Camargo Lab Publications
Featured Publications
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2023. A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells. Cell. 186(23):5183-5199.e22. DOI:10.1016/j.cell.2023.09.019 Li L, Bowling S, McGeary SE, Yu Q, Lemke B, Alcedo K, Jia Y, Liu X, Ferreira M, Klein AM, Wang SW, Camargo FD. 2023. A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells. Cell. 186(23):5183-5199.e22. DOI:10.1016/j.cell.2023.09.019 Cellular lineage histories and their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have insufficient barcode diversity and single-cell lineage coverage for profiling tissues composed of millions of cells. Here, we developed DARLIN, an inducible Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) and 30 CRISPR target sites. DARLIN is inducible, generates massive lineage barcodes across tissues, and enables the detection of edited barcodes in ∼70% of profiled single cells. Using DARLIN, we examined fate bias within developing hematopoietic stem cells (HSCs) and revealed unique features of HSC migration. Additionally, we established a protocol for joint transcriptomic and epigenomic single-cell measurements with DARLIN and found that cellular clonal memory is associated with genome-wide DNA methylation rather than gene expression or chromatin accessibility. DARLIN will enable the high-resolution study of lineage relationships and their molecular signatures in diverse tissues and physiological contexts. -
Kim KM, Mura-Meszaros A, Tollot M, Krishnan MS, Gründl M, Neubert L, Groth M, Rodriguez-Fraticelli A, Svendsen AF, Campaner S, Andreas N, Kamradt T, Hoffmann S, Camargo FD, Heidel FH, Bystrykh LV, de Haan G, von Eyss B. 2022. Taz protects hematopoietic stem cells from an aging-dependent decrease in PU.1 activity. Nature Communications. 13(1):5187. DOI:10.1038/s41467-022-32970-1 Kim KM, Mura-Meszaros A, Tollot M, Krishnan MS, Gründl M, Neubert L, Groth M, Rodriguez-Fraticelli A, Svendsen AF, Campaner S, Andreas N, Kamradt T, Hoffmann S, Camargo FD, Heidel FH, Bystrykh LV, de Haan G, von Eyss B. 2022. Taz protects hematopoietic stem cells from an aging-dependent decrease in PU.1 activity. Nature Communications. 13(1):5187. DOI:10.1038/s41467-022-32970-1 Specific functions of the immune system are essential to protect us from infections caused by pathogens such as viruses and bacteria. However, as we age, the immune system shows a functional decline that can be attributed in large part to age-associated defects in hematopoietic stem cells (HSCs)—the cells at the apex of the immune cell hierarchy. Here, we find that the Hippo pathway coactivator TAZ is potently induced in old HSCs and protects these cells from functional decline. We identify Clca3a1 as a TAZ-induced gene that allows us to trace TAZ activity in vivo. Using CLCA3A1 as a marker, we can isolate “young-like” HSCs from old mice. Mechanistically, Taz acts as coactivator of PU.1 and to some extent counteracts the gradual loss of PU.1 expression during HSC aging. Our work thus uncovers an essential role for Taz in a previously undescribed fail-safe mechanism in aging HSCs. -
He S, Lei P, Kang W, Cheung P, Xu T, Mana M, Park CY, Wang H, Imada S, Russell JO, Wang J, Wang R, Zhou Z, Chetal K, Stas E, Mohad V, Bruun-Rasmussen P, Sadreyev RI, Hodin RA, Zhang Y, Breault DT, Camargo FD, Yilmaz ÖH, Fredberg JJ, Saeidi N. 2023. Stiffness Restricts the Stemness of the Intestinal Stem Cells and Skews Their Differentiation Toward Goblet Cells. Gastroenterology. 164(7):1137-1151.e15. DOI:10.1053/j.gastro.2023.02.030 He S, Lei P, Kang W, Cheung P, Xu T, Mana M, Park CY, Wang H, Imada S, Russell JO, Wang J, Wang R, Zhou Z, Chetal K, Stas E, Mohad V, Bruun-Rasmussen P, Sadreyev RI, Hodin RA, Zhang Y, Breault DT, Camargo FD, Yilmaz ÖH, Fredberg JJ, Saeidi N. 2023. Stiffness Restricts the Stemness of the Intestinal Stem Cells and Skews Their Differentiation Toward Goblet Cells. Gastroenterology. 164(7):1137-1151.e15. DOI:10.1053/j.gastro.2023.02.030 Background & aims: Fibrosis and tissue stiffening are hallmarks of inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). Methods: We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs in vivo. Results: We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+-proliferating cells. Conversely, cells expressing the stem cell marker, olfactomedin-4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of olfactomedin-4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs toward goblet cells. Furthermore, analysis of colon samples from murine colitis models and patients with IBD demonstrated cellular and molecular remodeling reminiscent of those observed in vitro. Conclusions: Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD.
All Publications
2023
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He S, Lei P, Kang W, Cheung P, Xu T, Mana M, Park CY, Wang H, Imada S, Russell JO, Wang J, Wang R, Zhou Z, Chetal K, Stas E, Mohad V, Bruun-Rasmussen P, Sadreyev RI, Hodin RA, Zhang Y, Breault DT, Camargo FD, Yilmaz ÖH, Fredberg JJ, Saeidi N. 2023. Stiffness Restricts the Stemness of the Intestinal Stem Cells and Skews Their Differentiation Toward Goblet Cells. Gastroenterology. 164(7):1137-1151.e15. DOI:10.1053/j.gastro.2023.02.030 He S, Lei P, Kang W, Cheung P, Xu T, Mana M, Park CY, Wang H, Imada S, Russell JO, Wang J, Wang R, Zhou Z, Chetal K, Stas E, Mohad V, Bruun-Rasmussen P, Sadreyev RI, Hodin RA, Zhang Y, Breault DT, Camargo FD, Yilmaz ÖH, Fredberg JJ, Saeidi N. 2023. Stiffness Restricts the Stemness of the Intestinal Stem Cells and Skews Their Differentiation Toward Goblet Cells. Gastroenterology. 164(7):1137-1151.e15. DOI:10.1053/j.gastro.2023.02.030 Background & aims: Fibrosis and tissue stiffening are hallmarks of inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). Methods: We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs in vivo. Results: We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+-proliferating cells. Conversely, cells expressing the stem cell marker, olfactomedin-4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of olfactomedin-4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs toward goblet cells. Furthermore, analysis of colon samples from murine colitis models and patients with IBD demonstrated cellular and molecular remodeling reminiscent of those observed in vitro. Conclusions: Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD. -
Li L, Bowling S, McGeary SE, Yu Q, Lemke B, Alcedo K, Jia Y, Liu X, Ferreira M, Klein AM, Wang SW, Camargo FD. 2023. A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells. Cell. 186(23):5183-5199.e22. DOI:10.1016/j.cell.2023.09.019 Li L, Bowling S, McGeary SE, Yu Q, Lemke B, Alcedo K, Jia Y, Liu X, Ferreira M, Klein AM, Wang SW, Camargo FD. 2023. A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells. Cell. 186(23):5183-5199.e22. DOI:10.1016/j.cell.2023.09.019 Cellular lineage histories and their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have insufficient barcode diversity and single-cell lineage coverage for profiling tissues composed of millions of cells. Here, we developed DARLIN, an inducible Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) and 30 CRISPR target sites. DARLIN is inducible, generates massive lineage barcodes across tissues, and enables the detection of edited barcodes in ∼70% of profiled single cells. Using DARLIN, we examined fate bias within developing hematopoietic stem cells (HSCs) and revealed unique features of HSC migration. Additionally, we established a protocol for joint transcriptomic and epigenomic single-cell measurements with DARLIN and found that cellular clonal memory is associated with genome-wide DNA methylation rather than gene expression or chromatin accessibility. DARLIN will enable the high-resolution study of lineage relationships and their molecular signatures in diverse tissues and physiological contexts. 2022
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Kim KM, Mura-Meszaros A, Tollot M, Krishnan MS, Gründl M, Neubert L, Groth M, Rodriguez-Fraticelli A, Svendsen AF, Campaner S, Andreas N, Kamradt T, Hoffmann S, Camargo FD, Heidel FH, Bystrykh LV, de Haan G, von Eyss B. 2022. Taz protects hematopoietic stem cells from an aging-dependent decrease in PU.1 activity. Nature Communications. 13(1):5187. DOI:10.1038/s41467-022-32970-1 Kim KM, Mura-Meszaros A, Tollot M, Krishnan MS, Gründl M, Neubert L, Groth M, Rodriguez-Fraticelli A, Svendsen AF, Campaner S, Andreas N, Kamradt T, Hoffmann S, Camargo FD, Heidel FH, Bystrykh LV, de Haan G, von Eyss B. 2022. Taz protects hematopoietic stem cells from an aging-dependent decrease in PU.1 activity. Nature Communications. 13(1):5187. DOI:10.1038/s41467-022-32970-1 Specific functions of the immune system are essential to protect us from infections caused by pathogens such as viruses and bacteria. However, as we age, the immune system shows a functional decline that can be attributed in large part to age-associated defects in hematopoietic stem cells (HSCs)—the cells at the apex of the immune cell hierarchy. Here, we find that the Hippo pathway coactivator TAZ is potently induced in old HSCs and protects these cells from functional decline. We identify Clca3a1 as a TAZ-induced gene that allows us to trace TAZ activity in vivo. Using CLCA3A1 as a marker, we can isolate “young-like” HSCs from old mice. Mechanistically, Taz acts as coactivator of PU.1 and to some extent counteracts the gradual loss of PU.1 expression during HSC aging. Our work thus uncovers an essential role for Taz in a previously undescribed fail-safe mechanism in aging HSCs. -
Patel S, Christodoulou C, Weinreb C, Yu Q, Lummertz da Rocha E, Pepe-Mooney BP, Bowling S, Li L, Garcia-Osorio F, Daley GQ, Camargo FD. 2022. Embryonic multipotent progenitors specified during the definitive wave of hematopoiesis represent a significant source of adult blood. Nature. In press. Patel S, Christodoulou C, Weinreb C, Yu Q, Lummertz da Rocha E, Pepe-Mooney BP, Bowling S, Li L, Garcia-Osorio F, Daley GQ, Camargo FD. 2022. Embryonic multipotent progenitors specified during the definitive wave of hematopoiesis represent a significant source of adult blood. Nature. In press. -
Russell JO, Camargo FD. 2022. Hippo signalling in the liver: role in development, regeneration and disease. Nat Rev Gastroenterol Hepatol. 19(5):297-312. DOI:10.1038/s41575-021-00571-w Russell JO, Camargo FD. 2022. Hippo signalling in the liver: role in development, regeneration and disease. Nat Rev Gastroenterol Hepatol. 19(5):297-312. DOI:10.1038/s41575-021-00571-w The Hippo signaling pathway has emerged as a major player in many aspects of liver biology, such as liver development, cell fate determination, homeostatic function, and regeneration from injury. The regulation of Hippo signaling is complex, with activation of the pathway coming from diverse upstream inputs including signals from cellular adhesion, mechanotransduction, and crosstalk with other signaling pathways. Pathological activation of the downstream transcriptional co-activators yes-associated protein 1 (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), which are negatively regulated by Hippo signaling, has been implicated in multiple aspects of chronic liver disease, such as the development of liver fibrosis and tumorigenesis. As such, development of pharmacological inhibitors of YAP/TAZ signaling has been an area of great interest. In this review, we summarize the diverse roles of Hippo signaling in liver biology and highlight areas where outstanding questions remain to be investigated. Greater understanding of the mechanisms of Hippo signaling in liver function should help to facilitate the development of novel therapies for the treatment of liver disease. 2021
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Van Egeren D, Escabi J, Nguyen M, Liu S, Reilly CR, Patel S, Kamaz B, Kalyva M, DeAngelo DJ, Galinsky I, Wadleigh M, Winer ES, Luskin MR, Stone RM, Garcia JS, Hobbs GS, Camargo FD, Michor F, Mullally A, Cortes-Ciriano I, Hormoz S. 2021. Reconstructing the Lineage Histories and Differentiation Trajectories of Individual Cancer Cells in Myeloproliferative Neoplasms. Cell Stem Cell. 28(3):514-523.e9. DOI:10.1016/j.stem.2021.02.001 Van Egeren D, Escabi J, Nguyen M, Liu S, Reilly CR, Patel S, Kamaz B, Kalyva M, DeAngelo DJ, Galinsky I, Wadleigh M, Winer ES, Luskin MR, Stone RM, Garcia JS, Hobbs GS, Camargo FD, Michor F, Mullally A, Cortes-Ciriano I, Hormoz S. 2021. Reconstructing the Lineage Histories and Differentiation Trajectories of Individual Cancer Cells in Myeloproliferative Neoplasms. Cell Stem Cell. 28(3):514-523.e9. DOI:10.1016/j.stem.2021.02.001 Some cancers originate from a single mutation event in a single cell. Blood cancers known as myeloproliferative neoplasms (MPNs) are thought to originate when a driver mutation is acquired by a hematopoietic stem cell (HSC). However, when the mutation first occurs in individuals and how it affects the behavior of HSCs in their native context is not known. Here we quantified the effect of the JAK2-V617F mutation on the self-renewal and differentiation dynamics of HSCs in treatment-naive individuals with MPNs and reconstructed lineage histories of individual HSCs using somatic mutation patterns. We found that JAK2-V617F mutations occurred in a single HSC several decades before MPN diagnosis-at age 9 ± 2 years in a 34-year-old individual and at age 19 ± 3 years in a 63-year-old individual-and found that mutant HSCs have a selective advantage in both individuals. These results highlight the potential of harnessing somatic mutations to reconstruct cancer lineages. -
Shalhout SZ, Yang PY, Grzelak EM, Nutsch K, Shao S, Zambaldo C, Iaconelli J, Ibrahim L, Stanton C, Chadwick SR, Wu X, Chatterjee AK, Shen W, Camargo FD*, Schultz PG*, Bollong MJ. 2021. YAP-dependent proliferation by a small molecule targeting annexin A2. Nature Chemical Biology. 17(7):767-775. DOI:10.1038/s41589-021-00755-0 Shalhout SZ, Yang PY, Grzelak EM, Nutsch K, Shao S, Zambaldo C, Iaconelli J, Ibrahim L, Stanton C, Chadwick SR, Wu X, Chatterjee AK, Shen W, Camargo FD*, Schultz PG*, Bollong MJ. 2021. YAP-dependent proliferation by a small molecule targeting annexin A2. Nature Chemical Biology. 17(7):767-775. DOI:10.1038/s41589-021-00755-0 -
Sommerkamp P, Romero-Mulero MC, Narr A, Ladel L, Hustin L, Rodriguez-Fraticelli A, Camargo FD, Perié L, Trumpp A, Cabezas-Wallscheid N. 2021. Mouse multipotent progenitor 5 cells are located at the interphase between hematopoietic stem and progenitor cells. Blood. 137(23):3218-3224. DOI:10.1182/blood.2020007876 Sommerkamp P, Romero-Mulero MC, Narr A, Ladel L, Hustin L, Rodriguez-Fraticelli A, Camargo FD, Perié L, Trumpp A, Cabezas-Wallscheid N. 2021. Mouse multipotent progenitor 5 cells are located at the interphase between hematopoietic stem and progenitor cells. Blood. 137(23):3218-3224. DOI:10.1182/blood.2020007876 Hematopoietic stem cells (HSCs) and distinct multipotent progenitor (MPP) populations (MPP1-4) contained within the Lin-Sca-1+c-Kit+ (LSK) compartment have previously been identified using diverse surface-marker panels. Here, we phenotypically define and functionally characterize MPP5 (LSK CD34+CD135-CD48-CD150-). Upon transplantation, MPP5 supports initial emergency myelopoiesis followed by stable contribution to the lymphoid lineage. MPP5, capable of generating MPP1-4 but not HSCs, represents a dynamic and versatile component of the MPP network. To characterize all hematopoietic stem and progenitor cells, we performed RNA-sequencing (RNA-seq) analysis to identify specific transcriptomic landscapes of HSCs and MPP1-5. This was complemented by single-cell RNA-seq analysis of LSK cells to establish the differentiation trajectories from HSCs to MPP1-5. In agreement with functional reconstitution activity, MPP5 is located immediately downstream of HSCs but upstream of the more committed MPP2-4. This study provides a comprehensive analysis of the LSK compartment, focusing on the functional and molecular characteristics of the newly defined MPP5 subset. -
