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Melton Lab
The many stages of beta cell development.
Melton Lab Publications
Featured Publications
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2019. A Peninsular Structure Coordinates Asynchronous Differentiation with Morphogenesis to Generate Pancreatic Islets. Cell. 176(4):790-804.e13. Pubmed: 30661759 Sharon N, Chawla R, Mueller J, Vanderhooft J, Whitehorn LJ, Rosenthal B, Gürtler M, Estanboulieh RR, Shvartsman D, Gifford DK, Trapnell C, Melton D. 2019. A Peninsular Structure Coordinates Asynchronous Differentiation with Morphogenesis to Generate Pancreatic Islets. Cell. 176(4):790-804.e13. Pubmed: 30661759 The pancreatic islets of Langerhans regulate glucose homeostasis. The loss of insulin-producing β cells within islets results in diabetes, and islet transplantation from cadaveric donors can cure the disease. In vitro production of whole islets, not just β cells, will benefit from a better understanding of endocrine differentiation and islet morphogenesis. We used single-cell mRNA sequencing to obtain a detailed description of pancreatic islet development. Contrary to the prevailing dogma, we find islet morphology and endocrine differentiation to be directly related. As endocrine progenitors differentiate, they migrate in cohesion and form bud-like islet precursors, or "peninsulas" (literally "almost islands"). α cells, the first to develop, constitute the peninsular outer layer, and β cells form later, beneath them. This spatiotemporal collinearity leads to the typical core-mantle architecture of the mature, spherical islet. Finally, we induce peninsula-like structures in differentiating human embryonic stem cells, laying the ground for the generation of entire islets in vitro.Copyright © 2018 Elsevier Inc. All rights reserved. -
Veres A, Faust AL, Bushnell HL, Engquist EN, Kenty JH, Harb G, Poh YC, Sintov E, Gürtler M, Pagliuca FW, Peterson QP, Melton DA. 2019. Charting cellular identity during human in vitro β-cell differentiation. Nature. 569(7756):368-373. Pubmed: 31068696 DOI:10.1038/s41586-019-1168-5 Veres A, Faust AL, Bushnell HL, Engquist EN, Kenty JH, Harb G, Poh YC, Sintov E, Gürtler M, Pagliuca FW, Peterson QP, Melton DA. 2019. Charting cellular identity during human in vitro β-cell differentiation. Nature. 569(7756):368-373. Pubmed: 31068696 DOI:10.1038/s41586-019-1168-5 In vitro differentiation of human stem cells can produce pancreatic β-cells; the loss of this insulin-secreting cell type underlies type 1 diabetes. Here, as a step towards understanding this differentiation process, we report the transcriptional profiling of more than 100,000 human cells undergoing in vitro β-cell differentiation, and describe the cells that emerged. We resolve populations that correspond to β-cells, α-like poly-hormonal cells, non-endocrine cells that resemble pancreatic exocrine cells and a previously unreported population that resembles enterochromaffin cells. We show that endocrine cells maintain their identity in culture in the absence of exogenous growth factors, and that changes in gene expression associated with in vivo β-cell maturation are recapitulated in vitro. We implement a scalable re-aggregation technique to deplete non-endocrine cells and identify CD49a (also known as ITGA1) as a surface marker of the β-cell population, which allows magnetic sorting to a purity of 80%. Finally, we use a high-resolution sequencing time course to characterize gene-expression dynamics during the induction of human pancreatic endocrine cells, from which we develop a lineage model of in vitro β-cell differentiation. This study provides a perspective on human stem-cell differentiation, and will guide future endeavours that focus on the differentiation of pancreatic islet cells, and their applications in regenerative medicine. -
Sharon N, Vanderhooft J, Straubhaar J, Mueller J, Chawla R, Zhou Q, Engquist EN, Trapnell C, Gifford DK, Melton DA. 2019. Wnt Signaling Separates the Progenitor and Endocrine Compartments during Pancreas Development. Cell reports. 27(8):2281-2291.e5. Pubmed: 31116975 Sharon N, Vanderhooft J, Straubhaar J, Mueller J, Chawla R, Zhou Q, Engquist EN, Trapnell C, Gifford DK, Melton DA. 2019. Wnt Signaling Separates the Progenitor and Endocrine Compartments during Pancreas Development. Cell reports. 