employed to amplify fragments from Xenopus genomic DNA that are related to Ig light chains and TCR. One fragment was identified that appeared to represent a novel type of light chain and was used as a probe to recover the corresponding cDNA from a spleen cDNA library. We designate these light chains type III. Using an iterative screening procedure, six families of VL genes, two distinct JL and two distinct CL sequences, were identified. In a comparison of phylogenetically diverse light chains, the type III genes align with higher vertebrate lambda genes. Southern blot hybridization analyses with genomic DNA from different animals showed the VL and CL sequences to be both diverse and polymorphic. Intrafamily sequence comparisons of VL genes revealed additional diversity. Collectively, these studies confirm the existence of a third type of light chain gene in Xenopus, establish a high degree of genetic variation in the sequences encoding the light chain V regions, and provide the most significant evidence to date for the presence of a lambda-like light chain gene at the phylogenetic level of the amphibians. -
Rana A, Gallo K, Godowski P, Hirai S, Ohno S, Zon L, Kyriakis JM, Avruch J. 1996. The mixed lineage kinase SPRK phosphorylates and activates the stress-activated protein kinase activator, SEK-1. The Journal of biological chemistry. 271(32):19025-8. Pubmed: 8702571 Rana A, Gallo K, Godowski P, Hirai S, Ohno S, Zon L, Kyriakis JM, Avruch J. 1996. The mixed lineage kinase SPRK phosphorylates and activates the stress-activated protein kinase activator, SEK-1. The Journal of biological chemistry. 271(32):19025-8. Pubmed: 8702571 SPRK (also called PTK-1 and MLK-3), a member of the mixed lineage kinase subfamily of (Ser/Thr) protein kinases, encodes an amino-terminal SH3 domain followed by a kinase catalytic domain, two leucine zippers interrupted by a short spacer, a Rac/Cdc42 binding domain, and a long carboxyl-terminal proline-rich region. We report herein that SPRK activates the stress-activated protein kinases (SAPKs) but not ERK-1 during transient expression in COS cells; the p38 kinase is activated modestly (1.3-2 fold) but consistently. SPRK also activates cotransfected SEK-1/MKK-4, a dual specificity kinase which phosphorylates and activates SAPK. Reciprocally, expression of mutant, inactive SEK-1 inhibits completely the basal and SPRK-activated SAPK activity. Immunoprecipitated recombinant SPRK is able to phosphorylate and activate recombinant SEK-1 in vitro to an extent comparable to that achieved by MEK kinase-1. These results identify SPRK as a candidate upstream activator of the stress-activated protein kinases, acting through the phosphorylation and activation of SEK-1. -
Mead PE, Brivanlou IH, Kelley CM, Zon LI. 1996. BMP-4-responsive regulation of dorsal-ventral patterning by the homeobox protein Mix.1. Nature. 382(6589):357-60. Pubmed: 8684465 Mead PE, Brivanlou IH, Kelley CM, Zon LI. 1996. BMP-4-responsive regulation of dorsal-ventral patterning by the homeobox protein Mix.1. Nature. 382(6589):357-60. Pubmed: 8684465 In an expression screen for factors that pattern or induce ventral mesoderm, we isolated a complementary DNA encoding Mix.1, a paired class homeobox gene with no previously known function. Injection of Mix.1 messenger RNA results in extensive blood formation in the whole embryo and transforms dorsal mesoderm to a ventral fate. Mix.1 expression is induced by bone morphogenetic protein-4 (BMP-4), and a dominant inhibitory mutant of Mix.1 can restore a dorsal axis in embryos ventralized by ectopic BMP-4 expression. Mix.1 can form heterodimers with the dorsalizing gene siamois, which encodes a homeodomain protein that is structurally similar to Mix.1. Furthermore, Mix.1 blocks the duplicated axis induced by ectopic siamois expression. Our findings indicate that Mix.1 participates in a BMP-4 signalling pathway to pattern ventral mesoderm, and suggest a model whereby dimerization of homeodomain proteins regulates dorsal-ventral patterning. -
Bishop TR, Miller MW, Beall J, Zon LI, Dierks P. 1996. Genetic regulation of delta-aminolevulinate dehydratase during erythropoiesis. Nucleic acids research. 24(13):2511-8. Pubmed: 8692689 Bishop TR, Miller MW, Beall J, Zon LI, Dierks P. 1996. Genetic regulation of delta-aminolevulinate dehydratase during erythropoiesis. Nucleic acids research. 24(13):2511-8. Pubmed: 8692689 In an effort to understand how the heme biosynthetic pathway is uniquely regulated in erythroid cells, we examined the structure of the gene encoding murine delta-aminolevulinate dehydratase (ALAD; EC4.2.1.24), which is the second enzyme of the pathway. The gene contains two first exons, named 1A and 1B, which are alternatively spliced to exon 2, where the coding region begins. Each first exon has its own promoter. The promoter driving exon 1A expression is TATA-less and contains many GC boxes. In contrast, the exon 1B promoter bears regulatory sequences similar to those found for beta-globin and other erythroid-specific genes. Tissue distribution studies reveal that ALAD mRNA containing axon 1A is ubiquitous, whereas mRNA containing axon 1B is found only in erythroid tissues. This finding, together with our further observation that GATA-1 mRNA levels increase 3-fold during maturation of murine erythroid progenitor cells, may help explain simultaneous 3-fold increases in exon 1B expression. The unexpected result that axon 1A expression also increases 3-fold during CFU-E maturation may be attributable to the action of NF-E2, since there is a potential binding site in a position analogous to the NF-E2 site in the locus control region of the beta-globin gene cluster. -
White RA, Hughes RT, Adkison LR, Bruns G, Zon LI. 1996. The gene encoding protein kinase SEK1 maps to mouse chromosome 11 and human chromosome 17. Genomics. 34(3):430-2. Pubmed: 8786147 White RA, Hughes RT, Adkison LR, Bruns G, Zon LI. 1996. The gene encoding protein kinase SEK1 maps to mouse chromosome 11 and human chromosome 17. Genomics. 34(3):430-2. Pubmed: 8786147 We report the mapping of the human and mouse genes encoding SEK1 (SAPK/ERK kinase-1), a newly identified protein kinase that is a potent physiological activator of the stress-activated protein kinases. The human SERK1 gene was assigned to human chromosome 17 using genomic DNAs from human-rodent somatic cell hybrid lines. A specific human PCR product was observed solely in the somatic cell line containing human chromosome 17. The mouse Serk1 gene was mapped to chromosome 11, closely linked to D11Mit4, using genomic DNAs from a (C57BL/6J x Mus spretus)F1 x M. spretus backcross. -
Johnson NL, Gardner AM, Diener KM, Lange-Carter CA, Gleavy J, Jarpe MB, Minden A, Karin M, Zon LI, Johnson GL. 1996. Signal transduction pathways regulated by mitogen-activated/extracellular response kinase kinase kinase induce cell death. The Journal of biological chemistry. 271(6):3229-37. Pubmed: 8621725 Johnson NL, Gardner AM, Diener KM, Lange-Carter CA, Gleavy J, Jarpe MB, Minden A, Karin M, Zon LI, Johnson GL. 1996. Signal transduction pathways regulated by mitogen-activated/extracellular response kinase kinase kinase induce cell death. The Journal of biological chemistry. 271(6):3229-37. Pubmed: 8621725 Mitogen-activated/extracellular response kinase kinase (MEK) kinase (MEKK) is a serine-threonine kinase that regulates sequential protein phosphorylation pathways, leading to the activation of mitogen-activated protein kinases (MAPK), including members of the Jun kinase (JNK)/stress-activated protein kinase (SAPK) family. In Swiss 3T3 and REF52 fibroblasts, activated MEKK induces cell death involving cytoplasmic shrinkage, nuclear condensation, and DNA fragmentation characteristic of apoptosis. Expression of activated MEKK enhanced the apoptotic response to ultraviolet irradiation, indicating that MEKK-regulated pathways sensitize cells to apoptotic stimuli. Inducible expression of activated MEKK stimulated the transactivation of c-Myc and Elk-1. Activated Raf, the serine-threonine protein kinase that activates the ERK members of the MAPK family, stimulated Elk-1 transactivation but not c-Myc; expression of activated Raf does not induce any of the cellular changes associated with MEKK-mediated cell death. Thus, MEKK selectively regulates signal transduction pathways that contribute to the apoptotic response. -
Zanke BW, Boudreau K, Rubie E, Winnett E, Tibbles LA, Zon L, Kyriakis J, Liu FF, Woodgett JR. 1996. The stress-activated protein kinase pathway mediates cell death following injury induced by cis-platinum, UV irradiation or heat. Current biology : CB. 6(5):606-13. Pubmed: 8805279 Zanke BW, Boudreau K, Rubie E, Winnett E, Tibbles LA, Zon L, Kyriakis J, Liu FF, Woodgett JR. 1996. The stress-activated protein kinase pathway mediates cell death following injury induced by cis-platinum, UV irradiation or heat. Current biology : CB. 6(5):606-13. Pubmed: 8805279 Array -
Sen J, Kapeller R, Fragoso R, Sen R, Zon LI, Burakoff SJ. 1996. Intrathymic signals in thymocytes are mediated by p38 mitogen-activated protein kinase. Journal of immunology (Baltimore, Md. : 1950). 156(12):4535-8. Pubmed: 8648093 Sen J, Kapeller R, Fragoso R, Sen R, Zon LI, Burakoff SJ. 1996. Intrathymic signals in thymocytes are mediated by p38 mitogen-activated protein kinase. Journal of immunology (Baltimore, Md. : 1950). 156(12):4535-8. Pubmed: 8648093 Thymocytes develop into mature functional T cells in the inductive environment of the thymus where thymocyte-stromal cell interactions and cytokines provide survival and differentiation signals as cues for thymocyte maturation. Disruption of the thymic microenvironment results in attenuation of T cell maturation, suggesting that intrathymic signals are essential for differentiation and repertoire selection. We have previously shown that several inducible nuclear factors such as AP-1, NF-AT, and NF-kappaB are activated in response to intrathymic signals. Here we demonstrate that in thymocytes p38 mitogen-activated protein (MAP) kinase, a member of the MAP kinase family of proteins that include the extracellular-signal regulated kinases and Jun aminoterminal kinases, is highly activated in response to intrathymic signals in vivo. These studies suggest a role for p38 MAP kinase in T cell survival and differentiation. -