Gao R, Kalathur RKR, Coto-Llerena M, Ercan C, Buechel D, Shuang S, Piscuoglio S, Dill MT, Camargo FD, Christofori G, Tang F. 2021. YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis. EMBO Mol Med. 13(12):e14351. DOI:10.15252/emmm.202114351 Gao R, Kalathur RKR, Coto-Llerena M, Ercan C, Buechel D, Shuang S, Piscuoglio S, Dill MT, Camargo FD, Christofori G, Tang F. 2021. YAP/TAZ and ATF4 drive resistance to Sorafenib in hepatocellular carcinoma by preventing ferroptosis. EMBO Mol Med. 13(12):e14351. DOI:10.15252/emmm.202114351 Understanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In this study, a combination of shRNA-mediated synthetic lethality screening and transcriptomic analysis revealed the transcription factors YAP/TAZ as key drivers of Sorafenib resistance in hepatocellular carcinoma (HCC) by repressing Sorafenib-induced ferroptosis. Mechanistically, in a TEAD-dependent manner, YAP/TAZ induce the expression of SLC7A11, a key transporter maintaining intracellular glutathione homeostasis, thus enabling HCC cells to overcome Sorafenib-induced ferroptosis. At the same time, YAP/TAZ sustain the protein stability, nuclear localization, and transcriptional activity of ATF4 which in turn cooperates to induce SLC7A11 expression. Our study uncovers a critical role of YAP/TAZ in the repression of ferroptosis and thus in the establishment of Sorafenib resistance in HCC, highlighting YAP/TAZ-based rewiring strategies as potential approaches to overcome HCC therapy resistance. 2020
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Hurley K, Ding J, Villacorta-Martin C, Herriges MJ, Jacob A, Vedaie M, Alysandratos KD, Sun YL, Lin C, Werder RB, Huang J, Wilson AA, Mithal A, Mostoslavsky G, Oglesby I, Caballero IS, Guttentag SH, Ahangari F, Kaminski N, Rodriguez-Fraticelli A, Camargo FD, Bar-Joseph Z, Kotton DN. 2020. Reconstructed Single-Cell Fate Trajectories Define Lineage Plasticity Windows during Differentiation of Human PSC-Derived Distal Lung Progenitors. Cell Stem Cell. 26(4):593-608.e8. DOI:10.1016/j.stem.2019.12.009 Hurley K, Ding J, Villacorta-Martin C, Herriges MJ, Jacob A, Vedaie M, Alysandratos KD, Sun YL, Lin C, Werder RB, Huang J, Wilson AA, Mithal A, Mostoslavsky G, Oglesby I, Caballero IS, Guttentag SH, Ahangari F, Kaminski N, Rodriguez-Fraticelli A, Camargo FD, Bar-Joseph Z, Kotton DN. 2020. Reconstructed Single-Cell Fate Trajectories Define Lineage Plasticity Windows during Differentiation of Human PSC-Derived Distal Lung Progenitors. Cell Stem Cell. 26(4):593-608.e8. DOI:10.1016/j.stem.2019.12.009 Alveolar epithelial type 2 cells (AEC2s) are the facultative progenitors responsible for maintaining lung alveoli throughout life but are difficult to isolate from patients. Here, we engineer AEC2s from human pluripotent stem cells (PSCs) in vitro and use time-series single-cell RNA sequencing with lentiviral barcoding to profile the kinetics of their differentiation in comparison to primary fetal and adult AEC2 benchmarks. We observe bifurcating cell-fate trajectories as primordial lung progenitors differentiate in vitro, with some progeny reaching their AEC2 fate target, while others diverge to alternative non-lung endodermal fates. We develop a Continuous State Hidden Markov model to identify the timing and type of signals, such as overexuberant Wnt responses, that induce some early multipotent NKX2-1+ progenitors to lose lung fate. Finally, we find that this initial developmental plasticity is regulatable and subsides over time, ultimately resulting in PSC-derived AEC2s that exhibit a stable phenotype and nearly limitless self-renewal capacity. -
Christodoulou C, Spencer JA, Yeh SA, Turcotte R, Kokkaliaris KD, Panero R, Ramos A, Guo G, Seyedhassantehrani N, Esipova TV, Vinogradov SA, Rudzinskas S, Zhang Y, Perkins AS, Orkin SH, Calogero RA, Schroeder T, Lin CP*, Camargo FD*. 2020. Live-animal imaging of native haematopoietic stem and progenitor cells. Nature. 578(7794): 278-283. DOI:doi.org/10.1038/s41586-020-1971-z Christodoulou C, Spencer JA, Yeh SA, Turcotte R, Kokkaliaris KD, Panero R, Ramos A, Guo G, Seyedhassantehrani N, Esipova TV, Vinogradov SA, Rudzinskas S, Zhang Y, Perkins AS, Orkin SH, Calogero RA, Schroeder T, Lin CP*, Camargo FD*. 2020. Live-animal imaging of native haematopoietic stem and progenitor cells. Nature. 578(7794): 278-283. DOI:doi.org/10.1038/s41586-020-1971-z The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow3,4,5. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches. -
Weinreb C, Rodriguez-Fraticelli A, Camargo FD*, Klein AM*. 2020. Lineage tracing on transcriptional landscapes links state to fate during differentiation. Science. 367(6479). DOI:10.1126/science.aaw3381 Weinreb C, Rodriguez-Fraticelli A, Camargo FD*, Klein AM*. 2020. Lineage tracing on transcriptional landscapes links state to fate during differentiation. Science. 367(6479). DOI:10.1126/science.aaw3381 Biologists have long attempted to understand how stem and progenitor cells in regenerating and embryonic tissues differentiate into mature cell types. Through the use of recent technical advances to sequence the genes expressed in thousands of individual cells, differentiation mechanisms are being revealed. Weinreb et al. extended these methods to track clones of cells (cell families) across time. Their approach reveals differences in cellular gene expression as cells progress through hematopoiesis, which is the process of blood production. Using machine learning, they tested how well gene expression measurements account for the choices that cells make. This work reveals that a considerable gap still exists in understanding differentiation mechanisms, and future methods are needed to fully understand—and ultimately control—cell differentiation. -
Mooring M, Fowl BH, Lum SZC, Liu Y, Yao K, Softic S, Kirchner R, Bernstein A, Singhi AD, Jay DG, Kahn CR, Camargo FD, Yimlamai D. 2020. Hepatocyte Stress Increases Expression of Yes-Associated Protein and Transcriptional Coactivator With PDZ-Binding Motif in Hepatocytes to Promote Parenchymal Inflammation and Fibrosis. Hepatology. 71(5): 1813-1830. DOI:10.1002/hep.30928 Mooring M, Fowl BH, Lum SZC, Liu Y, Yao K, Softic S, Kirchner R, Bernstein A, Singhi AD, Jay DG, Kahn CR, Camargo FD, Yimlamai D. 2020. Hepatocyte Stress Increases Expression of Yes-Associated Protein and Transcriptional Coactivator With PDZ-Binding Motif in Hepatocytes to Promote Parenchymal Inflammation and Fibrosis. Hepatology. 71(5): 1813-1830. DOI:10.1002/hep.30928 Background and aims: Activated hepatocytes are hypothesized to be a major source of signals that drive cirrhosis, but the biochemical pathways that convert hepatocytes into such a state are unclear. We examined the role of the Hippo pathway transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in hepatocytes to facilitate cell-cell interactions that stimulate liver inflammation and fibrosis. Approach and results: Using a variety of genetic, metabolic, and liver injury models in mice, we manipulated Hippo signaling in hepatocytes and examined its effects in nonparenchymal cells to promote liver inflammation and fibrosis. YAP-expressing hepatocytes rapidly and potently activate the expression of proteins that promote fibrosis (collagen type I alpha 1 chain, tissue inhibitor of metalloproteinase 1, platelet-derived growth factor c, transforming growth factor β2) and inflammation (tumor necrosis factor, interleukin 1β). They stimulate expansion of myofibroblasts and immune cells, followed by aggressive liver fibrosis. In contrast, hepatocyte-specific YAP and YAP/TAZ knockouts exhibit limited myofibroblast expansion, less inflammation, and decreased fibrosis after CCl4 injury despite a similar degree of necrosis as controls. We identified cellular communication network factor 1 (CYR61) as a chemokine that is up-regulated by hepatocytes during liver injury but is expressed at significantly lower levels in mice with hepatocyte-specific deletion of YAP or TAZ. Gain-of-function and loss-of-function experiments with CYR61 in vivo point to it being a key chemokine controlling liver fibrosis and inflammation in the context of YAP/TAZ. There is a direct correlation between levels of YAP/TAZ and CYR61 in liver tissues of patients with high-grade nonalcoholic steatohepatitis. Conclusions: Liver injury in mice and humans increases levels of YAP/TAZ/CYR61 in hepatocytes, thus attracting macrophages to the liver to promote inflammation and fibrosis. -
Li Q, Sun Y, Jarugumilli GK, Liu S, Dang K, Cotton JL, Xiol J, Chan PY, DeRan M, Ma L, Li R, Zhu LJ, Li JH, Leiter AB, Ip YT, Camargo FD, Luo X, Johnson RL, Wu X, Mao J. 2020. Lats1/2 Sustain Intestinal Stem Cells and Wnt Activation through TEAD-Dependent and Independent Transcription. Cell Stem Cell. 26(5): 675-692.e8. DOI:10.1016/j.stem.2020.03.002 Li Q, Sun Y, Jarugumilli GK, Liu S, Dang K, Cotton JL, Xiol J, Chan PY, DeRan M, Ma L, Li R, Zhu LJ, Li JH, Leiter AB, Ip YT, Camargo FD, Luo X, Johnson RL, Wu X, Mao J. 2020. Lats1/2 Sustain Intestinal Stem Cells and Wnt Activation through TEAD-Dependent and Independent Transcription. Cell Stem Cell. 26(5): 675-692.e8. DOI:10.1016/j.stem.2020.03.002 Intestinal homeostasis is tightly regulated by complex yet poorly understood signaling networks. Here, we demonstrate that Lats1/2, the core Hippo kinases, are essential to maintain Wnt pathway activity and intestinal stem cells. Lats1/2 deletion leads to loss of intestinal stem cells but drives Wnt-uncoupled crypt expansion. To explore the function of downstream transcriptional enhanced associate domain (TEAD) transcription factors, we identified a selective small-molecule reversible inhibitor of TEAD auto-palmitoylation that directly occupies its lipid-binding site and inhibits TEAD-mediated transcription in vivo. Combining this chemical tool with genetic and proteomics approaches, we show that intestinal Wnt inhibition by Lats deletion is Yes-associated protein (YAP)/transcriptional activator with PDZ-binding domain (TAZ) dependent but TEAD independent. Mechanistically, nuclear YAP/TAZ interact with Groucho/Transducin-Like Enhancer of Split (TLE) to block Wnt/T-cell factor (TCF)-mediated transcription, and dual inhibition of TEAD and Lats suppresses Wnt-uncoupled Myc upregulation and epithelial over-proliferation in Adenomatous polyposis coli (APC)-mutated intestine. Our studies highlight a pharmacological approach to inhibit TEAD palmitoylation and have important implications for targeting Wnt and Hippo signaling in human malignancies. -
Bowling S, Sritharan D, Osorio FG, Nguyen M, Cheung P, Rodriguez-Fraticelli A, Patel S, Yuan WC, Fujiwara Y, Li BE, Orkin SH, Hormoz S*, Camargo FD*. 2020. An Engineered CRISPR-Cas9 Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression Profiles in Single Cells. Cell. 181(6):1410-1422.e27. DOI:10.1016/j.cell.2020.04.048 Bowling S, Sritharan D, Osorio FG, Nguyen M, Cheung P, Rodriguez-Fraticelli A, Patel S, Yuan WC, Fujiwara Y, Li BE, Orkin SH, Hormoz S*, Camargo FD*. 2020. An Engineered CRISPR-Cas9 Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression Profiles in Single Cells. Cell. 181(6):1410-1422.e27. DOI:10.1016/j.cell.2020.04.048 Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Coupling of cell ancestry information with other molecular readouts represents an important goal in the field. Here, we describe the CRISPR array repair lineage tracing (CARLIN) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures associated with HSC activity without cell sorting. -
Cheung P, Xiol J, Dill MT, Yuan WC, Panero R, Roper J, Osorio FG, Maglic D, Li Q, Gurung B, Calogero RA, Yilmaz ÖH, Mao J, Camargo FD. 2020. Regenerative Reprogramming of the Intestinal Stem Cell State via Hippo Signaling Suppresses Metastatic Colorectal Cancer. Cell Stem Cell. 27(4):590-604.e9. DOI:10.1016/j.stem.2020.07.003 Cheung P, Xiol J, Dill MT, Yuan WC, Panero R, Roper J, Osorio FG, Maglic D, Li Q, Gurung B, Calogero RA, Yilmaz ÖH, Mao J, Camargo FD. 2020. Regenerative Reprogramming of the Intestinal Stem Cell State via Hippo Signaling Suppresses Metastatic Colorectal Cancer. Cell Stem Cell. 27(4):590-604.e9. DOI:10.1016/j.stem.2020.07.003 Although the Hippo transcriptional coactivator YAP is considered oncogenic in many tissues, its roles in intestinal homeostasis and colorectal cancer (CRC) remain controversial. Here, we demonstrate that the Hippo kinases LATS1/2 and MST1/2, which inhibit YAP activity, are required for maintaining Wnt signaling and canonical stem cell function. Hippo inhibition induces a distinct epithelial cell state marked by low Wnt signaling, a wound-healing response, and transcription factor Klf6 expression. Notably, loss of LATS1/2 or overexpression of YAP is sufficient to reprogram Lgr5+ cancer stem cells to this state and thereby suppress tumor growth in organoids, patient-derived xenografts, and mouse models of primary and metastatic CRC. Finally, we demonstrate that genetic deletion of YAP and its paralog TAZ promotes the growth of these tumors. Collectively, our results establish the role of YAP as a tumor suppressor in the adult colon and implicate Hippo kinases as therapeutic vulnerabilities in colorectal malignancies. -
Rodriguez-Fraticelli AE, Weinreb C, Wang SW, Migueles RP, Jankovic M, Usart M, Klein AM, Lowell S, Camargo FD. 2020. Single-cell lineage tracing unveils a role for TCF15 in haematopoiesis. Nature. 583(7817):585-589. DOI:doi.org/10.1038/s41586-020-2503-6 Rodriguez-Fraticelli AE, Weinreb C, Wang SW, Migueles RP, Jankovic M, Usart M, Klein AM, Lowell S, Camargo FD. 2020. Single-cell lineage tracing unveils a role for TCF15 in haematopoiesis. Nature. 583(7817):585-589. DOI:doi.org/10.1038/s41586-020-2503-6 Bone marrow transplantation therapy relies on the life-long regenerative capacity of haematopoietic stem cells (HSCs)1,2. HSCs present a complex variety of regenerative behaviours at the clonal level, but the mechanisms underlying this diversity are still undetermined3,4,5,6,7,8,9,10,11. Recent advances in single-cell RNA sequencing have revealed transcriptional differences among HSCs, providing a possible explanation for their functional heterogeneity12,13,14,15,16,17. However, the destructive nature of sequencing assays prevents simultaneous observation of stem cell state and function. To solve this challenge, we implemented expressible lentiviral barcoding, which enabled simultaneous analysis of lineages and transcriptomes from single adult HSCs and their clonal trajectories during long-term bone marrow reconstitution. Analysis of differential gene expression between clones with distinct behaviour revealed an intrinsic molecular signature that characterizes functional long-term repopulating HSCs. Probing this signature through in vivo CRISPR screening, we found the transcription factor TCF15 to be required and sufficient to drive HSC quiescence and long-term self-renewal. In situ, Tcf15 expression labels the most primitive subset of true multipotent HSCs. In conclusion, our work elucidates clone-intrinsic molecular programmes associated with functional stem cell heterogeneity and identifies a mechanism for the maintenance of the self-renewing HSC state. 2019