27(8):2281-2291.e5. Pubmed: 31116975 In vitro differentiation of pluripotent cells into β cells is a promising alternative to cadaveric-islet transplantation as a cure for type 1 diabetes (T1D). During the directed differentiation of human embryonic stem cells (hESCS) by exogenous factors, numerous genes that affect the differentiation process are turned on and off autonomously. Manipulating these reactions could increase the efficiency of differentiation and provide a more complete control over the final composition of cell populations. To uncover in vitro autonomous responses, we performed single-cell RNA sequencing on hESCs as they differentiate in spherical clusters. We observed that endocrine cells and their progenitors exist beside one another in separate compartments that activate distinct genetic pathways. WNT pathway inhibition in the endocrine domain of the differentiating clusters reveals a necessary role for the WNT inhibitor APC during islet formation in vivo. Accordingly, WNT inhibition in vitro causes an increase in the proportion of differentiated endocrine cells.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved. -
Baron M, Veres A, Wolock SL, Faust AL, Gaujoux R, Vetere A, Ryu JH, Wagner BK, Shen-Orr SS, Klein AM, Melton DA, Yanai I. 2016. A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-cell Population Structure. Cell Syst. 3. DOI:10.1016/j.cels.2016.08.011 Baron M, Veres A, Wolock SL, Faust AL, Gaujoux R, Vetere A, Ryu JH, Wagner BK, Shen-Orr SS, Klein AM, Melton DA, Yanai I. 2016. A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-cell Population Structure. Cell Syst. 3. DOI:10.1016/j.cels.2016.08.011 Although the function of the mammalian pancreas hinges on complex interactions of distinct cell types, gene expression profiles have primarily been described with bulk mixtures. Here we implemented a droplet-based, single-cell RNA-seq method to determine the transcriptomes of over 12,000 individual pancreatic cells from four human donors and two mouse strains. Cells could be divided into 15 clusters that matched previously characterized cell types: all endocrine cell types, including rare epsilon-cells; exocrine cell types; vascular cells; Schwann cells; quiescent and activated stellate cells; and four types of immune cells. We detected subpopulations of ductal cells with distinct expression profiles and validated their existence with immuno-histochemistry stains. Moreover, among human beta- cells, we detected heterogeneity in the regulation of genes relating to functional maturation and levels of ER stress. Finally, we deconvolved bulk gene expression samples using the single-cell data to detect disease-associated differential expression. Our dataset provides a resource for the discovery of novel cell type-specific transcription factors, signaling receptors, and medically relevant genes. Data available in BioStudies: https://www.ebi.ac.uk/biostudies/studies/S-EPMC5228327?xr=true -
Millman JR, Xie C, Van Dervort A, Gürtler M, Pagliuca FW, Melton DA. 2016. Generation of stem cell-derived β-cells from patients with type 1 diabetes. Nature communications. 7:11463. Pubmed: 27163171 DOI:10.1038/ncomms11463 Millman JR, Xie C, Van Dervort A, Gürtler M, Pagliuca FW, Melton DA. 2016. Generation of stem cell-derived β-cells from patients with type 1 diabetes. Nature communications. 7:11463. Pubmed: 27163171 DOI:10.1038/ncomms11463 We recently reported the scalable in vitro production of functional stem cell-derived β-cells (SC-β cells). Here we extend this approach to generate the first SC-β cells from type 1 diabetic patients (T1D). β-cells are destroyed during T1D disease progression, making it difficult to extensively study them in the past. These T1D SC-β cells express β-cell markers, respond to glucose both in vitro and in vivo, prevent alloxan-induced diabetes in mice and respond to anti-diabetic drugs. Furthermore, we use an in vitro disease model to demonstrate the cells respond to different forms of β-cell stress. Using these assays, we find no major differences in T1D SC-β cells compared with SC-β cells derived from non-diabetic patients. These results show that T1D SC-β cells could potentially be used for the treatment of diabetes, drug screening and the study of β-cell biology. -
Vegas AJ, Veiseh O, Gürtler M, Millman JR, Pagliuca FW, Bader AR, Doloff JC, Li J, Chen M, Olejnik K, Tam HH, Jhunjhunwala S, Langan E, Aresta-Dasilva S, Gandham S, McGarrigle JJ, Bochenek MA, Hollister-Lock J, Oberholzer J, Greiner DL, Weir GC, Melton DA, Langer R, Anderson DG. 2016. Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice. Nature medicine. 22(3):306-11. Pubmed: 26808346 DOI:10.1038/nm.4030 Vegas AJ, Veiseh O, Gürtler M, Millman JR, Pagliuca FW, Bader AR, Doloff JC, Li J, Chen M, Olejnik K, Tam HH, Jhunjhunwala S, Langan E, Aresta-Dasilva S, Gandham S, McGarrigle JJ, Bochenek MA, Hollister-Lock J, Oberholzer J, Greiner DL, Weir GC, Melton DA, Langer R, Anderson DG. 2016. Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice. Nature medicine. 22(3):306-11. Pubmed: 26808346 DOI:10.1038/nm.4030 The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-β cells), which may represent an unlimited source of human cells for pancreas replacement therapy. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier. However, clinical implementation has been challenging because of host immune responses to the implant materials. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-β cells. SC-β cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells. -