Woodgett JR, Kyriakis JM, Avruch J, Zon LI, Zanke B, Templeton DJ. 1996. Reconstitution of novel signalling cascades responding to cellular stresses. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 351(1336):135-41; discussion 142. Pubmed: 8650259 Woodgett JR, Kyriakis JM, Avruch J, Zon LI, Zanke B, Templeton DJ. 1996. Reconstitution of novel signalling cascades responding to cellular stresses. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 351(1336):135-41; discussion 142. Pubmed: 8650259 Mammalian cells respond to their immediate environment by inducing signal transduction cascades that regulate metabolism, secretion and gene expression. Several of these signalling pathways are structurally and organizationally related insofar as they require activation of a protein-serine kinase via it's phosphorylation on tyrosine and threonine; the archetype being mitogen-activated protein kinase (MAPK) which responds primarily to mitogenic stimuli via Ras. In contrast, two more recently identified cascades are responsive to cellular stresses such as heat, inflammatory cytokines, ischaemia and metabolic poisons. The recent identification of the components of these pathways has allowed manipulation of the stress-responsive pathways and evaluation of their physiological roles. These studies reveal a high degree of independence between the pathways not apparent from in vitro studies. Manipulation of the pathways in vivo will likely result in novel therapies for inflammatory disease and reperfusion injury. -
Watanabe G, Howe A, Lee RJ, Albanese C, Shu IW, Karnezis AN, Zon L, Kyriakis J, Rundell K, Pestell RG. 1996. Induction of cyclin D1 by simian virus 40 small tumor antigen. Proceedings of the National Academy of Sciences of the United States of America. 93(23):12861-6. Pubmed: 8917510 Watanabe G, Howe A, Lee RJ, Albanese C, Shu IW, Karnezis AN, Zon L, Kyriakis J, Rundell K, Pestell RG. 1996. Induction of cyclin D1 by simian virus 40 small tumor antigen. Proceedings of the National Academy of Sciences of the United States of America. 93(23):12861-6. Pubmed: 8917510 Cell-cycle progression is mediated by a co-ordinated interaction between cyclin-dependent kinases and their target proteins including the pRB and E2F/DP-1 complexes. Immunoneutralization and antisense experiments have established that the abundance of cyclin D1, a regulatory subunit of the cyclin-dependent kinases, may be rate-limiting for G1 phase progression of the cell cycle. Simian virus 40 (SV40) small tumor (t) antigen is capable of promoting G1 phase progression and augments substantially the efficiency of SV40 transformation through several distinct domains. In these studies, small t antigen stimulated cyclin D1 promoter activity 7-fold, primarily through an AP-1 binding site at -954 with additional contributions from a CRE site at -57. The cyclin D1 AP-1 and CRE sites were sufficient for activation by small t antigen when linked to an heterologous promoter. Point mutations of small t antigen between residues 97-103 that reduced PP2A binding were partially defective in the induction of the cyclin D1 promoter. These mutations also reduced activation of MEK1 and two distinct members of the mitogen-activated protein kinase family, the ERKs (extracellular signal regulated kinases) and the SAPKs (stress-activated protein kinases), in transfected cells. Dominant negative mutants of either MEK1, ERK or SEK1, reduced small t-dependent induction of the cyclin D1 promoter. SV40 small t induction of the cyclin D1 promoter involves both the ERK and SAPK pathways that together may contribute to the proliferative and transformation enhancing activity of small t antigen. -
Maeno M, Mead PE, Kelley C, Xu RH, Kung HF, Suzuki A, Ueno N, Zon LI. 1996. The role of BMP-4 and GATA-2 in the induction and differentiation of hematopoietic mesoderm in Xenopus laevis. Blood. 88(6):1965-72. Pubmed: 8822915 Maeno M, Mead PE, Kelley C, Xu RH, Kung HF, Suzuki A, Ueno N, Zon LI. 1996. The role of BMP-4 and GATA-2 in the induction and differentiation of hematopoietic mesoderm in Xenopus laevis. Blood. 88(6):1965-72. Pubmed: 8822915 Vertebrate embryonic blood formation is regulated by factors that participate in dorsal-ventral patterning and mesoderm induction. The GATA-binding transcription factors are required for normal hematopoiesis and are expressed during gastrulation when ventral mesoderm (VM) is induced to form blood. Based on the recent demonstration that bone morphogenetic protein (BMP-4) is a potent ventralizing factor and inducer of hematopoietic tissue, we hypothesized that GATA-2 could be induced or activated by BMP-4. Here we demonstrate that BMP-4 can stimulate GATA-2 expression, and that expression of a dominant negative BMP-4 receptor can suppress GATA-2 induction by BMP-4 in ventral mesoderm. Over-expression of GATA-2 in ventral mesoderm leads to increased globin production and forced expression of GATA-2 in primitive ectoderm adjacent to ventral mesoderm also stimulates globin expression. Our results suggest that BMP-4 and GATA-2 can function in two adjacent germ layers, mesoderm and ectoderm, to participate in blood cell formation during embryogenesis. -
Pestell RG, Albanese C, Watanabe G, Lee RJ, Lastowiecki P, Zon L, Ostrowski M, Jameson JL. 1996. Stimulation of the P-450 side chain cleavage enzyme (CYP11A1) promoter through ras- and Ets-2-signaling pathways. Molecular endocrinology (Baltimore, Md.). 10(9):1084-94. Pubmed: 8885243 Pestell RG, Albanese C, Watanabe G, Lee RJ, Lastowiecki P, Zon L, Ostrowski M, Jameson JL. 1996. Stimulation of the P-450 side chain cleavage enzyme (CYP11A1) promoter through ras- and Ets-2-signaling pathways. Molecular endocrinology (Baltimore, Md.). 10(9):1084-94. Pubmed: 8885243 Expression of the ovine P-450 side-chain cleavage enzyme gene (CYP11A1) is stimulated by epidermal growth factor (EGF) through a pathway that involves c-Jun in JEG-3 placental cells. Growth factor signaling involves ras-dependent and ras-independent signaling pathways, which in turn regulate gene transcription through related but distinct mitogen-activated protein kinase pathways (MAPKs) including the extracellular signal-regulated kinases (ERKs) and the stress-activated protein kinases (SAPKs). We investigated the intracellular signaling pathways governing EGF induction of the CYP11A1 promoter. EGF stimulation of the CYP11A1 promoter (4-fold) was reduced 60% by a dominant negative mutant of ras (N17), and 30-40% by antisense ras. EGF induced both ERK and SAPK activity in JEG-3 cells. EGF-induced CYP11A1 promoter activity was reduced 60% by the MEK1 inhibitor PD098059 and 50% by a dominant negative mutant of the ERK-specific regulator MEK1. In contrast, dominant negative mutants of the SAPK-specific activator, SEK1, induced a further increase in EGF-induced CYP11A1 promoter activity. Constitutively active mutants of ras (V12 or L61) increased CYP11A1 promoter activity 6- to 8-fold. Deletion of the EGF response element (EGF-RE) between -92 and -77 bp reduced ras induction by 60%; however, a residual 3-fold induction remained through the proximal -77 bp. Mutation of the EGF-RE AP-1-like sequence in the context of the native promoter reduced CYP11A1 promoter activation by ras 60%. The EGF-RE sequence was sufficient for 6-fold activation by ras in the context of an heterologous thymidine kinase promoter. Candidate transcription factor targets (c-Jun, c-Ets-2) for the ras-signaling cascade were examined for their effects on CYP11A1 promoter activity. Overexpression of c-Jun induced the CYP11A1 promoter through the EGF-RE; however, c-Ets-2 activation of the CYP11A1 promoter (12-fold) required the proximal ras-responsive promoter sequences that are distinct from the EGF/MEK/c-Jun-responsive element. Induction of the CYP11A1 promoter by EGF involves a ras/MEK1/AP-1-dependent pathway that is distinct from induction by ras/c-Ets-2. -
Kieran MW, Perkins AC, Orkin SH, Zon LI. 1996. Thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor. Proceedings of the National Academy of Sciences of the United States of America. 93(17):9126-31. Pubmed: 8799165 Kieran MW, Perkins AC, Orkin SH, Zon LI. 1996. Thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor. Proceedings of the National Academy of Sciences of the United States of America. 93(17):9126-31. Pubmed: 8799165 The interaction of the hormone erythropoietin and its receptor (EpoR) is though to be required for normal hematopoiesis. To define the role of EpoR in this process, the murine EpoR was disrupted by homologous recombination. Mice lacking the EpoR died in utero at embryonic day 11-12.5 with severe anemia. Embryonic erythropoiesis was markedly diminished, while fetal liver hematopoiesis was blocked at the proerythroblast stage. Other cell types known to express EpoR, including megakaryocytes, mast, and neural cells were morphologically normal. Reverse transcription-coupled PCR analysis of RNA from embryonic yolk sac, peripheral blood, and fetal liver demonstrated near normal transcripts levels for EKLF, thrombopoietin (Tpo), c-MPL, GATA-1, GATA-2, and alpha- and embryonic beta H1-globin but non for adult beta maj-globin. While colony-forming unit-erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) colonies were not present in cultures derived from EpoR-/- liver or yolk sac cells, hemoglobin-containing BFU-E colonies were detected in cultures treated with recombinant Tpo and Kit ligand or with Tpo and interleukin 3 and 11. Rescued BFU-E colonies expressed adult beta-globin and c-MPL and appeared morphologically normal. Thus, erythroid progenitors are formed in vivo in mice lacking the EpoR, and our studies demonstrate that a signal transmitted through the Tpo receptor c-MPL stimulates proliferation and terminal differentiation of these progenitors in vitro. -