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Pepe-Mooney BJ, Dill MT, Alemany A, Ordovas-Montanes J, Matsushita Y, Rao A, Sen A, Miyazaki M, Anakk S, Dawson PA, Ono N, Shalek AK, van Oudenaarden A, Camargo FD. 2019. Single-Cell Analysis of the Liver Epithelium Reveals Dynamic Heterogeneity and an Essential Role for YAP in Homeostasis and Regeneration. Cell stem cell. Pubmed: 31080134 Pepe-Mooney BJ, Dill MT, Alemany A, Ordovas-Montanes J, Matsushita Y, Rao A, Sen A, Miyazaki M, Anakk S, Dawson PA, Ono N, Shalek AK, van Oudenaarden A, Camargo FD. 2019. Single-Cell Analysis of the Liver Epithelium Reveals Dynamic Heterogeneity and an Essential Role for YAP in Homeostasis and Regeneration. Cell stem cell. Pubmed: 31080134 The liver can substantially regenerate after injury, with both main epithelial cell types, hepatocytes and biliary epithelial cells (BECs), playing important roles in parenchymal regeneration. Beyond metabolic functions, BECs exhibit substantial plasticity and in some contexts can drive hepatic repopulation. Here, we performed single-cell RNA sequencing to examine BEC and hepatocyte heterogeneity during homeostasis and after injury. Instead of evidence for a transcriptionally defined progenitor-like BEC cell, we found significant homeostatic BEC heterogeneity that reflects fluctuating activation of a YAP-dependent program. This transcriptional signature defines a dynamic cellular state during homeostasis and is highly responsive to injury. YAP signaling is induced by physiological bile acids (BAs), required for BEC survival in response to BA exposure, and is necessary for hepatocyte reprogramming into biliary progenitors upon injury. Together, these findings uncover molecular heterogeneity within the ductal epithelium and reveal YAP as a protective rheostat and regenerative regulator in the mammalian liver.Copyright © 2019 Elsevier Inc. All rights reserved. 2018
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Wang Y, Xu X, Maglic D, Dill MT, Mojumdar K, Ng PK, Jeong KJ, Tsang YH, Moreno D, Bhavana VH, Peng X, Ge Z, Chen H, Li J, Chen Z, Zhang H, Han L, Du D, Creighton CJ, Mills GB, Camargo F, Liang H. 2018. Comprehensive Molecular Characterization of the Hippo Signaling Pathway in Cancer. Cell reports. 25(5):1304-1317.e5. Pubmed: 30380420 Wang Y, Xu X, Maglic D, Dill MT, Mojumdar K, Ng PK, Jeong KJ, Tsang YH, Moreno D, Bhavana VH, Peng X, Ge Z, Chen H, Li J, Chen Z, Zhang H, Han L, Du D, Creighton CJ, Mills GB, Camargo F, Liang H. 2018. Comprehensive Molecular Characterization of the Hippo Signaling Pathway in Cancer. Cell reports. 25(5):1304-1317.e5. Pubmed: 30380420 Hippo signaling has been recognized as a key tumor suppressor pathway. Here, we perform a comprehensive molecular characterization of 19 Hippo core genes in 9,125 tumor samples across 33 cancer types using multidimensional "omic" data from The Cancer Genome Atlas. We identify somatic drivers among Hippo genes and the related microRNA (miRNA) regulators, and using functional genomic approaches, we experimentally characterize YAP and TAZ mutation effects and miR-590 and miR-200a regulation for TAZ. Hippo pathway activity is best characterized by a YAP/TAZ transcriptional target signature of 22 genes, which shows robust prognostic power across cancer types. Our elastic-net integrated modeling further reveals cancer-type-specific pathway regulators and associated cancer drivers. Our results highlight the importance of Hippo signaling in squamous cell cancers, characterized by frequent amplification of YAP/TAZ, high expression heterogeneity, and significant prognostic patterns. This study represents a systems-biology approach to characterizing key cancer signaling pathways in the post-genomic era.Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved. -
Cox AG, Tsomides A, Yimlamai D, Hwang KL, Miesfeld J, Galli GG, Fowl BH, Fort M, Ma KY, Sullivan MR, Hosios AM, Snay E, Yuan M, Brown KK, Lien EC, Chhangawala S, Steinhauser ML, Asara JM, Houvras Y, Link B, Vander Heiden MG, Camargo FD, Goessling W. 2018. Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth. The EMBO journal. 37(22). Pubmed: 30348863 DOI:10.15252/embj.2018100294 Cox AG, Tsomides A, Yimlamai D, Hwang KL, Miesfeld J, Galli GG, Fowl BH, Fort M, Ma KY, Sullivan MR, Hosios AM, Snay E, Yuan M, Brown KK, Lien EC, Chhangawala S, Steinhauser ML, Asara JM, Houvras Y, Link B, Vander Heiden MG, Camargo FD, Goessling W. 2018. Yap regulates glucose utilization and sustains nucleotide synthesis to enable organ growth. The EMBO journal. 37(22). Pubmed: 30348863 DOI:10.15252/embj.2018100294 The Hippo pathway and its nuclear effector Yap regulate organ size and cancer formation. While many modulators of Hippo activity have been identified, little is known about the Yap target genes that mediate these growth effects. Here, we show that mutant zebrafish exhibit defects in hepatic progenitor potential and liver growth due to impaired glucose transport and nucleotide biosynthesis. Transcriptomic and metabolomic analyses reveal that Yap regulates expression of glucose transporter glut1, causing decreased glucose uptake and use for nucleotide biosynthesis in mutants, and impaired glucose tolerance in adults. Nucleotide supplementation improves Yap deficiency phenotypes, indicating functional importance of glucose-fueled nucleotide biosynthesis. Yap-regulated expression and glucose uptake are conserved in mammals, suggesting that stimulation of anabolic glucose metabolism is an evolutionarily conserved mechanism by which the Hippo pathway controls organ growth. Together, our results reveal a central role for Hippo signaling in glucose metabolic homeostasis.© 2018 The Authors. -
Maglic D, Schlegelmilch K, Dost AF, Panero R, Dill MT, Calogero RA, Camargo FD. 2018. YAP-TEAD signaling promotes basal cell carcinoma development via a c-JUN/AP1 axis. The EMBO journal. 37(17). Pubmed: 30037824 Maglic D, Schlegelmilch K, Dost AF, Panero R, Dill MT, Calogero RA, Camargo FD. 2018. YAP-TEAD signaling promotes basal cell carcinoma development via a c-JUN/AP1 axis. The EMBO journal. 37(17). Pubmed: 30037824 The mammalian Hippo signaling pathway, through its effectors YAP and TAZ, coerces epithelial progenitor cell expansion for appropriate tissue development or regeneration upon damage. Its ability to drive rapid tissue growth explains why many oncogenic events frequently exploit this pathway to promote cancer phenotypes. Indeed, several tumor types including basal cell carcinoma (BCC) show genetic aberrations in the Hippo (or YAP/TAZ) regulators. Here, we uncover that while YAP is dispensable for homeostatic epidermal regeneration, it is required for BCC development. Our clonal analyses further demonstrate that the few emerging Yap-null dysplasia have lower fitness and thus are diminished as they progress to invasive BCC Mechanistically, YAP depletion in BCC tumors leads to effective impairment of the JNK-JUN signaling, a well-established tumor-driving cascade. Importantly, in this context, YAP does not influence canonical Wnt or Hedgehog signaling. Overall, we reveal Hippo signaling as an independent promoter of BCC pathogenesis and thereby a viable target for drug-resistant BCC.© 2018 The Authors. -
Rodriguez-Fraticelli AE, Wolock SL, Weinreb CS, Panero R, Patel SH, Jankovic M, Sun J, Calogero RA, Klein AM, Camargo FD. 2018. Clonal analysis of lineage fate in native haematopoiesis. Nature. 553(7687):212-216. Pubmed: 29323290 DOI:10.1038/nature25168 Rodriguez-Fraticelli AE, Wolock SL, Weinreb CS, Panero R, Patel SH, Jankovic M, Sun J, Calogero RA, Klein AM, Camargo FD. 2018. Clonal analysis of lineage fate in native haematopoiesis. Nature. 553(7687):212-216. Pubmed: 29323290 DOI:10.1038/nature25168 Haematopoiesis, the process of mature blood and immune cell production, is functionally organized as a hierarchy, with self-renewing haematopoietic stem cells and multipotent progenitor cells sitting at the very top. Multiple models have been proposed as to what the earliest lineage choices are in these primitive haematopoietic compartments, the cellular intermediates, and the resulting lineage trees that emerge from them. Given that the bulk of studies addressing lineage outcomes have been performed in the context of haematopoietic transplantation, current models of lineage branching are more likely to represent roadmaps of lineage potential than native fate. Here we use transposon tagging to clonally trace the fates of progenitors and stem cells in unperturbed haematopoiesis. Our results describe a distinct clonal roadmap in which the megakaryocyte lineage arises largely independently of other haematopoietic fates. Our data, combined with single-cell RNA sequencing, identify a functional hierarchy of unilineage- and oligolineage-producing clones within the multipotent progenitor population. Finally, our results demonstrate that traditionally defined long-term haematopoietic stem cells are a significant source of megakaryocyte-restricted progenitors, suggesting that the megakaryocyte lineage is the predominant native fate of long-term haematopoietic stem cells. Our study provides evidence for a substantially revised roadmap for unperturbed haematopoiesis, and highlights unique properties of multipotent progenitors and haematopoietic stem cells in situ. -
Osorio FG, Rosendahl Huber A, Oka R, Verheul M, Patel SH, Hasaart K, de la Fonteijne L, Varela I, Camargo FD, van Boxtel R. 2018. Somatic Mutations Reveal Lineage Relationships and Age-Related Mutagenesis in Human Hematopoiesis. Cell reports. 25(9):2308-2316.e4. Pubmed: 30485801 Osorio FG, Rosendahl Huber A, Oka R, Verheul M, Patel SH, Hasaart K, de la Fonteijne L, Varela I, Camargo FD, van Boxtel R. 2018. Somatic Mutations Reveal Lineage Relationships and Age-Related Mutagenesis in Human Hematopoiesis. Cell reports. 25(9):2308-2316.e4. Pubmed: 30485801 Mutation accumulation during life can contribute to hematopoietic dysfunction; however, the underlying dynamics are unknown. Somatic mutations in blood progenitors can provide insight into the rate and processes underlying this accumulation, as well as the developmental lineage tree and stem cell division numbers. Here, we catalog mutations in the genomes of human-bone-marrow-derived and umbilical-cord-blood-derived hematopoietic stem and progenitor cells (HSPCs). We find that mutations accumulate gradually during life with approximately 14 base substitutions per year. The majority of mutations were acquired after birth and could be explained by the constant activity of various endogenous mutagenic processes, which also explains the mutation load in acute myeloid leukemia (AML). Using these mutations, we construct a developmental lineage tree of human hematopoiesis, revealing a polyclonal architecture and providing evidence that developmental clones exhibit multipotency. Our approach highlights features of human native hematopoiesis and its implications for leukemogenesis.Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved. -
Yuan WC, Pepe-Mooney B, Galli GG, Dill MT, Huang HT, Hao M, Wang Y, Liang H, Calogero RA, Camargo FD. 2018. NUAK2 is a critical YAP target in liver cancer. Nature communications. 9(1):4834. Pubmed: 30446657 DOI:10.1038/s41467-018-07394-5 Yuan WC, Pepe-Mooney B, Galli GG, Dill MT, Huang HT, Hao M, Wang Y, Liang H, Calogero RA, Camargo FD. 2018. NUAK2 is a critical YAP target in liver cancer. Nature communications. 9(1):4834. Pubmed: 30446657 DOI:10.1038/s41467-018-07394-5 The Hippo-YAP signaling pathway is a critical regulator of proliferation, apoptosis, and cell fate. The main downstream effector of this pathway, YAP, has been shown to be misregulated in human cancer and has emerged as an attractive target for therapeutics. A significant insufficiency in our understanding of the pathway is the identity of transcriptional targets of YAP that drive its potent growth phenotypes. Here, using liver cancer as a model, we identify NUAK2 as an essential mediator of YAP-driven hepatomegaly and tumorigenesis in vivo. By evaluating several human cancer cell lines we determine that NUAK2 is selectively required for YAP-driven growth. Mechanistically, we found that NUAK2 participates in a feedback loop to maximize YAP activity via promotion of actin polymerization and myosin activity. Additionally, pharmacological inactivation of NUAK2 suppresses YAP-dependent cancer cell proliferation and liver overgrowth. Importantly, our work here identifies a specific, potent, and actionable target for YAP-driven malignancies. 2017
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Katsuda T, Kawamata M, Hagiwara K, Takahashi RU, Yamamoto Y, Camargo FD, Ochiya T. 2017. Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity. Cell stem cell. 20(1):41-55. Pubmed: 27840021 Katsuda T, Kawamata M, Hagiwara K, Takahashi RU, Yamamoto Y, Camargo FD, Ochiya T. 2017. Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity. Cell stem cell. 20(1):41-55. Pubmed: 27840021 A challenge for advancing approaches to liver regeneration is loss of functional differentiation capacity when hepatocyte progenitors are maintained in culture. Recent lineage-tracing studies have shown that mature hepatocytes (MHs) convert to an immature state during chronic liver injury, and we investigated whether this conversion could be recapitulated in vitro and whether such converted cells could represent a source of expandable hepatocytes. We report that a cocktail of small molecules, Y-27632, A-83-01, and CHIR99021, can convert rat and mouse MHs in vitro into proliferative bipotent cells, which we term chemically induced liver progenitors (CLiPs). CLiPs can differentiate into both MHs and biliary epithelial cells that can form functional ductal structures. CLiPs in long-term culture did not lose their proliferative capacity or their hepatic differentiation ability, and rat CLiPs were shown to extensively repopulate chronically injured liver tissue. Thus, our study advances the goals of liver regenerative medicine.Copyright © 2017 Elsevier Inc. All rights reserved. -
Hu Y, Shin DJ, Pan H, Lin Z, Dreyfuss JM, Camargo FD, Miao J, Biddinger SB. 2017. YAP suppresses gluconeogenic gene expression through PGC1α. Hepatology (Baltimore, Md.). 66(6):2029-2041. Pubmed: 28714135 DOI:10.1002/hep.29373 Hu Y, Shin DJ, Pan H, Lin Z, Dreyfuss JM, Camargo FD, Miao J, Biddinger SB. 2017. YAP suppresses gluconeogenic gene expression through PGC1α. Hepatology (Baltimore, Md.). 66(6):2029-2041. Pubmed: 28714135 DOI:10.1002/hep.29373 Array© 2017 by the American Association for the Study of Liver Diseases. -
Myant KB, Cammareri P, Hodder MC, Wills J, Von Kriegsheim A, Győrffy B, Rashid M, Polo S, Maspero E, Vaughan L, Gurung B, Barry E, Malliri A, Camargo F, Adams DJ, Iavarone A, Lasorella A, Sansom OJ. 2017. HUWE1 is a critical colonic tumour suppressor gene that prevents MYC signalling, DNA damage accumulation and tumour initiation. EMBO molecular medicine. 9(2):181-197. Pubmed: 28003334 DOI:10.15252/emmm.201606684 Myant KB, Cammareri P, Hodder MC, Wills J, Von Kriegsheim A, Győrffy B, Rashid M, Polo S, Maspero E, Vaughan L, Gurung B, Barry E, Malliri A, Camargo F, Adams DJ, Iavarone A, Lasorella A, Sansom OJ. 2017. HUWE1 is a critical colonic tumour suppressor gene that prevents MYC signalling, DNA damage accumulation and tumour initiation. EMBO molecular medicine. 9(2):181-197. Pubmed: 28003334 DOI:10.15252/emmm.201606684 Cancer genome sequencing projects have identified hundreds of genetic alterations, often at low frequencies, raising questions as to their functional relevance. One exemplar gene is HUWE1, which has been found to be mutated in numerous studies. However, due to the large size of this gene and a lack of functional analysis of identified mutations, their significance to carcinogenesis is unclear. To determine the importance of HUWE1, we chose to examine its function in colorectal cancer, where it is mutated in up to 15 per cent of tumours. Modelling of identified mutations showed that they inactivate the E3 ubiquitin ligase activity of HUWE1. Genetic deletion of Huwe1 rapidly accelerated tumourigenic in mice carrying loss of the intestinal tumour suppressor gene Apc, with a dramatic increase in tumour initiation. Mechanistically, this phenotype was driven by increased MYC and rapid DNA damage accumulation leading to loss of the second copy of Apc The increased levels of DNA damage sensitised Huwe1-deficient tumours to DNA-damaging agents and to deletion of the anti-apoptotic protein MCL1. Taken together, these data identify HUWE1 as a bona fide tumour suppressor gene in the intestinal epithelium and suggest a potential vulnerability of HUWE1-mutated tumours to DNA-damaging agents and inhibitors of anti-apoptotic proteins.© 2016 Cancer Research UK Beatson Institute. Published under the terms of the CC BY 4.0 license. -