Melton DA. 2016. Applied Developmental Biology: Making Human Pancreatic Beta Cells for Diabetics. Current topics in developmental biology. 117:65-73. Pubmed: 26969972 Melton DA. 2016. Applied Developmental Biology: Making Human Pancreatic Beta Cells for Diabetics. Current topics in developmental biology. 117:65-73. Pubmed: 26969972 Understanding the genes and signaling pathways that determine the differentiation and fate of a cell is a central goal of developmental biology. Using that information to gain mastery over the fates of cells presents new approaches to cell transplantation and drug discovery for human diseases including diabetes.© 2016 Elsevier Inc. All rights reserved. -
Kenty JH, Melton DA. 2015. Testing pancreatic islet function at the single cell level by calcium influx with associated marker expression. PloS one. 10(4):e0122044. Pubmed: 25853429 DOI:10.1371/journal.pone.0122044 Kenty JH, Melton DA. 2015. Testing pancreatic islet function at the single cell level by calcium influx with associated marker expression. PloS one. 10(4):e0122044. Pubmed: 25853429 DOI:10.1371/journal.pone.0122044 Studying the response of islet cells to glucose stimulation is important for understanding cell function in healthy and disease states. Most functional assays are performed on whole islets or cell populations, resulting in averaged observations and loss of information at the single cell level. We demonstrate methods to examine calcium fluxing in individual cells of intact islets in response to multiple glucose challenges. Wild-type mouse islets predominantly contained cells that responded to three (out of three) sequential high glucose challenges, whereas cells of diabetic islets (db/db or NOD) responded less frequently or not at all. Imaged islets were also immunostained for endocrine markers to associate the calcium flux profile of individual cells with gene expression. Wild-type mouse islet cells that robustly fluxed calcium expressed β cell markers (INS/NKX6.1), whereas islet cells that inversely fluxed at low glucose expressed α cell markers (GCG). Diabetic mouse islets showed a higher proportion of dysfunctional β cells that responded poorly to glucose challenges. Most of the failed calcium influx responses in β cells were observed in the second and third high glucose challenges, emphasizing the importance of multiple sequential glucose challenges for assessing the full function of islet cells. Human islet cells were also assessed and showed functional α and β cells. This approach to analyze islet responses to multiple glucose challenges in correlation with gene expression assays expands the understanding of β cell function and the diseased state. -
Ben-Zvi D, Melton DA. 2015. Modeling human nutrition using human embryonic stem cells. Cell. 161(1):12-17. Pubmed: 25815980 Ben-Zvi D, Melton DA. 2015. Modeling human nutrition using human embryonic stem cells. Cell. 161(1):12-17. Pubmed: 25815980 Nutrition presents unanswered scientific questions of high public health importance. We envision model systems composed of interacting gastrointestinal and metabolic tissues derived from human embryonic stem cells, populated by gut microbiota. The culture will be embedded in 3D scaffolds, creating a controlled experimental system that enables tissue sampling and imaging.Copyright © 2015 Elsevier Inc. All rights reserved. -
Pagliuca FW, Millman JR, Gürtler M, Segel M, Van Dervort A, Ryu JH, Peterson QP, Greiner D, Melton DA. 2014. Generation of functional human pancreatic β cells in vitro. Cell. 159(2):428-39. Pubmed: 25303535 Pagliuca FW, Millman JR, Gürtler M, Segel M, Van Dervort A, Ryu JH, Peterson QP, Greiner D, Melton DA. 2014. Generation of functional human pancreatic β cells in vitro. Cell. 159(2):428-39. Pubmed: 25303535 The generation of insulin-producing pancreatic β cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However, insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide β cells. Here, we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive β cells from hPSC in vitro. These stem-cell-derived β cells (SC-β) express markers found in mature β cells, flux Ca(2+) in response to glucose, package insulin into secretory granules, and secrete quantities of insulin comparable to adult β cells in response to multiple sequential glucose challenges in vitro. Furthermore, these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner, and transplantation of these cells ameliorates hyperglycemia in diabetic mice.Copyright © 2014 Elsevier Inc. All rights reserved. -