Verheij M, Bose R, Lin XH, Yao B, Jarvis WD, Grant S, Birrer MJ, Szabo E, Zon LI, Kyriakis JM, Haimovitz-Friedman A, Fuks Z, Kolesnick RN. 1996. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature. 380(6569):75-9. Pubmed: 8598911 Verheij M, Bose R, Lin XH, Yao B, Jarvis WD, Grant S, Birrer MJ, Szabo E, Zon LI, Kyriakis JM, Haimovitz-Friedman A, Fuks Z, Kolesnick RN. 1996. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature. 380(6569):75-9. Pubmed: 8598911 The induction of programmed cell death, or apoptosis, involves activation of a signalling system, many elements of which remain unknown. The sphingomyelin pathway, initiated by hydrolysis of the phospholipid sphingomyelin in the cell membrane to generate the second messenger ceramide, is thought to mediate apoptosis in response to tumour-necrosis factor (TNF)-alpha, to Fas ligand and to X-rays. It is not known whether it plays a role in the stimulation of other forms of stress-induced apoptosis. Given that environmental stresses also stimulate a stress-activated protein kinase (SAPK/JNK), the sphingomyelin and SAPK/JNK signalling systems may be coordinated in induction of apoptosis. Here we report that ceramide initiates apoptosis through the SAPK cascade and provide evidence for a signalling mechanism that integrates cytokine- and stress-activated apoptosis. 1995
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Winardi R, Discher D, Kelley C, Zon L, Mays K, Mohandas N, Conboy JG. 1995. Evolutionarily conserved alternative pre-mRNA splicing regulates structure and function of the spectrin-actin binding domain of erythroid protein 4.1. Blood. 86(11):4315-22. Pubmed: 7492792 Winardi R, Discher D, Kelley C, Zon L, Mays K, Mohandas N, Conboy JG. 1995. Evolutionarily conserved alternative pre-mRNA splicing regulates structure and function of the spectrin-actin binding domain of erythroid protein 4.1. Blood. 86(11):4315-22. Pubmed: 7492792 A developmental alternative splicing switch, involving exon 16 of protein 4.1 pre-mRNA, occurs during mammalian erythropoiesis. By controlling expression of a 21-amino acid peptide required for high-affinity interaction of protein 4.1 with spectrin and actin, this switch helps to regulate erythrocyte membrane mechanical stability. Here we show that key aspects of protein 4.1 structure and function are conserved in nucleated erythroid cells of the amphibian Xenopus laevis. Analysis of protein 4.1 cDNA sequences cloned from Xenopus erythrocytes and oocytes showed that tissue-specific alternative splicing of exon 16 also occurs in frogs. Importantly, functional studies with recombinant Xenopus erythroid 4.1 demonstrated specific binding to and mechanical stabilization of 4.1-deficient human erythrocyte membranes. Phylogenetic sequence comparison showed two evolutionarily conserved peptides that represent candidate spectrin-actin binding sites. Finally, in situ hybridization of early embryos showed high expression of 4.1 mRNA in ventral blood islands and in developing brain structures. These results demonstrate that regulated expression of structurally and functionally distinct protein 4.1 isoforms, mediated by tissue-specific alternative splicing, has been highly evolutionarily conserved. Moreover, both nucleated amphibian erythrocytes and their enucleated mammalian counterparts express 4.1 isoforms functionally competent for spectrin-actin binding. -
Kharbanda S, Pandey P, Ren R, Mayer B, Zon L, Kufe D. 1995. c-Abl activation regulates induction of the SEK1/stress-activated protein kinase pathway in the cellular response to 1-beta-D-arabinofuranosylcytosine. The Journal of biological chemistry. 270(51):30278-81. Pubmed: 8530447 Kharbanda S, Pandey P, Ren R, Mayer B, Zon L, Kufe D. 1995. c-Abl activation regulates induction of the SEK1/stress-activated protein kinase pathway in the cellular response to 1-beta-D-arabinofuranosylcytosine. The Journal of biological chemistry. 270(51):30278-81. Pubmed: 8530447 Previous work has shown that treatment of cells with the antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) is associated with induction of the c-jun gene. The present studies demonstrate that ara-C activates the c-Abl non-receptor tyrosine kinase. We also demonstrate that activity of the stress-activated protein kinase (SAP kinase/JNK) is increased in ara-C-treated cells. Using cells deficient in c-Abl (Abl-/-) and after introduction of the c-abl gene, we show that ara-C-induced c-Abl activity is necessary for the stimulation of SAP kinase. Other studies using cells transfected with a SEK1 dominant negative demonstrate that ara-C-induced SAP kinase activity is SEK1-dependent. Furthermore, we show that overexpression of truncated c-Abl results in activation of the SEK1/SAP kinase cascade. -
Richardson PM, Zon LI. 1995. Molecular cloning of a cDNA with a novel domain present in the tre-2 oncogene and the yeast cell cycle regulators BUB2 and cdc16. Oncogene. 11(6):1139-48. Pubmed: 7566974 Richardson PM, Zon LI. 1995. Molecular cloning of a cDNA with a novel domain present in the tre-2 oncogene and the yeast cell cycle regulators BUB2 and cdc16. Oncogene. 11(6):1139-48. Pubmed: 7566974 In an effort to identify genes that are differentially regulated during mast cell development, subtracted cDNA prepared from wild-type murine P815 mastocytoma cells and a P815 subline that exhibits properties of mast cell differentiation was used to screen mast cell cDNA libraries. Several known mast cell-specific cDNAs were isolated including mast cell carboxypeptidase A (MC-CPA), murine mast cell protease-5 (MMCP-5), and gp49. A novel cDNA, designated Tbc1, was identified that showed differential expression in the two mast cell lines. The amino acid sequence predicted from the cDNA contains a 200 amino acid domain that is homologous to regions in the tre-2 oncogene and the yeast regulators of mitosis, BUB2 and cdc16. The N-terminal region contains a number of cysteine and histidine residues, potentially encoding a zinc finger domain. Tbc1 is a nuclear protein and is expressed in highest levels in hematopoietic cells, testis and kidney. Within these tissues, expression of Tbc1 is cell- and stage-specific. Based on sequence similarity, pattern of expression and subcellular localization, Tbc1 may play a role in the cell cycle and differentiation of various tissues. -
Hughes R, Chan FY, White RA, Zon LI. 1995. Cloning and chromosomal localization of a mouse cDNA with homology to the Saccharomyces cerevisiae gene zuotin. Genomics. 29(2):546-50. Pubmed: 8666407 Hughes R, Chan FY, White RA, Zon LI. 1995. Cloning and chromosomal localization of a mouse cDNA with homology to the Saccharomyces cerevisiae gene zuotin. Genomics. 29(2):546-50. Pubmed: 8666407 The eukaryotic DnaJ homologs form a family of proteins with diverse functions. One member of the family, the Saccharomyces cerevisiae gene zuotin, was isolated for its ability to bind Z-DNA. Here, we have isolated a mouse cDNA called ZRF1 (for zuotin-related factor1) with significant homology to zuotin. The DnaJ domain and candidate phosphorylation sites of zuotin and ZRF1 are highly conserved. ZRF1 gene is localized on chromosome 5. The structural similarity of zuotin and ZRF1 suggests conservation of function of this DnaJ subfamily. -
Stainier DY, Weinstein BM, Detrich HW, Zon LI, Fishman MC. 1995. Cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages. Development (Cambridge, England). 121(10):3141-50. Pubmed: 7588049 Stainier DY, Weinstein BM, Detrich HW, Zon LI, Fishman MC. 1995. Cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages. Development (Cambridge, England). 121(10):3141-50. Pubmed: 7588049 Endothelial and hematopoietic cells appear synchronously on the extra-embryonic membranes of amniotes in structures known as blood islands. This observation has led to the suggestion that these two ventral lineages share a common progenitor. Recently, we have shown in the zebrafish, Danio rerio, that a single cell in the ventral marginal zone of the early blastula can give rise to both endothelial and blood cells as well as to other mesodermal cells (Stainier, D. Y. R., Lee, R. K. and Fishman, M. C. (1993). Development 119, 31-40; Lee, R. K. K., Stainier, D. Y. R., Weinstein, B. M. and Fishman, M. C. (1994). Development 120, 3361-3366). Here we describe a zebrafish mutation, cloche, that affects both the endothelial and hematopoietic lineages at a very early stage. The endocardium, the endothelial lining of the heart, is missing in mutant embryos. This deletion is selective as evidenced by the presence of other endothelial cells, for example those lining the main vessels of the trunk. Early cardiac morphogenesis proceeds normally even in the absence of the endocardium. The myocardial cells form a tube that is demarcated into chambers, beats rhythmically, but exhibits a reduced contractility. This functional deficit is likely due to the absence of the endocardial cells, although it may be a direct effect of the mutation on the myocardial cells. Cell transplantation studies reveal that the endothelial defect, i.e. the endocardial deletion, is a cell-autonomous lesion, consistent with the possibility that cloche is part of a signal transduction pathway. In addition, the number of blood cells is greatly reduced in cloche mutants and the hematopoietic tissues show no expression of GATA-1 or GATA-2, two key hematopoietic transcription factors that are first expressed during early embryogenesis. These results show that cloche is involved in the genesis and early diversification of the endothelial and blood lineages, possibly by affecting a common progenitor cell population. -