Patel SH, Camargo FD, Yimlamai D. 2017. Hippo Signaling in the Liver Regulates Organ Size, Cell Fate, and Carcinogenesis. Gastroenterology. 152(3):533-545. Pubmed: 28003097 Patel SH, Camargo FD, Yimlamai D. 2017. Hippo Signaling in the Liver Regulates Organ Size, Cell Fate, and Carcinogenesis. Gastroenterology. 152(3):533-545. Pubmed: 28003097 The Hippo signaling pathway, also known as the Salvador-Warts-Hippo pathway, is a regulator of organ size. The pathway takes its name from the Drosophila protein kinase, Hippo (STK4/MST1 and STK3/MST2 in mammals), which, when inactivated, leads to considerable tissue overgrowth. In mammals, MST1 and MST2 negatively regulate the transcriptional co-activators yes-associated protein 1 and WW domain containing transcription regulator 1 (WWTR1/TAZ), which together regulate expression of genes that control proliferation, survival, and differentiation. Yes-associated protein 1 and TAZ activation have been associated with liver development, regeneration, and tumorigenesis. How their activity is dynamically regulated in these contexts is just beginning to be elucidated. We review the mechanisms of Hippo signaling in the liver and explore outstanding questions for future research.Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved. 2016
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Cox AG, Hwang KL, Brown KK, Evason K, Beltz S, Tsomides A, O'Connor K, Galli GG, Yimlamai D, Chhangawala S, Yuan M, Lien EC, Wucherpfennig J, Nissim S, Minami A, Cohen DE, Camargo FD, Asara JM, Houvras Y, Stainier DYR, Goessling W. 2016. Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth. Nature cell biology. 18(8):886-896. Pubmed: 27428308 DOI:10.1038/ncb3389 Cox AG, Hwang KL, Brown KK, Evason K, Beltz S, Tsomides A, O'Connor K, Galli GG, Yimlamai D, Chhangawala S, Yuan M, Lien EC, Wucherpfennig J, Nissim S, Minami A, Cohen DE, Camargo FD, Asara JM, Houvras Y, Stainier DYR, Goessling W. 2016. Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth. Nature cell biology. 18(8):886-896. Pubmed: 27428308 DOI:10.1038/ncb3389 The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signalling provides the cellular building blocks required for rapid growth. Here, we demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumour formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase (glul) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppressing hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis. 2015
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Yimlamai D, Fowl BH, Camargo FD. 2015. Emerging evidence on the role of the Hippo/YAP pathway in liver physiology and cancer. Journal of hepatology. 63(6):1491-501. Pubmed: 26226451 Yimlamai D, Fowl BH, Camargo FD. 2015. Emerging evidence on the role of the Hippo/YAP pathway in liver physiology and cancer. Journal of hepatology. 63(6):1491-501. Pubmed: 26226451 The Hippo pathway and its regulatory target, YAP, has recently emerged as an important biochemical signaling pathway that tightly governs epithelial tissue growth. Initially defined in Drosophilia, this pathway has shown remarkable conservation in vertebrate systems with many components of the Hippo/YAP pathway showing biochemical and functional conservation. The liver is particularly sensitive to changes in Hippo/YAP signaling with rapid increases in liver size becoming manifest on the order of days to weeks after perturbation. The first identified direct targets of Hippo/YAP signaling were pro-proliferative and anti-apoptotic gene programs, but recent work has now implicated this pathway in cell fate choice, stem cell maintenance/renewal, epithelial to mesenchymal transition, and oncogenesis. The mechanisms by which Hippo/YAP signaling is changed endogenously are beginning to come to light as well as how this pathway interacts with other signaling pathways, and important details for designing new therapeutic interventions. This review focuses on the known roles for Hippo/YAP signaling in the liver and promising avenues for future study.Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. -
Gentner B, Pochert N, Rouhi A, Boccalatte F, Plati T, Berg T, Sun SM, Mah SM, Mirkovic-Hösle M, Ruschmann J, Muranyi A, Leierseder S, Argiropoulos B, Starczynowski DT, Karsan A, Heuser M, Hogge D, Camargo FD, Engelhardt S, Döhner H, Buske C, Jongen-Lavrencic M, Naldini L, Humphries RK, Kuchenbauer F. 2015. MicroRNA-223 dose levels fine tune proliferation and differentiation in human cord blood progenitors and acute myeloid leukemia. Experimental hematology. 43(10):858-868.e7. Pubmed: 26163797 Gentner B, Pochert N, Rouhi A, Boccalatte F, Plati T, Berg T, Sun SM, Mah SM, Mirkovic-Hösle M, Ruschmann J, Muranyi A, Leierseder S, Argiropoulos B, Starczynowski DT, Karsan A, Heuser M, Hogge D, Camargo FD, Engelhardt S, Döhner H, Buske C, Jongen-Lavrencic M, Naldini L, Humphries RK, Kuchenbauer F. 2015. MicroRNA-223 dose levels fine tune proliferation and differentiation in human cord blood progenitors and acute myeloid leukemia. Experimental hematology. 43(10):858-868.e7. Pubmed: 26163797 A precise understanding of the role of miR-223 in human hematopoiesis and in the pathogenesis of acute myeloid leukemia (AML) is still lacking. By measuring miR-223 expression in blasts from 115 AML patients, we found significantly higher miR-223 levels in patients with favorable prognosis, whereas patients with low miR-223 expression levels were associated with worse outcome. Furthermore, miR-223 was hierarchically expressed in AML subpopulations, with lower expression in leukemic stem cell-containing fractions. Genetic depletion of miR-223 decreased the leukemia initiating cell (LIC) frequency in a myelomonocytic AML mouse model, but it was not mandatory for rapid-onset AML. To relate these observations to physiologic myeloid differentiation, we knocked down or ectopically expressed miR-223 in cord-blood CD34⁺ cells using lentiviral vectors. Although miR-223 knockdown delayed myeloerythroid precursor differentiation in vitro, it increased myeloid progenitors in vivo following serial xenotransplantation. Ectopic miR-223 expression increased erythropoiesis, T lymphopoiesis, and early B lymphopoiesis in vivo. These findings broaden the role of miR-223 as a regulator of the expansion/differentiation equilibrium in hematopoietic stem and progenitor cells where its impact is dose- and differentiation-stage-dependent. This also explains the complex yet minor role of miR-223 in AML, a heterogeneous disease with variable degree of myeloid differentiation.Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved. -
Galli GG, Carrara M, Yuan WC, Valdes-Quezada C, Gurung B, Pepe-Mooney B, Zhang T, Geeven G, Gray NS, de Laat W, Calogero RA, Camargo FD. 2015. YAP Drives Growth by Controlling Transcriptional Pause Release from Dynamic Enhancers. Molecular cell. 60(2):328-37. Pubmed: 26439301 Galli GG, Carrara M, Yuan WC, Valdes-Quezada C, Gurung B, Pepe-Mooney B, Zhang T, Geeven G, Gray NS, de Laat W, Calogero RA, Camargo FD. 2015. YAP Drives Growth by Controlling Transcriptional Pause Release from Dynamic Enhancers. Molecular cell. 60(2):328-37. Pubmed: 26439301 The Hippo/YAP signaling pathway is a crucial regulator of tissue growth, stem cell activity, and tumorigenesis. However, the mechanism by which YAP controls transcription remains to be fully elucidated. Here, we utilize global chromatin occupancy analyses to demonstrate that robust YAP binding is restricted to a relatively small number of distal regulatory elements in the genome. YAP occupancy defines a subset of enhancers and superenhancers with the highest transcriptional outputs. YAP modulates transcription from these elements predominantly by regulating promoter-proximal polymerase II (Pol II) pause release. Mechanistically, YAP interacts and recruits the Mediator complex to enhancers, allowing the recruitment of the CDK9 elongating kinase. Genetic and chemical perturbation experiments demonstrate the requirement for Mediator and CDK9 in YAP-driven phenotypes of overgrowth and tumorigenesis. Our results here uncover the molecular mechanisms employed by YAP to exert its growth and oncogenic functions, and suggest strategies for intervention.Copyright © 2015 Elsevier Inc. All rights reserved. 2014
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Tremblay AM, Missiaglia E, Galli GG, Hettmer S, Urcia R, Carrara M, Judson RN, Thway K, Nadal G, Selfe JL, Murray G, Calogero RA, De Bari C, Zammit PS, Delorenzi M, Wagers AJ, Shipley J, Wackerhage H, Camargo FD. 2014. The Hippo transducer YAP1 transforms activated satellite cells and is a potent effector of embryonal rhabdomyosarcoma formation. Cancer cell. 26(2):273-87. Pubmed: 25087979 DOI:S1535-6108(14)00230-X Tremblay AM, Missiaglia E, Galli GG, Hettmer S, Urcia R, Carrara M, Judson RN, Thway K, Nadal G, Selfe JL, Murray G, Calogero RA, De Bari C, Zammit PS, Delorenzi M, Wagers AJ, Shipley J, Wackerhage H, Camargo FD. 2014. The Hippo transducer YAP1 transforms activated satellite cells and is a potent effector of embryonal rhabdomyosarcoma formation. Cancer cell. 26(2):273-87. Pubmed: 25087979 DOI:S1535-6108(14)00230-X The role of the Hippo pathway effector YAP1 in soft tissue sarcomas is poorly defined. Here we report that YAP1 activity is elevated in human embryonal rhabdomyosarcoma (ERMS). In mice, sustained YAP1 hyperactivity in activated, but not quiescent, satellite cells induces ERMS with high penetrance and short latency. Via its transcriptional program with TEAD1, YAP1 directly regulates several major hallmarks of ERMS. YAP1-TEAD1 upregulate pro-proliferative and oncogenic genes and maintain the ERMS differentiation block by interfering with MYOD1 and MEF2 pro-differentiation activities. Normalization of YAP1 expression reduces tumor burden in human ERMS xenografts and allows YAP1-driven ERMS to differentiate in situ. Collectively, our results identify YAP1 as a potent ERMS oncogenic driver and a promising target for differentiation therapy.Copyright © 2014 Elsevier Inc. All rights reserved. -
Sun J, Ramos A, Chapman B, Johnnidis JB, Le L, Ho YJ, Klein A, Hofmann O, Camargo FD. 2014. Clonal dynamics of native haematopoiesis. Nature. 514(7522):322-7. Pubmed: 25296256 DOI:10.1038/nature13824 Sun J, Ramos A, Chapman B, Johnnidis JB, Le L, Ho YJ, Klein A, Hofmann O, Camargo FD. 2014. Clonal dynamics of native haematopoiesis. Nature. 514(7522):322-7. Pubmed: 25296256 DOI:10.1038/nature13824 It is currently thought that life-long blood cell production is driven by the action of a small number of multipotent haematopoietic stem cells. Evidence supporting this view has been largely acquired through the use of functional assays involving transplantation. However, whether these mechanisms also govern native non-transplant haematopoiesis is entirely unclear. Here we have established a novel experimental model in mice where cells can be uniquely and genetically labelled in situ to address this question. Using this approach, we have performed longitudinal analyses of clonal dynamics in adult mice that reveal unprecedented features of native haematopoiesis. In contrast to what occurs following transplantation, steady-state blood production is maintained by the successive recruitment of thousands of clones, each with a minimal contribution to mature progeny. Our results demonstrate that a large number of long-lived progenitors, rather than classically defined haematopoietic stem cells, are the main drivers of steady-state haematopoiesis during most of adulthood. Our results also have implications for understanding the cellular origin of haematopoietic disease. -
Mori M, Triboulet R, Mohseni M, Schlegelmilch K, Shrestha K, Camargo FD, Gregory RI. 2014. Hippo signaling regulates microprocessor and links cell-density-dependent miRNA biogenesis to cancer. Cell. 156(5):893-906. Pubmed: 24581491 Mori M, Triboulet R, Mohseni M, Schlegelmilch K, Shrestha K, Camargo FD, Gregory RI. 2014. Hippo signaling regulates microprocessor and links cell-density-dependent miRNA biogenesis to cancer. Cell. 156(5):893-906. Pubmed: 24581491 Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis. The mechanisms responsible for this phenomenon remain poorly understood. Here, we show that YAP, the downstream target of the tumor-suppressive Hippo-signaling pathway regulates miRNA biogenesis in a cell-density-dependent manner. At low cell density, nuclear YAP binds and sequesters p72 (DDX17), a regulatory component of the miRNA-processing machinery. At high cell density, Hippo-mediated cytoplasmic retention of YAP facilitates p72 association with Microprocessor and binding to a specific sequence motif in pri-miRNAs. Inactivation of the Hippo pathway or expression of constitutively active YAP causes widespread miRNA suppression in cells and tumors and a corresponding posttranscriptional induction of MYC expression. Thus, the Hippo pathway links contact-inhibition regulation to miRNA biogenesis and may be responsible for the widespread miRNA repression observed in cancer.Copyright © 2014 Elsevier Inc. All rights reserved. -
Zhang H, von Gise A, Liu Q, Hu T, Tian X, He L, Pu W, Huang X, He L, Cai CL, Camargo FD, Pu WT, Zhou B. 2014. Yap1 is required for endothelial to mesenchymal transition of the atrioventricular cushion. The Journal of biological chemistry. 289(27):18681-92. Pubmed: 24831012 DOI:10.1074/jbc.M114.554584 Zhang H, von Gise A, Liu Q, Hu T, Tian X, He L, Pu W, Huang X, He L, Cai CL, Camargo FD, Pu WT, Zhou B. 2014. Yap1 is required for endothelial to mesenchymal transition of the atrioventricular cushion. The Journal of biological chemistry. 289(27):18681-92. Pubmed: 24831012 DOI:10.1074/jbc.M114.554584 Cardiac malformations due to aberrant development of the atrioventricular (AV) valves are among the most common forms of congenital heart diseases. Normally, heart valve mesenchyme is formed from an endothelial to mesenchymal transition (EMT) of endothelial cells of the endocardial cushions. Yes-associated protein 1 (YAP1) has been reported to regulate EMT in vitro, in addition to its known role as a major regulator of organ size and cell proliferation in vertebrates, leading us to hypothesize that YAP1 is required for heart valve development. We tested this hypothesis by conditional inactivation of YAP1 in endothelial cells and their derivatives. This resulted in markedly hypocellular endocardial cushions due to impaired formation of heart valve mesenchyme by EMT and to reduced endocardial cell proliferation. In endothelial cells, TGFβ induces nuclear localization of Smad2/3/4 complex, which activates expression of Snail, Twist1, and Slug, key transcription factors required for EMT. YAP1 interacts with this complex, and loss of YAP1 disrupts TGFβ-induced up-regulation of Snail, Twist1, and Slug. Together, our results identify a role of YAP1 in regulating EMT through modulation of TGFβ-Smad signaling and through proliferative activity during cardiac cushion development.© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. -