Hrvatin S, Deng F, O'Donnell CW, Gifford DK, Melton DA. 2014. MARIS: method for analyzing RNA following intracellular sorting. PloS one. 9(3):e89459. Pubmed: 24594682 DOI:10.1371/journal.pone.0089459 Hrvatin S, Deng F, O'Donnell CW, Gifford DK, Melton DA. 2014. MARIS: method for analyzing RNA following intracellular sorting. PloS one. 9(3):e89459. Pubmed: 24594682 DOI:10.1371/journal.pone.0089459 Transcriptional profiling is a key technique in the study of cell biology that is limited by the availability of reagents to uniquely identify specific cell types and isolate high quality RNA from them. We report a Method for Analyzing RNA following Intracellular Sorting (MARIS) that generates high quality RNA for transcriptome profiling following cellular fixation, intracellular immunofluorescent staining and FACS. MARIS can therefore be used to isolate high quality RNA from many otherwise inaccessible cell types simply based on immunofluorescent tagging of unique intracellular proteins. As proof of principle, we isolate RNA from sorted human embryonic stem cell-derived insulin-expressing cells as well as adult human β cells. MARIS is a basic molecular biology technique that could be used across several biological disciplines. -
Blum B, Roose AN, Barrandon O, Maehr R, Arvanites AC, Davidow LS, Davis JC, Peterson QP, Rubin LL, Melton DA. 2014. Reversal of β cell de-differentiation by a small molecule inhibitor of the TGFβ pathway. eLife. 3:e02809. Pubmed: 25233132 DOI:10.7554/eLife.02809 Blum B, Roose AN, Barrandon O, Maehr R, Arvanites AC, Davidow LS, Davis JC, Peterson QP, Rubin LL, Melton DA. 2014. Reversal of β cell de-differentiation by a small molecule inhibitor of the TGFβ pathway. eLife. 3:e02809. Pubmed: 25233132 DOI:10.7554/eLife.02809 Dysfunction or death of pancreatic β cells underlies both types of diabetes. This functional decline begins with β cell stress and de-differentiation. Current drugs for type 2 diabetes (T2D) lower blood glucose levels but they do not directly alleviate β cell stress nor prevent, let alone reverse, β cell de-differentiation. We show here that Urocortin 3 (Ucn3), a marker for mature β cells, is down-regulated in the early stages of T2D in mice and when β cells are stressed in vitro. Using an insulin expression-coupled lineage tracer, with Ucn3 as a reporter for the mature β cell state, we screen for factors that reverse β cell de-differentiation. We find that a small molecule inhibitor of TGFβ receptor I (Alk5) protects cells from the loss of key β cell transcription factors and restores a mature β cell identity even after exposure to prolonged and severe diabetes. -
Li W, Nakanishi M, Zumsteg A, Shear M, Wright C, Melton DA, Zhou Q. 2014. In vivo reprogramming of pancreatic acinar cells to three islet endocrine subtypes. eLife. 3:e01846. Pubmed: 24714494 DOI:10.7554/eLife.01846 Li W, Nakanishi M, Zumsteg A, Shear M, Wright C, Melton DA, Zhou Q. 2014. In vivo reprogramming of pancreatic acinar cells to three islet endocrine subtypes. eLife. 3:e01846. Pubmed: 24714494 DOI:10.7554/eLife.01846 Direct lineage conversion of adult cells is a promising approach for regenerative medicine. A major challenge of lineage conversion is to generate specific cell subtypes. The pancreatic islets contain three major hormone-secreting endocrine subtypes: insulin(+) β-cells, glucagon(+) α-cells, and somatostatin(+) δ-cells. We previously reported that a combination of three transcription factors, Ngn3, Mafa, and Pdx1, directly reprograms pancreatic acinar cells to β-cells. We now show that acinar cells can be converted to δ-like and α-like cells by Ngn3 and Ngn3+Mafa respectively. Thus, three major islet endocrine subtypes can be derived by acinar reprogramming. Ngn3 promotes establishment of a generic endocrine state in acinar cells, and also promotes δ-specification in the absence of other factors. δ-specification is in turn suppressed by Mafa and Pdx1 during α- and β-cell induction. These studies identify a set of defined factors whose combinatorial actions reprogram acinar cells to distinct islet endocrine subtypes in vivo. DOI: http://dx.doi.org/10.7554/eLife.01846.001.