Zon LI. 1995. Developmental biology of hematopoiesis. Blood. 86(8):2876-91. Pubmed: 7579378 Zon LI. 1995. Developmental biology of hematopoiesis. Blood. 86(8):2876-91. Pubmed: 7579378 The cellular and environmental regulation of hematopoiesis has been generally conserved throughout vertebrate evolution, although subtle species differences exist. The factors that regulate hematopoietic stem cell homeostasis may closely resemble the inducers of embryonic patterning, rather than the factors that stimulate hematopoietic cell proliferation and differentiation. Comparative study of embryonic hematopoiesis in lower vertebrates can generate testable hypotheses that similar mechanisms occur during hematopoiesis in higher species. -
Detrich HW, Kieran MW, Chan FY, Barone LM, Yee K, Rundstadler JA, Pratt S, Ransom D, Zon LI. 1995. Intraembryonic hematopoietic cell migration during vertebrate development. Proceedings of the National Academy of Sciences of the United States of America. 92(23):10713-7. Pubmed: 7479870 Detrich HW, Kieran MW, Chan FY, Barone LM, Yee K, Rundstadler JA, Pratt S, Ransom D, Zon LI. 1995. Intraembryonic hematopoietic cell migration during vertebrate development. Proceedings of the National Academy of Sciences of the United States of America. 92(23):10713-7. Pubmed: 7479870 Vertebrate hematopoietic stem cells are derived from vental mesoderm, which is postulated to migrate to both extra- and intraembryonic positions during gastrula and neurula stages. Extraembryonic migration has previously been documented, but the origin and migration of intraembryonic hematopoietic cells have not been visualized. The zebrafish and most other teleosts do not form yolk sac blood islands during early embryogenesis, but instead hematopoiesis occurs solely in a dorsal location known as the intermediate cell mass (IM) or Oellacher. In this report, we have isolated cDNAs encoding zebrafish homologs of the hematopoietic transcription factors GATA-1 and GATA-2 and have used these markers to determine that the IM is formed from mesodermal cells in a posterior-lateral position on the yolk syncytial layer of the gastrula yolk sac. Surprisingly, cells of the IM then migrate anteriorly through most of the body length prior to the onset of active circulation and exit onto the yolk sac. These findings support a hypothesis in which the hematopoietic program of vertebrates is established by variations in homologous migration pathways of extra- and intraembryonic progenitors. -
Pombo CM, Kehrl JH, Sánchez I, Katz P, Avruch J, Zon LI, Woodgett JR, Force T, Kyriakis JM. 1995. Activation of the SAPK pathway by the human STE20 homologue germinal centre kinase. Nature. 377(6551):750-4. Pubmed: 7477268 Pombo CM, Kehrl JH, Sánchez I, Katz P, Avruch J, Zon LI, Woodgett JR, Force T, Kyriakis JM. 1995. Activation of the SAPK pathway by the human STE20 homologue germinal centre kinase. Nature. 377(6551):750-4. Pubmed: 7477268 Eukaryotic cells respond to different extracellular stimuli by recruiting homologous signalling pathways that use members of the MEKK, MEK and ERK families of protein kinases. The MEKK-->MEK-->ERK core pathways of Saccharomyces cerevisiae may themselves be regulated by members of the STE20 family of protein kinases. Here we report specific activation of the mammalian stress-activated protein kinase (SAPK) pathway by germinal centre kinase (GCK), a human STE20 homologue. SAPKs, members of the ERK family, are activated in situ by inflammatory stimuli, including tumour-necrosis factor (TNF) and interleukin-1, and phosphorylate and probably stimulate the transactivation function of c-Jun. Although GCK is found in many tissues, its expression in lymphoid follicles is restricted to the cells of the germinal centre, where it may participate in B-cell differentiation. Activation of the SAPK pathway by GCK illustrates further the striking conservation of eukaryotic signalling mechanisms and defines the first physiological function of a mammalian Ste20. 1994
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Kelley C, Yee K, Harland R, Zon LI. 1994. Ventral expression of GATA-1 and GATA-2 in the Xenopus embryo defines induction of hematopoietic mesoderm. Developmental biology. 165(1):193-205. Pubmed: 8088437 Kelley C, Yee K, Harland R, Zon LI. 1994. Ventral expression of GATA-1 and GATA-2 in the Xenopus embryo defines induction of hematopoietic mesoderm. Developmental biology. 165(1):193-205. Pubmed: 8088437 The early embryonic events involved in the commitment of mesoderm to form blood have not been studied in detail for lack of molecular markers. We have studied the expression of the hematopoietic transcription factors GATA-1 and GATA-2 during Xenopus embryogenesis. During development GATA-1 expression is localized to the ventral region of the embryo and precedes the expression of embryonic globins. GATA-2 is highly expressed in the ventral region of the embryo by the end of gastrulation and later is expressed in the blood island region and the central nervous system. Lithium-induced dorsalization of embryos abrogates GATA-2 expression, and uv-induced ventralization of embryos leads to a radially symmetrical expression of GATA-2. Therefore, GATA-2 expression reflects the ventral character of the embryo. The expression of the GATA-binding proteins and globin in ventral marginal zone explants demonstrates that hematopoiesis is programmed as early as the blastula stage. GATA-1 and GATA-2 are also expressed in cultured animal cap explants, suggesting that these cells have hematopoietic potential. The developmental expression of GATA-1 and GATA-2 is consistent with their role in hematopoiesis in higher organisms and defines the ventral regions of the early embryo that give rise to hematopoietic progenitors. Our studies indicate that these genes will be useful in defining the inductive events that lead to the formation of hematopoietic mesoderm. 1993
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Yashar BM, Kelley C, Yee K, Errede B, Zon LI. 1993. Novel members of the mitogen-activated protein kinase activator family in Xenopus laevis. Molecular and cellular biology. 13(9):5738-48. Pubmed: 8395011 Yashar BM, Kelley C, Yee K, Errede B, Zon LI. 1993. Novel members of the mitogen-activated protein kinase activator family in Xenopus laevis. Molecular and cellular biology. 13(9):5738-48. Pubmed: 8395011 Mitogen-activated protein (MAP) kinases comprise an evolutionarily conserved family of proteins that includes at least three vertebrate protein kinases (p42, p44, and p55 MAPK) and five yeast protein kinases (SPK1, MPK1, HOG1, FUS3, and KSS1). Members of this family are activated by a variety of extracellular agents that influence cellular proliferation and differentiation. In Saccharomyces cerevisiae, there are multiple physiologically distinct MAP kinase activation pathways composed of structurally related kinases. The recently cloned vertebrate MAP kinase activators are structurally related to MAP kinase activators in these yeast pathways. These similarities suggest that homologous kinase cascades are utilized for signal transduction in many, if not all, eukaryotes. We have identified additional members of the MAP kinase activator family in Xenopus laevis by a polymerase chain reaction-based analysis of embryonic cDNAs. One of the clones identified (XMEK2) encodes a unique predicted protein kinase that is similar to the previously reported activator (MAPKK) in X. laevis. XMEK2, a highly expressed maternal mRNA, is developmentally regulated during embryogenesis and expressed in brain and muscle. Expression of XMEK2 in yeast cells suppressed the growth defect associated with loss of the yeast MAP kinase activator homologs, MKK1 and MKK2. Partial sequence of a second cDNA clone (XMEK3) identified yet another potential MAP kinase activator. The pattern of expression of XMEK3 is distinct from that of p42 MAPK and XMEK2. The high degree of amino acid sequence similarity of XMEK2, XMEK3, and MAPKK suggests that these three are related members of an amphibian family of protein kinases involved in the activation of MAP kinase. Discovery of this family suggests that multiple MAP kinase activation pathways similar to those in yeast cells exist in vertebrates. -
Kelley C, Blumberg H, Zon LI, Evans T. 1993. GATA-4 is a novel transcription factor expressed in endocardium of the developing heart. Development (Cambridge, England). 118(3):817-27. Pubmed: 8076520 Kelley C, Blumberg H, Zon LI, Evans T. 1993. GATA-4 is a novel transcription factor expressed in endocardium of the developing heart. Development (Cambridge, England). 118(3):817-27. Pubmed: 8076520 We have isolated and characterized Xenopus cDNA clones for a new transcription factor that represents an early marker for the developing heart. The cDNAs encode a protein that we have designated GATA-4; it contains the highly conserved DNA-binding domain that characterizes this family of cell-type restricted transcriptional activators. Whole-embryo in situ analysis of Xenopus embryos demonstrates that the GATA-4 gene is transcribed in presumptive cardiac ventral mesoderm at the time that bilateral progenitors fuse and form the cardiac tube. GATA-4 is therefore the earliest molecular marker of cardiogenesis yet characterized. By stage 30, the GATA-4 mRNA is expressed in the developing atria and ventricles; at stage 38, cross-sections reveal that the gene is active in the endocardial layer, but not in myocardium. By stage 40, GATA-4 message is detected in the great vessels. In the adult frog, the GATA-4 gene is highly transcribed in heart and gut; lower levels of message are detected in various endoderm-derived tissues and gonads. Expression in the stomach is largely confined to the epithelium. The GATA-4 gene is first activated at stage 11; mRNA is initially present throughout the marginal zone of explants and later partially localized to the ventral marginal zone. GATA-4 mRNA is also detected at high levels in cultured endodermal explants derived from the vegetal region of early embryos. In mesoderm induction experiments, GATA-4 transcription is not induced in animal caps treated with activin or bFGF. The GATA-4 gene may provide a new early marker for studying the inductive processes that lead to the formation of the cardiovascular system and for the specification of the endocardial lineage. -