Grijalva JL, Huizenga M, Mueller K, Rodriguez S, Brazzo J, Camargo F, Sadri-Vakili G, Vakili K. 2014. Dynamic alterations in Hippo signaling pathway and YAP activation during liver regeneration. American journal of physiology. Gastrointestinal and liver physiology. 307(2):G196-204. Pubmed: 24875096 DOI:10.1152/ajpgi.00077.2014 Grijalva JL, Huizenga M, Mueller K, Rodriguez S, Brazzo J, Camargo F, Sadri-Vakili G, Vakili K. 2014. Dynamic alterations in Hippo signaling pathway and YAP activation during liver regeneration. American journal of physiology. Gastrointestinal and liver physiology. 307(2):G196-204. Pubmed: 24875096 DOI:10.1152/ajpgi.00077.2014 The Hippo signaling pathway has been implicated in mammalian organ size regulation and tumor suppression. Specifically, the Hippo pathway plays a critical role regulating the activity of transcriptional coactivator Yes-associated protein (YAP), which modulates a proliferative transcriptional program. Recent investigations have demonstrated that while this pathway is activated in quiescent livers, its inhibition leads to liver overgrowth and tumorigenesis. However, the role of the Hippo pathway during the natural process of liver regeneration remains unknown. Here we investigated alterations in the Hippo signaling pathway and YAP activation during liver regeneration using a 70% partial hepatectomy (PH) rat model. Our results indicate an increase in YAP activation by 1 day following PH as demonstrated by increased YAP nuclear localization and increased YAP target gene expression. Investigation of the Hippo pathway revealed a decrease in the activation of core kinases Mst1/2 by 1 day as well as Lats1/2 and its adapter protein Mob1 by 3 days following PH. Evaluation of liver-to-body weight ratios indicated that the liver reaches its near normal size by 7 days following PH, which correlated with a return to baseline YAP nuclear levels and target gene expression. Additionally, when liver size was restored, Mst1/2 kinase activation returned to levels observed in quiescent livers indicating reactivation of the Hippo signaling pathway. These findings illustrate the dynamic changes in the Hippo signaling pathway and YAP activation during liver regeneration, which stabilize when the liver-to-body weight ratio reaches homeostatic levels.Copyright © 2014 the American Physiological Society. -
Yimlamai D, Christodoulou C, Galli GG, Yanger K, Pepe-Mooney B, Gurung B, Shrestha K, Cahan P, Stanger BZ, Camargo FD. 2014. Hippo pathway activity influences liver cell fate. Cell. 157(6):1324-38. Pubmed: 24906150 Yimlamai D, Christodoulou C, Galli GG, Yanger K, Pepe-Mooney B, Gurung B, Shrestha K, Cahan P, Stanger BZ, Camargo FD. 2014. Hippo pathway activity influences liver cell fate. Cell. 157(6):1324-38. Pubmed: 24906150 The Hippo-signaling pathway is an important regulator of cellular proliferation and organ size. However, little is known about the role of this cascade in the control of cell fate. Employing a combination of lineage tracing, clonal analysis, and organoid culture approaches, we demonstrate that Hippo pathway activity is essential for the maintenance of the differentiated hepatocyte state. Remarkably, acute inactivation of Hippo pathway signaling in vivo is sufficient to dedifferentiate, at very high efficiencies, adult hepatocytes into cells bearing progenitor characteristics. These hepatocyte-derived progenitor cells demonstrate self-renewal and engraftment capacity at the single-cell level. We also identify the NOTCH-signaling pathway as a functional important effector downstream of the Hippo transducer YAP. Our findings uncover a potent role for Hippo/YAP signaling in controlling liver cell fate and reveal an unprecedented level of phenotypic plasticity in mature hepatocytes, which has implications for the understanding and manipulation of liver regeneration.Copyright © 2014 Elsevier Inc. All rights reserved. -
Zhao R, Fallon TR, Saladi SV, Pardo-Saganta A, Villoria J, Mou H, Vinarsky V, Gonzalez-Celeiro M, Nunna N, Hariri LP, Camargo F, Ellisen LW, Rajagopal J. 2014. Yap tunes airway epithelial size and architecture by regulating the identity, maintenance, and self-renewal of stem cells. Developmental cell. 30(2):151-65. Pubmed: 25043474 Zhao R, Fallon TR, Saladi SV, Pardo-Saganta A, Villoria J, Mou H, Vinarsky V, Gonzalez-Celeiro M, Nunna N, Hariri LP, Camargo F, Ellisen LW, Rajagopal J. 2014. Yap tunes airway epithelial size and architecture by regulating the identity, maintenance, and self-renewal of stem cells. Developmental cell. 30(2):151-65. Pubmed: 25043474 Our understanding of how stem cells are regulated to maintain appropriate tissue size and architecture is incomplete. We show that Yap (Yes-associated protein 1) is required for the actual maintenance of an adult mammalian stem cell. Without Yap, adult airway basal stem cells are lost through their unrestrained differentiation, resulting in the simplification of a pseudostratified epithelium into a columnar one. Conversely, Yap overexpression increases stem cell self-renewal and blocks terminal differentiation, resulting in epithelial hyperplasia and stratification. Yap overexpression in differentiated secretory cells causes them to partially reprogram and adopt a stem cell-like identity. In contrast, Yap knockdown prevents the dedifferentiation of secretory cells into stem cells. We then show that Yap functionally interacts with p63, the cardinal transcription factor associated with myriad epithelial basal stem cells. In aggregate, we show that Yap regulates all of the cardinal behaviors of airway epithelial stem cells and determines epithelial architecture.Copyright © 2014 Elsevier Inc. All rights reserved. -
Ganem NJ, Cornils H, Chiu SY, O'Rourke KP, Arnaud J, Yimlamai D, Théry M, Camargo FD, Pellman D. 2014. Cytokinesis failure triggers hippo tumor suppressor pathway activation. Cell. 158(4):833-848. Pubmed: 25126788 Ganem NJ, Cornils H, Chiu SY, O'Rourke KP, Arnaud J, Yimlamai D, Théry M, Camargo FD, Pellman D. 2014. Cytokinesis failure triggers hippo tumor suppressor pathway activation. Cell. 158(4):833-848. Pubmed: 25126788 Genetically unstable tetraploid cells can promote tumorigenesis. Recent estimates suggest that ∼37% of human tumors have undergone a genome-doubling event during their development. This potentially oncogenic effect of tetraploidy is countered by a p53-dependent barrier to proliferation. However, the cellular defects and corresponding signaling pathways that trigger growth suppression in tetraploid cells are not known. Here, we combine RNAi screening and in vitro evolution approaches to demonstrate that cytokinesis failure activates the Hippo tumor suppressor pathway in cultured cells, as well as in naturally occurring tetraploid cells in vivo. Induction of the Hippo pathway is triggered in part by extra centrosomes, which alter small G protein signaling and activate LATS2 kinase. LATS2 in turn stabilizes p53 and inhibits the transcriptional regulators YAP and TAZ. These findings define an important tumor suppression mechanism and uncover adaptive mechanisms potentially available to nascent tumor cells that bypass this inhibitory regulation.Copyright © 2014 Elsevier Inc. All rights reserved. -
Mohseni M, Sun J, Lau A, Curtis S, Goldsmith J, Fox VL, Wei C, Frazier M, Samson O, Wong KK, Wong KK, Kim C, Camargo FD. 2014. A genetic screen identifies an LKB1-MARK signalling axis controlling the Hippo-YAP pathway. Nature cell biology. 16(1):108-17. Pubmed: 24362629 DOI:10.1038/ncb2884 Mohseni M, Sun J, Lau A, Curtis S, Goldsmith J, Fox VL, Wei C, Frazier M, Samson O, Wong KK, Wong KK, Kim C, Camargo FD. 2014. A genetic screen identifies an LKB1-MARK signalling axis controlling the Hippo-YAP pathway. Nature cell biology. 16(1):108-17. Pubmed: 24362629 DOI:10.1038/ncb2884 The Hippo-YAP pathway is an emerging signalling cascade involved in the regulation of stem cell activity and organ size. To identify components of this pathway, we performed an RNAi-based kinome screen in human cells. Our screen identified several kinases not previously associated with Hippo signalling that control multiple cellular processes. One of the hits, LKB1, is a common tumour suppressor whose mechanism of action is only partially understood. We demonstrate that LKB1 acts through its substrates of the microtubule affinity-regulating kinase family to regulate the localization of the polarity determinant Scribble and the activity of the core Hippo kinases. Our data also indicate that YAP is functionally important for the tumour suppressive effects of LKB1. Our results identify a signalling axis that links YAP activation with LKB1 mutations, and have implications for the treatment of LKB1-mutant human malignancies. In addition, our findings provide insight into upstream signals of the Hippo-YAP signalling cascade. -
Lau AN, Curtis SJ, Fillmore CM, Rowbotham SP, Mohseni M, Wagner DE, Beede AM, Montoro DT, Sinkevicius KW, Walton ZE, Barrios J, Weiss DJ, Camargo FD, Wong KK, Kim CF. 2014. Tumor-propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis. The EMBO journal. 33(5):468-81. Pubmed: 24497554 DOI:10.1002/embj.201386082 Lau AN, Curtis SJ, Fillmore CM, Rowbotham SP, Mohseni M, Wagner DE, Beede AM, Montoro DT, Sinkevicius KW, Walton ZE, Barrios J, Weiss DJ, Camargo FD, Wong KK, Kim CF. 2014. Tumor-propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis. The EMBO journal. 33(5):468-81. Pubmed: 24497554 DOI:10.1002/embj.201386082 Metastasis is the leading cause of morbidity for lung cancer patients. Here we demonstrate that murine tumor propagating cells (TPCs) with the markers Sca1 and CD24 are enriched for metastatic potential in orthotopic transplantation assays. CD24 knockdown decreased the metastatic potential of lung cancer cell lines resembling TPCs. In lung cancer patient data sets, metastatic spread and patient survival could be stratified with a murine lung TPC gene signature. The TPC signature was enriched for genes in the Hippo signaling pathway. Knockdown of the Hippo mediators Yap1 or Taz decreased in vitro cellular migration and transplantation of metastatic disease. Furthermore, constitutively active Yap was sufficient to drive lung tumor progression in vivo. These results demonstrate functional roles for two different pathways, CD24-dependent and Yap/Taz-dependent pathways, in lung tumor propagation and metastasis. This study demonstrates the utility of TPCs for identifying molecules contributing to metastatic lung cancer, potentially enabling the therapeutic targeting of this devastating disease. 2013
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Barry ER, Morikawa T, Butler BL, Shrestha K, de la Rosa R, Yan KS, Fuchs CS, Magness ST, Smits R, Ogino S, Kuo CJ, Camargo FD. 2013. Restriction of intestinal stem cell expansion and the regenerative response by YAP. Nature. 493(7430):106-10. Pubmed: 23178811 DOI:10.1038/nature11693 Barry ER, Morikawa T, Butler BL, Shrestha K, de la Rosa R, Yan KS, Fuchs CS, Magness ST, Smits R, Ogino S, Kuo CJ, Camargo FD. 2013. Restriction of intestinal stem cell expansion and the regenerative response by YAP. Nature. 493(7430):106-10. Pubmed: 23178811 DOI:10.1038/nature11693 A remarkable feature of regenerative processes is their ability to halt proliferation once an organ's structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)--a protein known for its powerful growth-inducing and oncogenic properties--has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN-APC-GSK-3β complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies. -
Barry ER, Camargo FD. 2013. The Hippo superhighway: signaling crossroads converging on the Hippo/Yap pathway in stem cells and development. Current opinion in cell biology. 25(2):247-53. Pubmed: 23312716 Barry ER, Camargo FD. 2013. The Hippo superhighway: signaling crossroads converging on the Hippo/Yap pathway in stem cells and development. Current opinion in cell biology. 25(2):247-53. Pubmed: 23312716 Tissue regeneration is vital to the form and function of an organ. At the core of an organs' ability to self-renew is the stem cell, which maintains homeostasis, and repopulates injured or aged tissue. Tissue damage can dramatically change the dimensions of an organ, and during regeneration, an organ must halt growth once the original tissue dimensions have been restored. Therefore, stem cells must give rise to the appropriate number of differentiated progeny to achieve homeostasis. How this tissue-size checkpoint is regulated and how tissue size information relayed to stem cell compartments is unclear, however, it is likely that these mechanisms are altered during the course of tumorigenesis. An emerging signaling cascade, the Hippo Signaling Pathway, is a broadly conserved potent organ size regulator [1]. However, this pathway does not act alone. A number of examples demonstrate crosstalk between Hippo and other signaling pathways including Wnt, Tgfβ and Notch, with implications for stem cell biology. Here, we focus on these interactions primarily in the context of well characterized stem cell populations.Copyright © 2013 Elsevier Ltd. All rights reserved. -
Wu H, Xiao Y, Zhang S, Ji S, Wei L, Fan F, Geng J, Tian J, Sun X, Qin F, Jin C, Lin J, Yin ZY, Zhang T, Luo L, Li Y, Song S, Lin SC, Deng X, Camargo F, Avruch J, Chen L, Zhou D. 2013. The Ets transcription factor GABP is a component of the hippo pathway essential for growth and antioxidant defense. Cell reports. 3(5):1663-77. Pubmed: 23684612 Wu H, Xiao Y, Zhang S, Ji S, Wei L, Fan F, Geng J, Tian J, Sun X, Qin F, Jin C, Lin J, Yin ZY, Zhang T, Luo L, Li Y, Song S, Lin SC, Deng X, Camargo F, Avruch J, Chen L, Zhou D. 2013. The Ets transcription factor GABP is a component of the hippo pathway essential for growth and antioxidant defense. Cell reports. 3(5):1663-77. Pubmed: 23684612 The transcriptional coactivator Yes-associated protein (YAP) plays an important role in organ-size control and tumorigenesis. However, how Yap gene expression is regulated remains unknown. This study shows that the Ets family member GABP binds to the Yap promoter and activates YAP transcription. The depletion of GABP downregulates YAP, resulting in a G1/S cell-cycle block and increased cell death, both of which are substantially rescued by reconstituting YAP. GABP can be inactivated by oxidative mechanisms, and acetaminophen-induced glutathione depletion inhibits GABP transcriptional activity and depletes YAP. In contrast, activating YAP by deleting Mst1/Mst2 strongly protects against acetaminophen-induced liver injury. Similar to its effects on YAP, Hippo signaling inhibits GABP transcriptional activity through several mechanisms. In human liver cancers, enhanced YAP expression is correlated with increased nuclear expression of GABP. Therefore, we conclude that GABP is an activator of Yap gene expression and a potential therapeutic target for cancers driven by YAP.Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved. -
Halder G, Camargo FD. 2013. The hippo tumor suppressor network: from organ size control to stem cells and cancer. Cancer research. 73(21):6389-92. Pubmed: 24022648 DOI:10.1158/0008-5472.CAN-13-2392 Halder G, Camargo FD. 2013. The hippo tumor suppressor network: from organ size control to stem cells and cancer. Cancer research. 73(21):6389-92. Pubmed: 24022648 DOI:10.1158/0008-5472.CAN-13-2392 -