Zon LI, Yamaguchi Y, Yee K, Albee EA, Kimura A, Bennett JC, Orkin SH, Ackerman SJ. 1993. Expression of mRNA for the GATA-binding proteins in human eosinophils and basophils: potential role in gene transcription. Blood. 81(12):3234-41. Pubmed: 8507862 Zon LI, Yamaguchi Y, Yee K, Albee EA, Kimura A, Bennett JC, Orkin SH, Ackerman SJ. 1993. Expression of mRNA for the GATA-binding proteins in human eosinophils and basophils: potential role in gene transcription. Blood. 81(12):3234-41. Pubmed: 8507862 The expression of the hematopoietic transcription factors GATA-1, GATA-2, and GATA-3 was studied in eosinophils and basophils. Eosinophils express mRNA for GATA-1, GATA-2, and GATA-3. Basophils express GATA-2 and GATA-3. Treatment of HL-60 eosinophilic sublines with either interleukin-5 or butyric acid increased the expression of GATA-1 mRNA concomitant with the expression of eosinophil-specific genes, whereas levels of GATA-2 mRNA remained relatively constant. The presence of mRNA for these proteins in eosinophils and basophils suggests that gene transcription in these lineages may be regulated by GATA-binding proteins. -
Yee K, Bishop TR, Mather C, Zon LI. 1993. Isolation of a novel receptor tyrosine kinase cDNA expressed by developing erythroid progenitors. Blood. 82(4):1335-43. Pubmed: 8394755 Yee K, Bishop TR, Mather C, Zon LI. 1993. Isolation of a novel receptor tyrosine kinase cDNA expressed by developing erythroid progenitors. Blood. 82(4):1335-43. Pubmed: 8394755 Activation of distinct receptor tyrosine kinases (RTK), such as the products of the c-fms and c-kit proto-oncogenes, profoundly affects hematopoietic development. We have isolated a novel RTK cDNA, called met-related kinase (MRK), which is expressed on early erythroid progenitors. MRK is also expressed in many hematopoietic cell lines, and is not lineage restricted. Several regions within the catalytic domain of MRK have amino acid similarity to the c-met proto-oncogene and v-sea oncogene. Specific polyclonal anti-MRK antisera detects an 84-Kd polypeptide in COS cells transfected with an expression vector containing the MRK cDNA. Further studies of this novel RTK will provide potential insight into its role during hematopoiesis. 1992
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Sposi NM, Zon LI, Carè A, Valtieri M, Testa U, Gabbianelli M, Mariani G, Bottero L, Mather C, Orkin SH. 1992. Cell cycle-dependent initiation and lineage-dependent abrogation of GATA-1 expression in pure differentiating hematopoietic progenitors. Proceedings of the National Academy of Sciences of the United States of America. 89(14):6353-7. Pubmed: 1631130 Sposi NM, Zon LI, Carè A, Valtieri M, Testa U, Gabbianelli M, Mariani G, Bottero L, Mather C, Orkin SH. 1992. Cell cycle-dependent initiation and lineage-dependent abrogation of GATA-1 expression in pure differentiating hematopoietic progenitors. Proceedings of the National Academy of Sciences of the United States of America. 89(14):6353-7. Pubmed: 1631130 The programmed activation/repression of transcription factors in early hematopoietic differentiation has not yet been explored. The DNA-binding protein GATA-1 is required for normal erythroid development and regulates erythroid-expressed genes in maturing erythroblasts. We analyzed GATA-1 expression in early human adult hematopoiesis by using an in vitro system in which "pure" early hematopoietic progenitors are induced to gradual and synchronized differentiation selectively along the erythroid or granulocyte-macrophage pathway by differential treatment with hematopoietic growth factors. The GATA-1 gene, though virtually silent in quiescent progenitors, is activated after entrance into the cell cycle upon stimulation with hematopoietic growth factors. Subsequently, increasing expression along the erythroid pathway contrasts with an abrupt downregulation in the granulocyte-macrophage lineage. These results suggest a microenvironment-directed, two-step model for GATA-1 expression in differentiating hematopoietic progenitors that involves (i) cycle-dependent initiation and (ii) lineage-dependent maintenance or suppression. Hypothetically, on/off switches of lineage-restricted transactivators may underlie the binary fate decisions of hematopoietic progenitors. -
Barut BA, Zon LI, Cochran MK, Paul SR, Chauhan D, Mohrbacher A, Fingeroth J, Anderson KC. 1992. Role of interleukin 6 in the growth of myeloma-derived cell lines. Leukemia research. 16(10):951-9. Pubmed: 1405708 Barut BA, Zon LI, Cochran MK, Paul SR, Chauhan D, Mohrbacher A, Fingeroth J, Anderson KC. 1992. Role of interleukin 6 in the growth of myeloma-derived cell lines. Leukemia research. 16(10):951-9. Pubmed: 1405708 The role of interleukin 6 (IL-6) in the growth of five multiple myeloma-derived cell lines was characterized. The U266 and RPMI 8226 cell lines demonstrated increased DNA synthesis when cultured with exogenous IL-6, expressed IL-6 cell surface receptors (IL-6Rs) and expressed mRNA for IL-6R. However, these cells did not secrete detectable IL-6 protein, and a neutralizing antibody to IL-6 did not inhibit their growth. Three other myeloma-derived cell lines ARH-77, IM-9 and HS-Sultan did not respond to exogenous IL-6, secrete IL-6 or express cell surface IL-6Rs. The IL-6 responsive cell lines bore late B-cell surface antigens (Ags), CD38 and PCA-1, whereas those lines which were non-IL-6 responsive strongly expressed B1 (CD20) and B4 (CD19) Ags, representing earlier stages in B-cell differentiation. Finally, the two IL-6 responsive cell lines did not express Epstein-Barr virus (EBV) proteins; in contrast, EBV encoded proteins typically expressed during latency could be detected in the three non-IL-6 responsive lines, confirming infection with virus. These studies clarify the heterogeneity observed in the myeloma cell line phenotype and biology and suggest that the U266 and RPMI 8226 cell lines, which express IL-6 cell surface receptors and are IL-6 responsive, may be useful for further study of IL-6 signal transduction in and related IL-6 mediated growth of myeloma in vivo. In contrast, those cell lines which are IL-6-independent provide a model for further study of EBV transformation and IL-6-dependent growth mechanisms in malignancy. -
Zon LI, Orkin SH. 1992. Sequence of the human GATA-1 promoter. Nucleic acids research. 20(7):1812. Pubmed: 1579480 Zon LI, Orkin SH. 1992. Sequence of the human GATA-1 promoter. Nucleic acids research. 20(7):1812. Pubmed: 1579480 -
Hromas R, Zon L, Friedman AD. 1992. Hematopoietic transcription regulators and the origins of leukemia. Critical reviews in oncology/hematology. 12(2):167-90. Pubmed: 1590941 Hromas R, Zon L, Friedman AD. 1992. Hematopoietic transcription regulators and the origins of leukemia. Critical reviews in oncology/hematology. 12(2):167-90. Pubmed: 1590941 -
Zon LI, Moreau JF, Koo JW, Mathey-Prevot B, D'Andrea AD. 1992. The erythropoietin receptor transmembrane region is necessary for activation by the Friend spleen focus-forming virus gp55 glycoprotein. Molecular and cellular biology. 12(7):2949-57. Pubmed: 1320192 Zon LI, Moreau JF, Koo JW, Mathey-Prevot B, D'Andrea AD. 1992. The erythropoietin receptor transmembrane region is necessary for activation by the Friend spleen focus-forming virus gp55 glycoprotein. Molecular and cellular biology. 12(7):2949-57. Pubmed: 1320192 The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated by binding either erythropoietin (EPO) or gp55, the Friend spleen focus-forming virus glycoprotein. The highly specific interaction between gp55 and EPO-R triggers cell proliferation and thereby causes the first stage of Friend virus-induced erythroleukemia. We have generated functional chimeric receptors containing regions of the EPO-R and the interleukin-3 receptor (AIC2A polypeptide), a related cytokine receptor which does not interact with gp55. All chimeric receptors were expressed at similar levels, had similar binding affinities for EPO, and conferred EPO-dependent cell growth. Only those chimeric receptors which contained the EPO-R transmembrane region were activated by gp55. These results demonstrate that the transmembrane region of the EPO-R is critical for activation by gp55. In addition, analysis of a soluble, secreted EPO-R and cysteine point mutants of the EPO-R show that the extracytoplasmic region of the EPO-R specifically interacts with gp55. 1991
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Zon LI, Gurish MF, Stevens RL, Mather C, Reynolds DS, Austen KF, Orkin SH. 1991. GATA-binding transcription factors in mast cells regulate the promoter of the mast cell carboxypeptidase A gene. The Journal of biological chemistry. 266(34):22948-53. Pubmed: 1744088 Zon LI, Gurish MF, Stevens RL, Mather C, Reynolds DS, Austen KF, Orkin SH. 1991. GATA-binding transcription factors in mast cells regulate the promoter of the mast cell carboxypeptidase A gene. The Journal of biological chemistry. 266(34):22948-53. Pubmed: 1744088 The transcription factors GATA-1, GATA-2, and GATA-3 were found to be expressed in several mouse and rat mast cell lines that contain mast cell carboxypeptidase A (MC-CPA) and other proteases in their cytoplasmic granules. GATA-1 mRNA was not detected in P815 cells, an immature mouse mastocytoma-derived cell line that lacks electron-dense granules and has low levels of secretory granule proteases. Because the 5'-flanking regions of the mouse and human MC-CPA genes contained a conserved GATA-binding motif 51 base pairs upstream of their translation initiation sites, the ability of GATA-binding proteins to regulate the promoter activity of the MC-CPA gene was examined in rat basophilic leukemia cells, mouse P815 cells, and transfected mouse P815 cells that expressed GATA-1. In all three mast cell lines, the promoter activity of the MC-CPA gene depended on the GATA binding site. GATA-1, GATA-2, and GATA-3 are thus the first DNA-binding proteins identified in mast cells which regulate the promoter activity of a gene that encodes a secretory granule protease. -
Zon LI, Mather C, Burgess S, Bolce ME, Harland RM, Orkin SH. 1991. Expression of GATA-binding proteins during embryonic development in Xenopus laevis. Proceedings of the National Academy of Sciences of the United States of America. 88(23):10642-6. Pubmed: 1961730 Zon LI, Mather C, Burgess S, Bolce ME, Harland RM, Orkin SH. 1991. Expression of GATA-binding proteins during embryonic development in Xenopus laevis. Proceedings of the National Academy of Sciences of the United States of America. 88(23):10642-6. Pubmed: 1961730 Proteins that recognize the core sequence GATA are important regulators of hematopoietic-specific gene transcription. We have characterized cDNAs encoding the Xenopus laevis homologues of three related transcription factors, designated GATA-1, -2, and -3. Comparative sequence analysis reveals strong conservation of the zinc-finger DNA-binding domain among all vertebrate GATA-binding proteins. GATA-2 and GATA-3 polypeptides are homologous throughout their entire sequences, whereas GATA-1 sequence is conserved only in the region responsible for DNA binding. In Xenopus, RNAs encoding GATA-binding proteins are expressed in both larval and adult erythroid cells. GATA-1, -2, and -3 RNAs are first detectable in early gastrula (Nieuwkoop developmental stage 11). This is earlier than the appearance of the early larval alpha T1 globin RNA (stage 15), beta T1 globin RNA (stage 26), or blood island formation (stage 30). The expression of GATA-1, -2, and -3 in early development may signal an early commitment of mesoderm to form hematopoietic tissue. -
Zon LI, Youssoufian H, Mather C, Lodish HF, Orkin SH. 1991. Activation of the erythropoietin receptor promoter by transcription factor GATA-1. Proceedings of the National Academy of Sciences of the United States of America. 88(23):10638-41. Pubmed: 1660143 Zon LI, Youssoufian H, Mather C, Lodish HF, Orkin SH. 1991. Activation of the erythropoietin receptor promoter by transcription factor GATA-1. Proceedings of the National Academy of Sciences of the United States of America. 88(23):10638-41. Pubmed: 1660143 Erythropoietin, a glycoprotein produced by the kidneys in response to anemia and hypoxia, is a major growth factor for cells of the erythroid lineage. Erythropoietin interacts with high-affinity cell surface receptors (EpoR) present on developing progenitors and is required for their survival. Previously we characterized the gene for EpoR and demonstrated that its promoter acts in a cell-specific manner. Here we show that the hematopoietic-specific transcription factor GATA-1 is necessary, and indeed is sufficient as the sole cell-restricted regulator, for activation of the EpoR promoter in fibroblast transfection assays. Hence, GATA-1, which participates in transcriptional control of the majority of erythroid-expressed genes, also acts on the promoter of an essential lineage-restricted receptor (EpoR). This central contribution of GATA-1 to EpoR promoter function provides a mechanism whereby a cell-restricted regulator may ensure the viability and subsequent maturation of progenitor cells during hematopoietic differentiation. -
D'Andrea AD, Yoshimura A, Youssoufian H, Zon LI, Koo JW, Lodish HF. 1991. The cytoplasmic region of the erythropoietin receptor contains nonoverlapping positive and negative growth-regulatory domains. Molecular and cellular biology. 11(4):1980-7. Pubmed: 1848667 D'Andrea AD, Yoshimura A, Youssoufian H, Zon LI, Koo JW, Lodish HF. 1991. The cytoplasmic region of the erythropoietin receptor contains nonoverlapping positive and negative growth-regulatory domains. Molecular and cellular biology. 11(4):1980-7. Pubmed: 1848667 The erythropoietin (EPO) receptor (EPO-R), a member of a large cytokine receptor superfamily, has a 236-amino-acid cytoplasmic region which contains no obvious tyrosine kinase or other catalytic domain. In order to delineate the linear functional domains of the cytoplasmic tail, we generated truncated mutant cDNAs which were transfected into a murine interleukin-3-dependent cell line, Ba/F3, and the EPO-dependent growth characteristics of the stable transfectants were assayed. We identified two unique domains of the cytoplasmic tail. A membrane-proximal positive signal transduction domain of less than or equal to 103 amino acids, in a region highly similar to the interleukin-2 receptor beta chain, was sufficient for EPO-mediated signal transduction. A carboxy-terminal negative-control domain, a serine-rich region of approximately 40 amino acids, increased the EPO requirement for the Ba/F3 transfectants without altering EPO-R cell surface expression, affinity for EPO, receptor oligosaccharide processing, or receptor endocytosis. Truncation of this negative-control domain allowed the Ba/F3 transfectants to grow maximally in only 1 pM EPO, 1/10 the concentration required for growth of cells expressing the wild-type EPO-R. All truncated EPO-R mutants which retained the transmembrane region of the EPO-R polypeptide bound to the gp55 envelope protein of Friend spleen focus-forming virus. Only the functional EPO-R mutants were activated by the gp55, however, suggesting that gp55- and EPO-mediated signaling occur via a similar mechanism. 1990
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Martin DI, Zon LI, Mutter G, Orkin SH. 1990. Expression of an erythroid transcription factor in megakaryocytic and mast cell lineages. Nature. 344(6265):444-7. Pubmed: 2320112 Martin DI, Zon LI, Mutter G, Orkin SH. 1990. Expression of an erythroid transcription factor in megakaryocytic and mast cell lineages. Nature. 344(6265):444-7. Pubmed: 2320112 The nuclear factor GF-1 (also known as NF-E1, Eryf-1; refs 1-3 respectively) is important in regulation of the transcription of globin and other genes that are specifically expressed in erythroid cells. We have previously shown that GF-1 of both mouse and human origin is a 413-amino-acid polypeptide with two novel zinc-finger domains whose expression is restricted to erythroid cells. Using in situ hybridization of mouse bone marrow cells and northern blot analysis of purified cell populations and permanent cell lines, we show here that GF-1 is expressed in two other hematopoietic lineages, megakaryocytes and bone marrow-derived mast cells. Our findings are consistent with results from hematopoietic progenitor culture which suggest a relationship between erythroid, megakaryocytic and mast cell lineages, and imply that GF-1 is expressed in committed multipotential cells and their progeny. Hence, the mere presence of this transcription factor is unlikely to be sufficient to programme differentiation of a single haematopoietic lineage. GF-1 may regulate the transcription of not only erythroid genes, but also many genes characteristic of megakaryocytes and mast cells, or genes shared among these lineages. -
Youssoufian H, Zon LI, Orkin SH, D'Andrea AD, Lodish HF. 1990. Structure and transcription of the mouse erythropoietin receptor gene. Molecular and cellular biology. 10(7):3675-82. Pubmed: 2162479 Youssoufian H, Zon LI, Orkin SH, D'Andrea AD, Lodish HF. 1990. Structure and transcription of the mouse erythropoietin receptor gene. Molecular and cellular biology. 10(7):3675-82. Pubmed: 2162479 The complete gene encoding the mouse erythropoietin receptor was isolated by using a cDNA probe derived from a mouse erythroleukemia (MEL) cell library. The gene spans approximately 5 kilobases and is present in a single copy per haploid genome. It contains eight exons, and the nucleotide sequence of the coding region from the genomic DNA is identical to the sequence of one of the MEL cDNA clones except for a single amino acid substitution (Leu----Val) at codon 163. There is a cluster of three major transcriptional start sites approximately 150 nucleotides upstream of the initiator ATG codon which is conserved in erythropoietin-dependent and -independent erythroleukemic cells, in MEL cells at different stages of differentiation, and in normal bone marrow cells. The promoter region contains a potential binding site for Sp1, erythroid-specific transcription factor GF-1, and several CACCC boxes, but not typical TATA or CAAT sequences. A fusion gene containing 452 nucleotides of 5' noncoding sequence linked to a promoterless human growth hormone gene directed the transcription of the latter in MEL cells but not in mouse fibroblasts, T cells, B cells, or macrophagelike cells, suggesting that this promoter functions in an erythroid-specific manner. -