Zhang Y, Owens K, Hatem L, Glass CH, Karuppaiah K, Camargo F, Perkins AS. 2013. Essential role of PR-domain protein MDS1-EVI1 in MLL-AF9 leukemia. Blood. 122(16):2888-92. Pubmed: 24021671 DOI:10.1182/blood-2012-08-453662 Zhang Y, Owens K, Hatem L, Glass CH, Karuppaiah K, Camargo F, Perkins AS. 2013. Essential role of PR-domain protein MDS1-EVI1 in MLL-AF9 leukemia. Blood. 122(16):2888-92. Pubmed: 24021671 DOI:10.1182/blood-2012-08-453662 A subgroup of leukemogenic mixed-lineage leukemia (MLL) fusion proteins (MFPs) including MLL-AF9 activates the Mecom locus and exhibits extremely poor clinical prognosis. Mecom encodes EVI1 and MDS1-EVI1 (ME) proteins via alternative transcription start sites; these differ by the presence of a PRDI-BF1-RIZ1 (PR) domain with histone methyltransferase activity in the ME isoform. Using an ME-deficient mouse, we show that ME is required for MLL-AF9-induced transformation both in vitro and in vivo. And, although Nup98-HOXA9, MEIS1-HOXA9, and E2A-Hlf could transform ME-deficient cells, both MLL-AF9 and MLL-ENL were ineffective, indicating that the ME requirement is specific to MLL fusion leukemia. Further, we show that the PR domain is essential for MFP-induced transformation. These studies clearly indicate an essential role of PR-domain protein ME in MFP leukemia, suggesting that ME may be a novel target for therapeutic intervention for this group of leukemias. 2012
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Ramos A, Camargo FD. 2012. The Hippo signaling pathway and stem cell biology. Trends in cell biology. 22(7):339-46. Pubmed: 22658639 DOI:10.1016/j.tcb.2012.04.006 Ramos A, Camargo FD. 2012. The Hippo signaling pathway and stem cell biology. Trends in cell biology. 22(7):339-46. Pubmed: 22658639 DOI:10.1016/j.tcb.2012.04.006 Stem cell (SC) activity fluctuates throughout an organism's lifetime to maintain homeostatic conditions in all tissues. As animals develop and age, their organs must remodel and regenerate themselves in response to environmental and physiological demands. Recently, the highly conserved Hippo signaling pathway, discovered in Drosophila melanogaster, has been implicated as a key regulator of organ size control across species. Deregulation is associated with substantial overgrowth phenotypes and eventual onset of cancer in various tissues. Importantly, emerging evidence suggests that the Hippo pathway can modulate its effects on tissue size by the direct regulation of SC proliferation and maintenance. These findings provide an attractive model for how this pathway might communicate physiological needs for growth to tissue-specific SC pools. In this review, we summarize the current and emerging data linking Hippo signaling to SC function.Copyright © 2012 Elsevier Ltd. All rights reserved. -
von Gise A, Lin Z, Schlegelmilch K, Honor LB, Pan GM, Buck JN, Ma Q, Ishiwata T, Zhou B, Camargo FD, Pu WT. 2012. YAP1, the nuclear target of Hippo signaling, stimulates heart growth through cardiomyocyte proliferation but not hypertrophy. Proceedings of the National Academy of Sciences of the United States of America. 109(7):2394-9. Pubmed: 22308401 DOI:10.1073/pnas.1116136109 von Gise A, Lin Z, Schlegelmilch K, Honor LB, Pan GM, Buck JN, Ma Q, Ishiwata T, Zhou B, Camargo FD, Pu WT. 2012. YAP1, the nuclear target of Hippo signaling, stimulates heart growth through cardiomyocyte proliferation but not hypertrophy. Proceedings of the National Academy of Sciences of the United States of America. 109(7):2394-9. Pubmed: 22308401 DOI:10.1073/pnas.1116136109 Heart growth is tightly controlled so that the heart reaches a predetermined size. Fetal heart growth occurs through cardiomyocyte proliferation, whereas postnatal heart growth involves primarily physiological cardiomyocyte hypertrophy. The Hippo kinase cascade is an important regulator of organ growth. A major target of this kinase cascade is YAP1, a transcriptional coactivator that is inactivated by Hippo kinase activity. Here, we used both genetic gain and loss of Yap1 function to investigate its role in regulating proliferative and physiologic hypertrophic heart growth. Fetal Yap1 inactivation caused marked, lethal myocardial hypoplasia and decreased cardiomyocyte proliferation, whereas fetal activation of YAP1 stimulated cardiomyocyte proliferation. Enhanced proliferation was particularly dramatic in trabecular cardiomyocytes that normally exit from the cell cycle. Remarkably, YAP1 activation was sufficient to stimulate proliferation of postnatal cardiomyocytes, both in culture and in the intact heart. A dominant negative peptide that blocked YAP1 binding to TEAD transcription factors inhibited YAP1 proliferative activity, indicating that this activity requires YAP1-TEAD interaction. Although Yap1 was a critical regulator of cardiomyocyte proliferation, it did not influence physiological hypertrophic growth of cardiomyocytes, because postnatal Yap1 gain or loss of function did not significantly alter cardiomyocyte size. These studies demonstrate that Yap1 is a crucial regulator of cardiomyocyte proliferation, cardiac morphogenesis, and myocardial trabeculation. Activation of Yap1 in postnatal cardiomyocytes may be a useful strategy to stimulate cardiomyocyte expansion in therapeutic myocardial regeneration. -
Tumaneng K, Schlegelmilch K, Russell RC, Yimlamai D, Basnet H, Mahadevan N, Fitamant J, Bardeesy N, Camargo FD, Guan KL. 2012. YAP mediates crosstalk between the Hippo and PI(3)K–TOR pathways by suppressing PTEN via miR-29. Nature cell biology. 14(12):1322-9. Pubmed: 23143395 Tumaneng K, Schlegelmilch K, Russell RC, Yimlamai D, Basnet H, Mahadevan N, Fitamant J, Bardeesy N, Camargo FD, Guan KL. 2012. YAP mediates crosstalk between the Hippo and PI(3)K–TOR pathways by suppressing PTEN via miR-29. Nature cell biology. 14(12):1322-9. Pubmed: 23143395 Organ development is a complex process governed by the interplay of several signalling pathways that have critical functions in the regulation of cell growth and proliferation. Over the past years, the Hippo pathway has emerged as a key regulator of organ size. Perturbation of this pathway has been shown to play important roles in tumorigenesis. YAP, the main downstream target of the mammalian Hippo pathway, promotes organ growth, yet the underlying molecular mechanism of this regulation remains unclear. Here we provide evidence that YAP activates the mammalian target of rapamycin (mTOR), a major regulator of cell growth. We have identified the tumour suppressor PTEN, an upstream negative regulator of mTOR, as a critical mediator of YAP in mTOR regulation. We demonstrate that YAP downregulates PTEN by inducing miR-29 to inhibit PTEN translation. Last, we show that PI(3)K–mTOR is a pathway modulated by YAP to regulate cell size, tissue growth and hyperplasia. Our studies reveal a functional link between Hippo and PI(3)K–mTOR, providing a molecular basis for the coordination of these two pathways in organ size regulation. -
Tremblay AM, Camargo FD. 2012. Hippo signaling in mammalian stem cells. Seminars in cell & developmental biology. 23(7):818-26. Pubmed: 23034192 Tremblay AM, Camargo FD. 2012. Hippo signaling in mammalian stem cells. Seminars in cell & developmental biology. 23(7):818-26. Pubmed: 23034192 Over the past decade, the Hippo signaling cascade has been linked to organ size regulation in mammals. Indeed, modulation of the Hippo pathway can have potent effects on cellular proliferation and/or apoptosis and a deregulation of the pathway often leads to tumor development. Importantly, emerging evidence indicates that the Hippo pathway can modulate its effects on tissue size by the regulation of stem and progenitor cell activity. This role has recently been associated with the central position of the pathway in sensing spatiotemporal or mechanical cues, and translating them into specific cellular outputs. These results provide an attractive model for how the Hippo cascade might sense and transduce cellular 'neighborhood' cues into activation of tissue-specific stem or progenitors cells. A further understanding of this process could allow the development of new therapies for various degenerative diseases and cancers. Here, we review current and emerging data linking Hippo signaling to progenitor cell function.Copyright © 2012 Elsevier Ltd. All rights reserved. -
Judson RN, Tremblay AM, Knopp P, White RB, Urcia R, De Bari C, Zammit PS, Camargo FD, Wackerhage H. 2012. The Hippo pathway member Yap plays a key role in influencing fate decisions in muscle satellite cells. Journal of cell science. 125(Pt 24):6009-19. Pubmed: 23038772 DOI:10.1242/jcs.109546 Judson RN, Tremblay AM, Knopp P, White RB, Urcia R, De Bari C, Zammit PS, Camargo FD, Wackerhage H. 2012. The Hippo pathway member Yap plays a key role in influencing fate decisions in muscle satellite cells. Journal of cell science. 125(Pt 24):6009-19. Pubmed: 23038772 DOI:10.1242/jcs.109546 Satellite cells are the resident stem cells of skeletal muscle. Mitotically quiescent in mature muscle, they can be activated to proliferate and generate myoblasts to supply further myonuclei to hypertrophying or regenerating muscle fibres, or self-renew to maintain the resident stem cell pool. Here, we identify the transcriptional co-factor Yap as a novel regulator of satellite cell fate decisions. Yap expression increases during satellite cell activation and Yap remains highly expressed until after the differentiation versus self-renewal decision is made. Constitutive expression of Yap maintains Pax7(+) and MyoD(+) satellite cells and satellite cell-derived myoblasts, promotes proliferation but prevents differentiation. In contrast, Yap knockdown reduces the proliferation of satellite cell-derived myoblasts by ≈40%. Consistent with the cellular phenotype, microarrays show that Yap increases expression of genes associated with Yap inhibition, the cell cycle, ribosome biogenesis and that it represses several genes associated with angiotensin signalling. We also identify known regulators of satellite cell function such as BMP4, CD34 and Myf6 (Mrf4) as genes whose expression is dependent on Yap activity. Finally, we confirm in myoblasts that Yap binds to Tead transcription factors and co-activates MCAT elements which are enriched in the proximal promoters of Yap-responsive genes. 2011
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Zhang Y, Stehling-Sun S, Lezon-Geyda K, Juneja SC, Coillard L, Chatterjee G, Wuertzer CA, Camargo F, Perkins AS. 2011. PR-domain-containing Mds1-Evi1 is critical for long-term hematopoietic stem cell function. Blood. 118(14):3853-61. Pubmed: 21666053 DOI:10.1182/blood-2011-02-334680 Zhang Y, Stehling-Sun S, Lezon-Geyda K, Juneja SC, Coillard L, Chatterjee G, Wuertzer CA, Camargo F, Perkins AS. 2011. PR-domain-containing Mds1-Evi1 is critical for long-term hematopoietic stem cell function. Blood. 118(14):3853-61. Pubmed: 21666053 DOI:10.1182/blood-2011-02-334680 The Mds1 and Evi1 complex locus (Mecom) gives rise to several alternative transcripts implicated in leukemogenesis. However, the contribution that Mecom-derived gene products make to normal hematopoiesis remains largely unexplored. To investigate the role of the upstream transcription start site of Mecom in adult hematopoiesis, we created a mouse model with a lacZ knock-in at this site, termed ME(m1), which eliminates Mds1-Evi1 (ME), the longer, PR-domain-containing isoform produced by the gene (also known as PRDM3). β-galactosidase-marking studies revealed that, within hematopoietic cells, ME is exclusively expressed in the stem cell compartment. ME deficiency leads to a reduction in the number of HSCs and a complete loss of long-term repopulation capacity, whereas the stem cell compartment is shifted from quiescence to active cycling. Genetic exploration of the relative roles of endogenous ME and EVI1 isoforms revealed that ME preferentially rescues long-term HSC defects. RNA-seq analysis in Lin(-)Sca-1(+)c-Kit(+) cells (LSKs) of ME(m1) documents near complete silencing of Cdkn1c, encoding negative cell-cycle regulator p57-Kip2. Reintroduction of ME into ME(m1) LSKs leads to normalization of both p57-Kip2 expression and growth control. Our results clearly demonstrate a critical role of PR-domain-containing ME in linking p57-kip2 regulation to long-term HSC function. -
Schlegelmilch K, Mohseni M, Kirak O, Pruszak J, Rodriguez JR, Zhou D, Kreger BT, Vasioukhin V, Avruch J, Brummelkamp TR, Camargo FD. 2011. Yap1 acts downstream of α-catenin to control epidermal proliferation. Cell. 144(5):782-95. Pubmed: 21376238 DOI:10.1016/j.cell.2011.02.031 Schlegelmilch K, Mohseni M, Kirak O, Pruszak J, Rodriguez JR, Zhou D, Kreger BT, Vasioukhin V, Avruch J, Brummelkamp TR, Camargo FD. 2011. Yap1 acts downstream of α-catenin to control epidermal proliferation. Cell. 144(5):782-95. Pubmed: 21376238 DOI:10.1016/j.cell.2011.02.031 During development and regeneration, proliferation of tissue-specific stem cells is tightly controlled to produce organs of a predetermined size. The molecular determinants of this process remain poorly understood. Here, we investigate the function of Yap1, the transcriptional effector of the Hippo signaling pathway, in skin biology. Using gain- and loss-of-function studies, we show that Yap1 is a critical modulator of epidermal stem cell proliferation and tissue expansion. Yap1 mediates this effect through interaction with TEAD transcription factors. Additionally, our studies reveal that α-catenin, a molecule previously implicated in tumor suppression and cell density sensing in the skin, is an upstream negative regulator of Yap1. α-catenin controls Yap1 activity and phosphorylation by modulating its interaction with 14-3-3 and the PP2A phosphatase. Together, these data identify Yap1 as a determinant of the proliferative capacity of epidermal stem cells and as an important effector of a "crowd control" molecular circuitry in mammalian skin.Copyright © 2011 Elsevier Inc. All rights reserved. -
Silvis MR, Kreger BT, Lien WH, Klezovitch O, Rudakova GM, Camargo FD, Lantz DM, Seykora JT, Vasioukhin V. 2011. α-catenin is a tumor suppressor that controls cell accumulation by regulating the localization and activity of the transcriptional coactivator Yap1. Science signaling. 4(174):ra33. Pubmed: 21610251 DOI:10.1126/scisignal.2001823 Silvis MR, Kreger BT, Lien WH, Klezovitch O, Rudakova GM, Camargo FD, Lantz DM, Seykora JT, Vasioukhin V. 2011. α-catenin is a tumor suppressor that controls cell accumulation by regulating the localization and activity of the transcriptional coactivator Yap1. Science signaling. 4(174):ra33. Pubmed: 21610251 DOI:10.1126/scisignal.2001823 The Hippo pathway regulates contact inhibition of cell proliferation and, ultimately, organ size in diverse multicellular organisms. Inactivation of the Hippo pathway promotes nuclear localization of the transcriptional coactivator Yap1, a Hippo pathway effector, and can cause cancer. Here, we show that deletion of αE (α epithelial) catenin in the hair follicle stem cell compartment resulted in the development of skin squamous cell carcinoma in mice. Tumor formation was accelerated by simultaneous deletion of αE-catenin and the tumor suppressor-encoding gene p53. A small interfering RNA screen revealed a functional connection between αE-catenin and Yap1. By interacting with Yap1, αE-catenin promoted its cytoplasmic localization, and Yap1 showed constitutive nuclear localization in αE-catenin-null cells. We also found an inverse correlation between αE-catenin abundance and Yap1 activation in human squamous cell carcinoma tumors. These findings identify αE-catenin as a tumor suppressor that inhibits Yap1 activity and sequesters it in the cytoplasm. -