Zon LI, Tsai SF, Burgess S, Matsudaira P, Bruns GA, Orkin SH. 1990. The major human erythroid DNA-binding protein (GF-1): primary sequence and localization of the gene to the X chromosome. Proceedings of the National Academy of Sciences of the United States of America. 87(2):668-72. Pubmed: 2300555 Zon LI, Tsai SF, Burgess S, Matsudaira P, Bruns GA, Orkin SH. 1990. The major human erythroid DNA-binding protein (GF-1): primary sequence and localization of the gene to the X chromosome. Proceedings of the National Academy of Sciences of the United States of America. 87(2):668-72. Pubmed: 2300555 Genes expressed in erythroid cells contain binding sites for a cell-specific nuclear factor, GF-1 (NF-E1, Eryf 1), believed to be an important transcriptional regulator. Previously we characterized murine GF-1 as a 413-amino acid polypeptide containing two cysteine-cysteine regions reminiscent of zinc-finger DNA-binding domains. By cross-hybridization to the finger domain of murine GF-1 we have isolated cDNA encoding the human homolog. Peptide sequencing of purified human GF-1 confirmed the authenticity of the human cDNA. The predicted primary sequence of human GF-1 is highly similar to that of murine GF-1, particularly in the DNA-binding region. Although the DNA-binding domains of human, murine, and chicken proteins are remarkably conserved, the mammalian polypeptides are strikingly divergent from the avian counterpart in other regions, most likely those responsible for transcriptional activation. By hybridization to panels of human-rodent DNAs we have assigned the human GF-1 locus to Xp21-11. The localization of the gene to the X chromosome has important implications for hereditary persistence of fetal hemoglobin syndromes unlinked to the beta-globin cluster and for genetic experiments designed to test the role of the factor in erythroid cell gene expression. -
Mignotte V, Navarro S, Eleouet JF, Zon LI, Romeo PH. 1990. The extinction of erythroid genes after tetradecanoylphorbol acetate treatment of erythroleukemic cells correlates with down-regulation of the tissue-specific factors NF-E1 and NF-E2. The Journal of biological chemistry. 265(36):22090-2. Pubmed: 2266112 Mignotte V, Navarro S, Eleouet JF, Zon LI, Romeo PH. 1990. The extinction of erythroid genes after tetradecanoylphorbol acetate treatment of erythroleukemic cells correlates with down-regulation of the tissue-specific factors NF-E1 and NF-E2. The Journal of biological chemistry. 265(36):22090-2. Pubmed: 2266112 We have studied the elements involved in the tetradecanoylphorbol acetate (TPA)-mediated extinction of erythroid-specific genes. We show that transcription driven by a -714/+78-base pair DNA fragment of the erythroid promoter of the human porphobilinogen deaminase gene is down-regulated upon TPA treatment of erythroleukemic cells. Examination of the DNA binding activity of trans-acting factors involved in the expression of the porphobilinogen deaminase erythroid promoter showed (i) a constitutive expression of the CACC binding proteins and (ii) a decrease in DNA binding activity of two tissue-specific factors, NF-E1 and NF-E2. Kinetics experiments indicated that NF-E2 was down-regulated after 1 h of TPA treatment whereas NF-E1 was down-regulated at the protein and mRNA levels only after 5 h of TPA treatment. These results suggest that different pathways, acting via different transcription factors, are involved in the TPA-mediated extinction of erythroid-specific genes. -
D'Andrea AD, Zon LI. 1990. Erythropoietin receptor. Subunit structure and activation. The Journal of clinical investigation. 86(3):681-7. Pubmed: 2168441 D'Andrea AD, Zon LI. 1990. Erythropoietin receptor. Subunit structure and activation. The Journal of clinical investigation. 86(3):681-7. Pubmed: 2168441 -
D'Andrea AD, Fasman GD, Zon LI, Li JP, Lodish HF. 1990. Structure of the erythropoietin receptor in stable fibroblast transfectants. Progress in clinical and biological research. 352:153-9. Pubmed: 2169626 D'Andrea AD, Fasman GD, Zon LI, Li JP, Lodish HF. 1990. Structure of the erythropoietin receptor in stable fibroblast transfectants. Progress in clinical and biological research. 352:153-9. Pubmed: 2169626 1989
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Zon LI, Johns WD, Stomper PC, Kaplan WD, Connolly JL, Morris JH, Harris JR, Henderson IC, Skarin AT. 1989. Breast carcinoma metastatic to a meningioma. Case report and review of the literature. Archives of internal medicine. 149(4):959-62. Pubmed: 2650648 Zon LI, Johns WD, Stomper PC, Kaplan WD, Connolly JL, Morris JH, Harris JR, Henderson IC, Skarin AT. 1989. Breast carcinoma metastatic to a meningioma. Case report and review of the literature. Archives of internal medicine. 149(4):959-62. Pubmed: 2650648 We describe a patient who developed an intracranial mass that consisted of a meningioma and metastatic breast cancer. A literature review revealed 12 similar cases. In epidemiologic studies, the incidence of meningioma in patients with breast cancer is higher than expected. Both tumors are more common in women, have been reported to flare during pregnancy, and express hormone receptors. In a patient with breast cancer, an intracranial mass with radiographic features suggestive, but atypical, of a meningioma should be evaluated surgically. The lesion may represent a metastasis, a meningioma, or both. -
Dorfman DM, Zon LI, Orkin SH. 1989. Rapid amplification of lambda gt11 bacteriophage library inserts from plaques using the polymerase chain reaction (PCR). BioTechniques. 7(6):568, 570. Pubmed: 2534350 Dorfman DM, Zon LI, Orkin SH. 1989. Rapid amplification of lambda gt11 bacteriophage library inserts from plaques using the polymerase chain reaction (PCR). BioTechniques. 7(6):568, 570. Pubmed: 2534350 -
Tsai SF, Martin DI, Zon LI, D'Andrea AD, Wong GG, Orkin SH. 1989. Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells. Nature. 339(6224):446-51. Pubmed: 2725678 Tsai SF, Martin DI, Zon LI, D'Andrea AD, Wong GG, Orkin SH. 1989. Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells. Nature. 339(6224):446-51. Pubmed: 2725678 Genes expressed in erythroid cells contain binding sites for a cell-specific factor believed to be an important regulator for this haematopoietic lineage. Using high-level transient expression in mammalian cells, we have identified complementary DNA encoding the murine protein. The factor, a new member of the zinc-finger family of DNA-binding proteins, is restricted to erythroid cells at the level of RNA expression and is closely homologous between mouse and man. -
Zon LI, Dorfman DM, Orkin SH. 1989. The polymerase chain reaction colony miniprep. BioTechniques. 7(7):696-8. Pubmed: 2631784 Zon LI, Dorfman DM, Orkin SH. 1989. The polymerase chain reaction colony miniprep. BioTechniques. 7(7):696-8. Pubmed: 2631784 -
Scadden DT, Zon LI, Groopman JE. 1989. Pathophysiology and management of HIV-associated hematologic disorders. Blood. 74(5):1455-63. Pubmed: 2676010 Scadden DT, Zon LI, Groopman JE. 1989. Pathophysiology and management of HIV-associated hematologic disorders. Blood. 74(5):1455-63. Pubmed: 2676010 1988
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Zon LI, Groopman JE. 1988. Hematologic manifestations of the human immune deficiency virus (HIV). Seminars in hematology. 25(3):208-18. Pubmed: 3043675 Zon LI, Groopman JE. 1988. Hematologic manifestations of the human immune deficiency virus (HIV). Seminars in hematology. 25(3):208-18. Pubmed: 3043675 1987
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Zon LI, Arkin C, Groopman JE. 1987. Haematologic manifestations of the human immune deficiency virus (HIV). British journal of haematology. 66(2):251-6. Pubmed: 3606961 Zon LI, Arkin C, Groopman JE. 1987. Haematologic manifestations of the human immune deficiency virus (HIV). British journal of haematology. 66(2):251-6. Pubmed: 3606961 A variety of haematologic abnormalities are associated with infection by HIV, the human retrovirus that is the primary aetiologic agent of the acquired immunodeficiency syndrome (AIDS). We have reviewed the haematologic findings in well-characterized cohorts of patients with AIDS, AIDS-related complex (ARC) and asymptomatic homosexual men at risk for this retrovirus. Anaemia, granulocytopenia and thrombocytopenia were found in increasing prevalence according to the severity of clinical disease associated with retroviral infection. Bone marrow aspirations and biopsies revealed frequent hypercellularity, dysplasia, plasmacytosis and lymphoid infiltrates. These marrow morphologic findings were strongly associated with anaemia and granulocytopenia. Review of transfusion records of patients with HIV antibodies revealed a 21% prevalence of a positive direct antiglobulin test. The pathophysiology of the observed haematologic abnormalities may involve direct retroviral infection of bone marrow progenitors, abnormal regulation of haematopoiesis and/or autoimmune phenomena. -
Clairmont GJ, Zon LI, Groopman JE. 1987. Hemophilus parainfluenzae prostatitis in a homosexual man with chronic lymphadenopathy syndrome and HTLV-III infection. The American journal of medicine. 82(1):175-8. Pubmed: 3799681 Clairmont GJ, Zon LI, Groopman JE. 1987. Hemophilus parainfluenzae prostatitis in a homosexual man with chronic lymphadenopathy syndrome and HTLV-III infection. The American journal of medicine. 82(1):175-8. Pubmed: 3799681 Hemophilus parainfluenzae, a common upper respiratory tract pathogen, has been reported to cause pharyngitis, epiglottitis, otitis media, conjunctivitis, and pneumonia. Rarely H. parainfluenzae infects the urinary tract, and is believed not to have been previously reported as a cause of prostatitis. A case of H. parainfluenzae in a young homosexual man infected with HTLV-III and chronic lymphadenopathy is described. Common clinical syndromes such as prostatitis may be associated with unusual pathogens in persons immunodeficient due to HTLV-III infection. -