Kuchenbauer F, Mah SM, Heuser M, McPherson A, Rüschmann J, Rouhi A, Berg T, Bullinger L, Argiropoulos B, Morin RD, Lai D, Starczynowski DT, Karsan A, Eaves CJ, Watahiki A, Wang Y, Aparicio SA, Ganser A, Krauter J, Döhner H, Döhner K, Marra MA, Camargo FD, Palmqvist L, Buske C, Humphries RK. 2011. Comprehensive analysis of mammalian miRNA* species and their role in myeloid cells. Blood. 118(12):3350-8. Pubmed: 21628414 DOI:10.1182/blood-2010-10-312454 Kuchenbauer F, Mah SM, Heuser M, McPherson A, Rüschmann J, Rouhi A, Berg T, Bullinger L, Argiropoulos B, Morin RD, Lai D, Starczynowski DT, Karsan A, Eaves CJ, Watahiki A, Wang Y, Aparicio SA, Ganser A, Krauter J, Döhner H, Döhner K, Marra MA, Camargo FD, Palmqvist L, Buske C, Humphries RK. 2011. Comprehensive analysis of mammalian miRNA* species and their role in myeloid cells. Blood. 118(12):3350-8. Pubmed: 21628414 DOI:10.1182/blood-2010-10-312454 Processing of pre-miRNA through Dicer1 generates an miRNA duplex that consists of an miRNA and miRNA* strand. Despite the general view that miRNA*s have no functional role, we further investigated miRNA* species in 10 deep-sequencing libraries from mouse and human tissue. Comparisons of miRNA/miRNA* ratios across the miRNA sequence libraries revealed that 50% of the investigated miRNA duplexes exhibited a highly dominant strand. Conversely, 10% of miRNA duplexes showed a comparable expression of both strands, whereas the remaining 40% exhibited variable ratios across the examined libraries, as exemplified by miR-223/miR-223* in murine and human cell lines. Functional analyses revealed a regulatory role for miR-223* in myeloid progenitor cells, which implies an active role for both arms of the miR-223 duplex. This was further underscored by the demonstration that miR-223 and miR-223* targeted the insulin-like growth factor 1 receptor/phosphatidylinositol 3-kinase axis and that high miR-223* levels were associated with increased overall survival in patients with acute myeloid leukemia. Thus, we found a supporting role for miR-223* in differentiating myeloid cells in normal and leukemic cell states. The fact that the miR-223 duplex acts through both arms extends the complexity of miRNA-directed gene regulation of this myeloid key miRNA. -
Zhou D, Zhang Y, Wu H, Barry E, Yin Y, Lawrence E, Dawson D, Willis JE, Markowitz SD, Camargo FD, Avruch J. 2011. Mst1 and Mst2 protein kinases restrain intestinal stem cell proliferation and colonic tumorigenesis by inhibition of Yes-associated protein (Yap) overabundance. Proceedings of the National Academy of Sciences of the United States of America. 108(49):E1312-20. Pubmed: 22042863 DOI:10.1073/pnas.1110428108 Zhou D, Zhang Y, Wu H, Barry E, Yin Y, Lawrence E, Dawson D, Willis JE, Markowitz SD, Camargo FD, Avruch J. 2011. Mst1 and Mst2 protein kinases restrain intestinal stem cell proliferation and colonic tumorigenesis by inhibition of Yes-associated protein (Yap) overabundance. Proceedings of the National Academy of Sciences of the United States of America. 108(49):E1312-20. Pubmed: 22042863 DOI:10.1073/pnas.1110428108 Ablation of the kinases Mst1 and Mst2, orthologs of the Drosophila antiproliferative kinase Hippo, from mouse intestinal epithelium caused marked expansion of an undifferentiated stem cell compartment and loss of secretory cells throughout the small and large intestine. Although median survival of mice lacking intestinal Mst1/Mst2 is 13 wk, adenomas of the distal colon are common by this age. Diminished phosphorylation, enhanced abundance, and nuclear localization of the transcriptional coactivator Yes-associated protein 1 (Yap1) is evident in Mst1/Mst2-deficient intestinal epithelium, as is strong activation of β-catenin and Notch signaling. Although biallelic deletion of Yap1 from intestinal epithelium has little effect on intestinal development, inactivation of a single Yap1 allele reduces Yap1 polypeptide abundance to nearly wild-type levels and, despite the continued Yap hypophosphorylation and preferential nuclear localization, normalizes epithelial structure. Thus, supraphysiologic Yap polypeptide levels are necessary to drive intestinal stem cell proliferation. Yap is overexpressed in 68 of 71 human colon cancers and in at least 30 of 36 colon cancer-derived cell lines. In colon-derived cell lines where Yap is overabundant, its depletion strongly reduces β-catenin and Notch signaling and inhibits proliferation and survival. These findings demonstrate that Mst1 and Mst2 actively suppress Yap1 abundance and action in normal intestinal epithelium, an antiproliferative function that frequently is overcome in colon cancer through Yap1 polypeptide overabundance. The dispensability of Yap1 in normal intestinal homeostasis and its potent proliferative and prosurvival actions when overexpressed in colon cancer make it an attractive therapeutic target. 2010
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Carette JE, Pruszak J, Varadarajan M, Blomen VA, Gokhale S, Camargo FD, Wernig M, Jaenisch R, Brummelkamp TR. 2010. Generation of iPSCs from cultured human malignant cells. Blood. 115(20):4039-42. Pubmed: 20233975 DOI:10.1182/blood-2009-07-231845 Carette JE, Pruszak J, Varadarajan M, Blomen VA, Gokhale S, Camargo FD, Wernig M, Jaenisch R, Brummelkamp TR. 2010. Generation of iPSCs from cultured human malignant cells. Blood. 115(20):4039-42. Pubmed: 20233975 DOI:10.1182/blood-2009-07-231845 Induced pluripotent stem cells (iPSCs) can be generated from various differentiated cell types by the expression of a set of defined transcription factors. So far, iPSCs have been generated from primary cells, but it is unclear whether human cancer cell lines can be reprogrammed. Here we describe the generation and characterization of iPSCs derived from human chronic myeloid leukemia cells. We show that, despite the presence of oncogenic mutations, these cells acquired pluripotency by the expression of 4 transcription factors and underwent differentiation into cell types derived of all 3 germ layers during teratoma formation. Interestingly, although the parental cell line was strictly dependent on continuous signaling of the BCR-ABL oncogene, also termed oncogene addiction, reprogrammed cells lost this dependency and became resistant to the BCR-ABL inhibitor imatinib. This finding indicates that the therapeutic agent imatinib targets cells in a specific epigenetic differentiated cell state, and this may contribute to its inability to fully eradicate disease in chronic myeloid leukemia patients. 2009
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Stehling-Sun S, Dade J, Nutt SL, DeKoter RP, Camargo FD. 2009. Regulation of lymphoid versus myeloid fate 'choice' by the transcription factor Mef2c. Nature immunology. 10(3):289-96. Pubmed: 19169261 DOI:10.1038/ni.1694 Stehling-Sun S, Dade J, Nutt SL, DeKoter RP, Camargo FD. 2009. Regulation of lymphoid versus myeloid fate 'choice' by the transcription factor Mef2c. Nature immunology. 10(3):289-96. Pubmed: 19169261 DOI:10.1038/ni.1694 Despite advances in the identification of lymphoid-restricted progenitor cells, the transcription factors essential for their generation remain to be identified. Here we describe an unexpected function for the myeloid oncogene product Mef2c in lymphoid development. Mef2c deficiency was associated with profound defects in the production of B cells, T cells, natural killer cells and common lymphoid progenitor cells and an enhanced myeloid output. In multipotent progenitors, Mef2c was required for the proper expression of several key lymphoid regulators and restriction of the myeloid fate. Our studies also show that Mef2c was a critical transcriptional target of the transcription factor PU.1 during lymphopoiesis. Thus, Mef2c is a crucial component of the transcriptional network that regulates cell fate 'choice' in multipotent progenitors. 2008
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Johnnidis JB, Harris MH, Wheeler RT, Stehling-Sun S, Lam MH, Kirak O, Brummelkamp TR, Fleming MD, Camargo FD. 2008. Regulation of progenitor cell proliferation and granulocyte function by microRNA-223. Nature. 451(7182):1125-9. Pubmed: 18278031 DOI:10.1038/nature06607 Johnnidis JB, Harris MH, Wheeler RT, Stehling-Sun S, Lam MH, Kirak O, Brummelkamp TR, Fleming MD, Camargo FD. 2008. Regulation of progenitor cell proliferation and granulocyte function by microRNA-223. Nature. 451(7182):1125-9. Pubmed: 18278031 DOI:10.1038/nature06607 MicroRNAs are abundant in animal genomes and have been predicted to have important roles in a broad range of gene expression programmes. Despite this prominence, there is a dearth of functional knowledge regarding individual mammalian microRNAs. Using a loss-of-function allele in mice, we report here that the myeloid-specific microRNA-223 (miR-223) negatively regulates progenitor proliferation and granulocyte differentiation and activation. miR-223 (also called Mirn223) mutant mice have an expanded granulocytic compartment resulting from a cell-autonomous increase in the number of granulocyte progenitors. We show that Mef2c, a transcription factor that promotes myeloid progenitor proliferation, is a target of miR-223, and that genetic ablation of Mef2c suppresses progenitor expansion and corrects the neutrophilic phenotype in miR-223 null mice. In addition, granulocytes lacking miR-223 are hypermature, hypersensitive to activating stimuli and display increased fungicidal activity. As a consequence of this neutrophil hyperactivity, miR-223 mutant mice spontaneously develop inflammatory lung pathology and exhibit exaggerated tissue destruction after endotoxin challenge. Our data support a model in which miR-223 acts as a fine-tuner of granulocyte production and the inflammatory response. -
Johnnidis JB, Camargo FD. 2008. Isolation and functional characterization of side population stem cells. Methods in molecular biology (Clifton, N.J.). 430:183-93. Pubmed: 18370300 DOI:10.1007/978-1-59745-182-6_13 Johnnidis JB, Camargo FD. 2008. Isolation and functional characterization of side population stem cells. Methods in molecular biology (Clifton, N.J.). 430:183-93. Pubmed: 18370300 DOI:10.1007/978-1-59745-182-6_13 The "side population" (SP) phenotype is a manifestation of primitive cells' ability to efficiently efflux the fluorescent DNA-staining dye Hoechst 33342 and can be used as the basis by which to isolate these cells using flow cytometry. In the bone marrow (BM), the SP defines a cell subset with a highly homogeneous content of hematopoietic stem cells (HSCs). In this chapter, we describe a protocol to reproducibly isolate murine BM SP cells, as well as analytic measures, such as single cell transplantation, that can be used to assess the functionality of SP-derived stem cells. -
Baek D, Villén J, Shin C, Camargo FD, Gygi SP, Bartel DP. 2008. The impact of microRNAs on protein output. Nature. 455(7209):64-71. Pubmed: 18668037 DOI:10.1038/nature07242 Baek D, Villén J, Shin C, Camargo FD, Gygi SP, Bartel DP. 2008. The impact of microRNAs on protein output. Nature. 455(7209):64-71. Pubmed: 18668037 DOI:10.1038/nature07242 MicroRNAs are endogenous approximately 23-nucleotide RNAs that can pair to sites in the messenger RNAs of protein-coding genes to downregulate the expression from these messages. MicroRNAs are known to influence the evolution and stability of many mRNAs, but their global impact on protein output had not been examined. Here we use quantitative mass spectrometry to measure the response of thousands of proteins after introducing microRNAs into cultured cells and after deleting mir-223 in mouse neutrophils. The identities of the responsive proteins indicate that targeting is primarily through seed-matched sites located within favourable predicted contexts in 3' untranslated regions. Hundreds of genes were directly repressed, albeit each to a modest degree, by individual microRNAs. Although some targets were repressed without detectable changes in mRNA levels, those translationally repressed by more than a third also displayed detectable mRNA destabilization, and, for the more highly repressed targets, mRNA destabilization usually comprised the major component of repression. The impact of microRNAs on the proteome indicated that for most interactions microRNAs act as rheostats to make fine-scale adjustments to protein output. 2007
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Lengner CJ, Camargo FD, Hochedlinger K, Welstead GG, Zaidi S, Gokhale S, Scholer HR, Tomilin A, Jaenisch R. 2007. Oct4 expression is not required for mouse somatic stem cell self-renewal. Cell stem cell. 1(4):403-15. Pubmed: 18159219 Lengner CJ, Camargo FD, Hochedlinger K, Welstead GG, Zaidi S, Gokhale S, Scholer HR, Tomilin A, Jaenisch R. 2007. Oct4 expression is not required for mouse somatic stem cell self-renewal. Cell stem cell. 1(4):403-15. Pubmed: 18159219 The Pou domain containing transcription factor Oct4 is a well-established regulator of pluripotency in the inner cell mass of the mammalian blastocyst as well as in embryonic stem cells. While it has been shown that the Oct4 gene is inactivated through a series of epigenetic modifications following implantation, recent studies have detected Oct4 activity in a variety of somatic stem cells and tumor cells. Based on these observations it has been suggested that Oct4 may also function in maintaining self-renewal of somatic stem cells and, in addition, may promote tumor formation. We employed a genetic approach to determine whether Oct4 is important for maintaining pluripotency in the stem cell compartments of several somatic tissues including the intestinal epithelium, bone marrow (hematopoietic and mesenchymal lineages), hair follicle, brain, and liver. Oct4 gene ablation in these tissues revealed no abnormalities in homeostasis or regenerative capacity. We conclude that Oct4 is dispensable for both self-renewal and maintenance of somatic stem cells in the adult mammal. -
Camargo FD, Gokhale S, Johnnidis JB, Fu D, Bell GW, Jaenisch R, Brummelkamp TR. 2007. YAP1 increases organ size and expands undifferentiated progenitor cells. Current biology : CB. 17(23):2054-60. Pubmed: 17980593 Camargo FD, Gokhale S, Johnnidis JB, Fu D, Bell GW, Jaenisch R, Brummelkamp TR. 2007. YAP1 increases organ size and expands undifferentiated progenitor cells. Current biology : CB. 17(23):2054-60. Pubmed: 17980593 The mechanisms that regulate mammalian organ size are poorly understood. It is unclear whether the pathways that control organ size also impinge on stem/progenitor cells. A highly expressed gene in stem cells is YAP1, the ortholog of Drosophila Yorkie, a downstream component of the Hippo pathway. Mutations in components of this pathway produce tissue overgrowth phenotypes in the fly whereas mammalian orthologs, like salvador, merlin, LATS, and YAP1, have been implicated in tumorigenesis. We report here that YAP1 increases organ size and causes aberrant tissue expansion in mice. YAP1 activation reversibly increases liver size more than 4-fold. In the intestine, expression of endogenous YAP1 is restricted to the progenitor/stem cell compartment, and activation of YAP1 expands multipotent undifferentiated progenitor cells, which differentiate upon cessation of YAP1 expression. YAP1 stimulates Notch signaling, and administration of gamma-secretase inhibitors suppressed the intestinal dysplasia caused by YAP1. Human colorectal cancers expressing higher levels of YAP1 share molecular aspects with YAP1-induced dysplastic growth in the mouse. Our data show that the Hippo signaling pathway regulates organ size in mammals and can act on stem cell compartments, indicating a potential link between stem/progenitor cells, organ size, and cancer. 2006
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Camargo FD, Chambers SM, Drew E, McNagny KM, Goodell MA. 2006. Hematopoietic stem cells do not engraft with absolute efficiencies. Blood. 107(2):501-7. Pubmed: 16204316 Camargo FD, Chambers SM, Drew E, McNagny KM, Goodell MA. 2006. Hematopoietic stem cells do not engraft with absolute efficiencies. Blood. 107(2):501-7. Pubmed: 16204316 Hematopoietic stem cells (HSCs) can be isolated from murine bone marrow by their ability to efflux the Hoechst 33342 dye. This method defines an extremely small and hematopoietically potent subset of cells known as the side population (SP). Recent studies suggest that transplanted single SP cells are capable of lymphohematopoietic repopulation at near absolute efficiencies. Here, we carefully reevaluate the hematopoietic potential of individual SP cells and find substantially lower rates of reconstitution. Our strategy involved the cotransplantation of single SP cells along with different populations of competitor cells that varied in their self-renewal capacity. Even with minimized HSC competition, SP cells were only able to reconstitute up to 35% of recipient mice. Furthermore, through immunophenotyping and clonal in vitro assays we find that SP cells are virtually homogeneous. Isolation of HSCs on the basis of Hoechst exclusion and a single cell-surface marker allows enrichment levels similar to that obtained with complex multicolor strategies. Altogether, our results indicate that even an extremely homogeneous HSC population, based on phenotype and dye efflux, cannot reconstitute mice at absolute efficiencies. 2005
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Goodell MA, McKinney-Freeman S, Camargo FD. 2005. Isolation and characterization of side population cells. Methods in molecular biology (Clifton, N.J.). 290:343-52. Pubmed: 15361673 Goodell MA, McKinney-Freeman S, Camargo FD. 2005. Isolation and characterization of side population cells. Methods in molecular biology (Clifton, N.J.). 290:343-52. Pubmed: 15361673 The protocol for isolation of side population (SP) cells was originally established for murine bone marrow hematopoietic stem cells (HSCs), but it has also been adapted for other species and tissues. This purification strategy offers a simple and reproducible strategy to obtain a highly homogeneous population of HSCs. The method is based on the differential efflux of the fluorescent DNA-binding dye Hoechst 33342 from stem cells relative to nonstem cells. The protocols outlined in this chapter describe the isolation of murine SP cells from both bone marrow and skeletal muscle using the fluorescent DNA-binding dye Hoechst 33342. In these tissues, the SP cells that are isolated are HSCs. 2004
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Camargo FD, Finegold M, Goodell MA. 2004. Hematopoietic myelomonocytic cells are the major source of hepatocyte fusion partners. The Journal of clinical investigation. 113(9):1266-70. Pubmed: 15124017 Camargo FD, Finegold M, Goodell MA. 2004. Hematopoietic myelomonocytic cells are the major source of hepatocyte fusion partners. The Journal of clinical investigation. 113(9):1266-70. Pubmed: 15124017 Several recent reports have demonstrated that transplantation of bone marrow cells can result in the generation of functional hepatocytes. Cellular fusion between bone marrow-derived cells and host hepatocytes has been shown to be the mechanism of this phenomenon. However, the exact identity of the bone marrow cells that mediate cellular fusion has remained undetermined. Here we demonstrate that the hematopoietic progeny of a single hematopoietic stem cell (HSC) is sufficient to produce functional hepatic repopulation. Furthermore, transplantation of lymphocyte-deficient bone marrow cells and in vivo fate mapping of the myeloid lineage revealed that HSC-derived hepatocytes are primarily derived from mature myelomonocytic cells. In addition, using a Cre/lox-based strategy, we directly demonstrate that myeloid cells spontaneously fuse with host hepatocytes. Our findings raise the possibility that differentiated myeloid cells may be useful for future therapeutic applications of in vivo cellular fusion. -
Camargo FD, Chambers SM, Goodell MA. 2004. Stem cell plasticity: from transdifferentiation to macrophage fusion. Cell proliferation. 37(1):55-65. Pubmed: 14871237 Camargo FD, Chambers SM, Goodell MA. 2004. Stem cell plasticity: from transdifferentiation to macrophage fusion. Cell proliferation. 37(1):55-65. Pubmed: 14871237 The past 5 years have witnessed an explosion of interest in using adult-derived stem cells for cell and gene therapy. This has been driven by a number of findings, in particular, the possibility that some adult stem cells can differentiate into non-autologous cell types, and also the discovery of multipotential stem cells in adult bone marrow. These discoveries suggested a quasi-alchemical nature of cells derived from adult organs, thus raising new and exciting therapeutic possibilities. Recent data, however, argue against the whole idea of stem cell 'plasticity', and bring into question the therapeutic strategies based upon this concept. Here, we will review the current state of knowledge in the field and discuss some of the clinical implications. 2003
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Olmsted-Davis EA, Gugala Z, Camargo F, Gannon FH, Jackson K, Kienstra KA, Shine HD, Lindsey RW, Hirschi KK, Goodell MA, Brenner MK, Davis AR. 2003. Primitive adult hematopoietic stem cells can function as osteoblast precursors. Proceedings of the National Academy of Sciences of the United States of America. 100(26):15877-82. Pubmed: 14673088 Olmsted-Davis EA, Gugala Z, Camargo F, Gannon FH, Jackson K, Kienstra KA, Shine HD, Lindsey RW, Hirschi KK, Goodell MA, Brenner MK, Davis AR. 2003. Primitive adult hematopoietic stem cells can function as osteoblast precursors. Proceedings of the National Academy of Sciences of the United States of America. 100(26):15877-82. Pubmed: 14673088 Osteoblasts are continually recruited from stem cell pools to maintain bone. Although their immediate precursor is a plastic-adherent mesenchymal stem cell able to generate tissues other than bone, increasing evidence suggests the existence of a more primitive cell that can differentiate to both hematopoietic and mesenchymal cells. We show here that the "side population" (SP) of marrow stem cells, defined by their ability to rapidly expel a DNA-binding dye and to regenerate the hematopoietic compartment, can differentiate to osteoblasts through a mesenchymal intermediate. When transplanted into lethally irradiated mice, single gene-marked murine SP cells reconstituted depleted osteoprogenitor pools, such that a large proportion of the osteogenic cells in the epiphysis of long bone carried the donor SP cell marker. These findings suggest that the developmental capacity of SP cells is not restricted to the hematopoietic lineages but extends to osteogenic differentiation. This property not only elucidates a previously unrecognized step in osteoblast development, but also has intriguing implications for the use of SP cells in clinical orthopedics and stem cell-based disorders of bone. -
Camargo FD, Green R, Capetanaki Y, Jackson KA, Goodell MA, Capetenaki Y. 2003. Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nature medicine. 9(12):1520-7. Pubmed: 14625546 Camargo FD, Green R, Capetanaki Y, Jackson KA, Goodell MA, Capetenaki Y. 2003. Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nature medicine. 9(12):1520-7. Pubmed: 14625546 Recent studies have shown that cells from the bone marrow can give rise to differentiated skeletal muscle fibers. However, the mechanisms and identities of the cell types involved have remained unknown, and the validity of the observation has been questioned. Here, we use transplantation of single CD45+ hematopoietic stem cells (HSCs) to demonstrate that the entire circulating myogenic activity in bone marrow is derived from HSCs and their hematopoietic progeny. We also show that ongoing muscle regeneration and inflammatory cell infiltration are required for HSC-derived contribution, which does not occur through a myogenic stem cell intermediate. Using a lineage tracing strategy, we show that myofibers are derived from mature myeloid cells in response to injury. Our results indicate that circulating myeloid cells, in response to inflammatory cues, migrate to regenerating skeletal muscle and stochastically incorporate into mature myofibers. 2002
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McKinney-Freeman SL, Jackson KA, Camargo FD, Ferrari G, Mavilio F, Goodell MA. 2002. Muscle-derived hematopoietic stem cells are hematopoietic in origin. Proceedings of the National Academy of Sciences of the United States of America. 99(3):1341-6. Pubmed: 11830662 McKinney-Freeman SL, Jackson KA, Camargo FD, Ferrari G, Mavilio F, Goodell MA. 2002. Muscle-derived hematopoietic stem cells are hematopoietic in origin. Proceedings of the National Academy of Sciences of the United States of America. 99(3):1341-6. Pubmed: 11830662 It has recently been shown that mononuclear cells from murine skeletal muscle contain the potential to repopulate all major peripheral blood lineages in lethally irradiated mice, but the origin of this activity is unknown. We have fractionated muscle cells on the basis of hematopoietic markers to show that the active population exclusively expresses the hematopoietic stem cell antigens Sca-1 and CD45. Muscle cells obtained from 6- to 8-week-old C57BL/6-CD45.1 mice and enriched for cells expressing Sca-1 and CD45 were able to generate hematopoietic but not myogenic colonies in vitro and repopulated multiple hematopoietic lineages of lethally irradiated C57BL/6-CD45.2 mice. These data show that muscle-derived hematopoietic stem cells are likely derived from the hematopoietic system and are a result not of transdifferentiation of myogenic stem cells but instead of the presence of substantial numbers of hematopoietic stem cells in the muscle. Although CD45-negative cells were highly myogenic in vitro and in vivo, CD45-positive muscle-derived cells displayed only very limited myogenic activity and only in vivo. 2001
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Camargo F, Erickson RP, Garver WS, Hossain GS, Carbone PN, Heidenreich RA, Blanchard J. 2001. Cyclodextrins in the treatment of a mouse model of Niemann-Pick C disease. Life sciences. 70(2):131-42. Pubmed: 11787939 Camargo F, Erickson RP, Garver WS, Hossain GS, Carbone PN, Heidenreich RA, Blanchard J. 2001. Cyclodextrins in the treatment of a mouse model of Niemann-Pick C disease. Life sciences. 70(2):131-42. Pubmed: 11787939 Niemann-Pick type C (NPC) is a neurodegenerative disorder characterized by greatly altered somatic cholesterol metabolism. The NPC1 gene has recently been cloned and shown to have sequence homology to other sterol-sensing proteins. We have used a mouse model with a disrupted npc1 gene to study the effects of the cholesterol-mobilizing compound, 2-hydroxypropyl-beta-cyclodextrins (HPBCD), on the clinical course of this disorder. Treatment with two HPBCDs, with varying levels of 2-hydroxypropyl substitution, had effects in delaying neurological symptoms and in decreasing liver cholesterol storage while a third HPBCD was without effect. The ameliorating effect was not improved by longer exposure times (commencement of exposure in utero), however, it is not known if there is transplacental transfer of HPBCDs. The combination of HPBCD with probucol or nifedipine (which have previously been shown to lower liver cholesterol in this animal model) markedly decreased liver storage of unesterified cholesterol without altering the depressed levels of esterified cholesterol. The slight effects of the HPBCDs on neurological symptoms may be partially due to their apparent non-permeation of the blood-brain barrier (BBB). This non-permeation was assayed with radioactive tracers and was also present in the mdr1a knockout mice which have greatly increased BBB permeability for many drugs. Intrathecal delivery of HPBCD by an Alzet osmotic minipump did not improve its efficacy in ameliorating neurological symptoms. 2000
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Camargo FD, Huey-Louie DA, Finn AV, Sassani AB, Cozen AE, Moriwaki H, Schneider DB, Agah R, Dichek DA. 2000. Germline incorporation of a replication-defective adenoviral vector in mice does not alter immune responses to adenoviral vectors. Molecular therapy : the journal of the American Society of Gene Therapy. 2(5):496-504. Pubmed: 11082323 Camargo FD, Huey-Louie DA, Finn AV, Sassani AB, Cozen AE, Moriwaki H, Schneider DB, Agah R, Dichek DA. 2000. Germline incorporation of a replication-defective adenoviral vector in mice does not alter immune responses to adenoviral vectors. Molecular therapy : the journal of the American Society of Gene Therapy. 2(5):496-504. Pubmed: 11082323 The utility of adenoviral vectors is limited by immune responses to adenoviral antigens. We sought to develop immune-competent mice in which the immune response to adenoviral antigens was selectively absent. To do so, we generated mice that were transgenic for a replication-defective vector. Adenoviral antigens might be seen as self-antigens by these mice, and the mice could exhibit immunologic tolerance after postnatal exposure to adenoviral vectors. In addition, characterization of these mice could reveal potential consequences of germline transmission of an adenoviral vector, as might occur in a gene therapy trial. Injection of a "null" (not containing a transgene) E1, E3-deleted vector genome into mouse zygotes yielded five founders that were capable of transmitting the vector genome. Among offspring of these mice, transgenic pups were significantly underrepresented: 108 of 255 pups (42%) were transgenic (P<0.02 versus expected frequency of 50%). Postnatal transgenic mice, however, had no apparent abnormalities. Persistence of an adenoviral vector after intravenous injection was equivalent in livers of transgenic mice and their nontransgenic littermates. Transgenic and nontransgenic mice also had equivalent humoral and cellular immune responses to adenoviral vector injection. Mice that are transgenic for an E1, E3-deleted adenoviral genome can be easily generated; however, they are not tolerant of adenovirus. Moreover, germline transmission of an adenoviral vector genome does not prevent generation of a robust immune response after exposure to adenoviral antigens. -
Erickson RP, Garver WS, Camargo F, Hossain GS, Heidenreich RA. 2000. Pharmacological and genetic modifications of somatic cholesterol do not substantially alter the course of CNS disease in Niemann-Pick C mice. Journal of inherited metabolic disease. 23(1):54-62. Pubmed: 10682308 Erickson RP, Garver WS, Camargo F, Hossain GS, Heidenreich RA. 2000. Pharmacological and genetic modifications of somatic cholesterol do not substantially alter the course of CNS disease in Niemann-Pick C mice. Journal of inherited metabolic disease. 23(1):54-62. Pubmed: 10682308 Niemann-Pick type C (NPC) is a neurodegenerative disorder with somatically altered cholesterol metabolism. The NPC1 gene has recently been cloned and shown to have sequences shared with known sterol-sensing proteins. We have used a mouse model with a disrupted Npc1 gene to study two cholesterol-lowering drugs (nifedipine and probucol) and the effects of introducing a null mutation in the low-density lipoprotein receptor (LDLR). Although these treatments significantly ameliorated liver cholesterol storage, little effect on the onset of neurological symptoms was found.