Canoso RT, Zon LI, Groopman JE. 1987. Anticardiolipin antibodies associated with HTLV-III infection. British journal of haematology. 65(4):495-8. Pubmed: 3580308 Canoso RT, Zon LI, Groopman JE. 1987. Anticardiolipin antibodies associated with HTLV-III infection. British journal of haematology. 65(4):495-8. Pubmed: 3580308 Anticardiolipin antibody levels were determined in 73 homosexual men. Thirty of these patients had acquired immunodeficiency syndrome (AIDS), 16 patients had the AIDS-related complex (ARC) and 27 were healthy. Antibodies to human T-lymphotropic virus type III (HTLV-III) were detected in all patients with AIDS and ARC and in 11 of the healthy homosexuals. Eight patients with positive fluorescent treponemal antibody absorption test were excluded from the study. High levels of IgG-anticardiolipin antibodies were present in 23 of the 28 patients with AIDS; 12 of the 14 with ARC; five of the 10 HTLV-III positive healthy homosexuals; and none of the 13 HTLV-III negative healthy homosexuals. High levels of IgM-ACA were detected only in four patients with AIDS. The IgG-anticardiolipin levels were higher in the HTLV-III positive patients than in the HTLV negative group (P = 0.013). None of the patients with anticardiolipin antibodies exhibited venous thrombosis. 1986
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Zon LI, Archibald DW, McLane MF, Essex M, Hepner MJ, Groopman JE. 1986. IgA deficiency and salivary transmission of human immunodeficiency virus. Lancet (London, England). 2(8514):1039-40. Pubmed: 2877203 Zon LI, Archibald DW, McLane MF, Essex M, Hepner MJ, Groopman JE. 1986. IgA deficiency and salivary transmission of human immunodeficiency virus. Lancet (London, England). 2(8514):1039-40. Pubmed: 2877203 -
Archibald DW, Zon LI, Groopman JE, Allan JS, McLane MF, Essex ME. 1986. Salivary antibodies as a means of detecting human T cell lymphotropic virus type III/lymphadenopathy-associated virus infection. Journal of clinical microbiology. 24(5):873-5. Pubmed: 3021816 Archibald DW, Zon LI, Groopman JE, Allan JS, McLane MF, Essex ME. 1986. Salivary antibodies as a means of detecting human T cell lymphotropic virus type III/lymphadenopathy-associated virus infection. Journal of clinical microbiology. 24(5):873-5. Pubmed: 3021816 Of 45 individuals seropositive for human T cell lymphotropic virus type III/lymphadenopathy-associated virus, 45 were found to have detectable salivary antibodies to viral antigens by a radioimmunoprecipitation assay. The results also showed that a Western blot assay for salivary antibodies may be possible. The feasibility of a diagnostic test for human T cell lymphotropic virus type III/lymphadenopathy-associated virus not requiring venipuncture is discussed. -
Archibald DW, Zon L, Groopman JE, McLane MF, Essex M. 1986. Antibodies to human T-lymphotropic virus type III (HTLV-III) in saliva of acquired immunodeficiency syndrome (AIDS) patients and in persons at risk for AIDS. Blood. 67(3):831-4. Pubmed: 3484980 Archibald DW, Zon L, Groopman JE, McLane MF, Essex M. 1986. Antibodies to human T-lymphotropic virus type III (HTLV-III) in saliva of acquired immunodeficiency syndrome (AIDS) patients and in persons at risk for AIDS. Blood. 67(3):831-4. Pubmed: 3484980 Whole saliva samples collected from available people at risk in Boston for infection with human T-lymphotropic virus type III (HTLV-III/LAV), from late 1984 through early 1985, were analyzed for the presence of antibodies to viral proteins. Fourteen of 20 (70%) acquired immunodeficiency syndrome (AIDS) patients and 14 of 15 (93%) AIDS-related complex (ARC) patients had salivary antibodies that reacted with the virus-encoded glycoproteins gp160 and gp120 of HTLV-III infected cells. All of the AIDS and ARC patients had serum antibodies to the same antigens. Of 20 sex partners of AIDS/ARC patients, nine (45%) showed anti-HTLV-III antibodies, and four of 18 (22%) healthy homosexual males also were positive for such antibodies. Serum and salivary antibody status were the same in these groups. A minority of those patients positive for salivary antibodies to env gene-encoded gp160 and gp120 also had salivary antibodies to gag gene-encoded proteins of 55,000, 24,000, and/or 17,000 daltons. Immunoglobulin A (IgA) class antibodies comprised the majority of the salivary antibody response. The spectrum of HTLV-III proteins detected by the salivary and serum antibodies was similar. The possibility that secretory IgA from the gut-associated lymphoid system may play a role to restrict salivary transmission of HTLV-III should be considered. 1983
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Caro J, Zon LI, Silver R, Miller O, Erslev AJ. 1983. Erythropoietin in liver tissue extracts and in liver perfusates from hypoxic rats. The American journal of physiology. 244(5):E431-4. Pubmed: 6846531 Caro J, Zon LI, Silver R, Miller O, Erslev AJ. 1983. Erythropoietin in liver tissue extracts and in liver perfusates from hypoxic rats. The American journal of physiology. 244(5):E431-4. Pubmed: 6846531 An attempt to evaluate the role of the liver in extrarenal erythropoietin production was made by measuring the content of erythropoietin in homogenates and perfusates from hypoxic rat livers. Extracts from livers from nephric or anephric animals rendered both anemic and hypoxic showed no detectable erythropoietin despite the fact that both plasma and kidney extracts contained large amounts of erythropoietin. This lack of measurable erythropoietin in the liver is not caused by degradation of erythropoietin during the extraction procedure because exogenously added rat erythropoietin was recovered to the same extent from livers or kidney homogenates. More likely, however, it is caused by the fact that extrarenal erythropoietin production accounts for only one-fifth of total erythropoietin production and that the liver mass is about six times that of both kidneys. Consequently, the erythropoietin content of 1 g of liver should be about one-thirtieth of that of 1 g of kidney, an amount that is below the limit of detection of the assay. On the other hand, the 2-h in situ perfusates of livers from similarly stimulated animals contained significant amounts of secreted erythropoietin. It is concluded that the liver participates actively in extrarenal erythropoietin production in the adult rat. However, the small amount expected to be present in tissue homogenates cannot be detected with our current bioassay. -
Sánchez I, Hughes RT, Mayer BJ, Yee K, Woodgett JR, Avruch J, Kyriakis JM, Zon LI. Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun. Nature. 372(6508):794-8. Pubmed: 7997269 Sánchez I, Hughes RT, Mayer BJ, Yee K, Woodgett JR, Avruch J, Kyriakis JM, Zon LI. Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun. Nature. 372(6508):794-8. Pubmed: 7997269 The stress-activated protein kinases (SAPKs), which are distantly related to the MAP kinases, are the dominant c-Jun amino-terminal protein kinases activated in response to a variety of cellular stresses, including treatment with tumour-necrosis factor-alpha and interleukin-beta (refs 1, 2). SAPK phosphorylation of c-Jun probably activates the c-Jun transactivation function. SAPKs are part of a signal transduction cascade related to, but distinct from, the MAPK pathway. We have now identified a novel protein kinase, called SAPK/ERK kinase-1 (SEK1), which is structurally related to the MAP kinase kinases (MEKs). SEK1 is a potent activator of the SAPKs in vitro and in vivo. An inactive SEK1 mutant blocks SAPK activation by extracellular stimuli without interfering with the MAPK pathway. Although alternative mechanisms of SAPK activation may exist, as an immediate upstream activator of the SAPKs, SEK1 further defines a signalling cascade that couples cellular stress agonists to the c-Jun transcription factor. -
Zon LI. Derivation of adult stem cells during embryogenesis. Harvey lectures. 102:117-32. Pubmed: 20166566 Zon LI. Derivation of adult stem cells during embryogenesis. Harvey lectures. 102:117-32. Pubmed: 20166566 -
Yan M, Dai T, Deak JC, Kyriakis JM, Zon LI, Woodgett JR, Templeton DJ. Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1. Nature. 372(6508):798-800. Pubmed: 7997270 Yan M, Dai T, Deak JC, Kyriakis JM, Zon LI, Woodgett JR, Templeton DJ. Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1. Nature. 372(6508):798-800. Pubmed: 7997270 A kinase distinct from the MEK activator Raf, termed MEK kinase-1 (MEKK), was originally identified by virtue of its homology to kinases involved in yeast mating signal cascades. Like Raf, MEKK is capable of activating MEK in vitro. High-level expression of MEKK in COS-7 cells or using vaccinia virus vectors also activates MEK and MAPK, indicating that MEKK and Raf provide alternative means of activating the MAPK signalling pathway. We have derived NIH3T3 cell sublines that can be induced to express active MEKK. Here we show that induction of MEKK does not result in the activation of MAPK, but instead stimulates the stress-activated protein kinases (SAPKs) which are identical to a Jun amino-terminal kinase. We find that MEKK regulates a new signalling cascade by phosphorylating an SAPK activator, SEK1 which in turn phosphorylates and activates SAPK. -
Donahue RE, Johnson MM, Zon LI, Clark SC, Groopman JE. Suppression of in vitro haematopoiesis following human immunodeficiency virus infection. Nature. 326(6109):200-3. Pubmed: 2434864 Donahue RE, Johnson MM, Zon LI, Clark SC, Groopman JE. Suppression of in vitro haematopoiesis following human immunodeficiency virus infection. Nature. 326(6109):200-3. Pubmed: 2434864 Viral infections are frequently associated with haematological disorders. Abnormalities including leukopenia, anaemia and thrombocytopenia are commonly observed in patients with the acquired immune deficiency syndrome (AIDS) or the AIDS-related complex (ARC). The underlying cause of these haematological abnormalities is poorly understood. We report here that bone marrow progenitors isolated from AIDS or ARC patients are responsive to recombinant human granulocyte-macrophage colony stimulating factor (rGM-CSF) and recombinant erythropoietin. Antibodies present in the serum of patients infected with the human immunodeficiency virus (HIV), however, could suppress the growth of these progenitors, but not the growth of progenitors from HIV seronegative controls. A component of this immune-mediated suppression appears to be antibodies directed towards the envelope glycoprotein (gp120) of HIV.