All Publications
2019
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Han X, Wang M, Duan S, Franco PJ, Kenty JH, Hedrick P, Xia Y, Allen A, Ferreira LMR, Strominger JL, Melton DA, Meissner TB, Cowan CA. 2019. Generation of hypoimmunogenic human pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America. 116(21):10441-10446. Pubmed: 31040209 DOI:10.1073/pnas.1902566116 Han X, Wang M, Duan S, Franco PJ, Kenty JH, Hedrick P, Xia Y, Allen A, Ferreira LMR, Strominger JL, Melton DA, Meissner TB, Cowan CA. 2019. Generation of hypoimmunogenic human pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America. 116(21):10441-10446. Pubmed: 31040209 DOI:10.1073/pnas.1902566116 Polymorphic HLAs form the primary immune barrier to cell therapy. In addition, innate immune surveillance impacts cell engraftment, yet a strategy to control both, adaptive and innate immunity, is lacking. Here we employed multiplex genome editing to specifically ablate the expression of the highly polymorphic HLA-A/-B/-C and HLA class II in human pluripotent stem cells. Furthermore, to prevent innate immune rejection and further suppress adaptive immune responses, we expressed the immunomodulatory factors PD-L1, HLA-G, and the macrophage "don't-eat me" signal CD47 from the safe harbor locus. Utilizing in vitro and in vivo immunoassays, we found that T cell responses were blunted. Moreover, NK cell killing and macrophage engulfment of our engineered cells were minimal. Our results describe an approach that effectively targets adaptive as well as innate immune responses and may therefore enable cell therapy on a broader scale. -
Rosado-Olivieri EA, Anderson K, Kenty JH, Melton DA. 2019. YAP inhibition enhances the differentiation of functional stem cell-derived insulin-producing β cells. Nature communications. 10(1):1464. Pubmed: 30931946 DOI:10.1038/s41467-019-09404-6 Rosado-Olivieri EA, Anderson K, Kenty JH, Melton DA. 2019. YAP inhibition enhances the differentiation of functional stem cell-derived insulin-producing β cells. Nature communications. 10(1):1464. Pubmed: 30931946 DOI:10.1038/s41467-019-09404-6 Stem cell-derived insulin-producing beta cells (SC-β) offer an inexhaustible supply of functional β cells for cell replacement therapies and disease modeling for diabetes. While successful directed differentiation protocols for this cell type have been described, the mechanisms controlling its differentiation and function are not fully understood. Here we report that the Hippo pathway controls the proliferation and specification of pancreatic progenitors into the endocrine lineage. Downregulation of YAP, an effector of the pathway, enhances endocrine progenitor differentiation and the generation of SC-β cells with improved insulin secretion. A chemical inhibitor of YAP acts as an inducer of endocrine differentiation and reduces the presence of proliferative progenitor cells. Conversely, sustained activation of YAP results in impaired differentiation, blunted glucose-stimulated insulin secretion, and increased proliferation of SC-β cells. Together these results support a role for YAP in controlling the self-renewal and differentiation balance of pancreatic progenitors and limiting endocrine differentiation in vitro. -
Alagpulinsa DA, Cao JJL, Driscoll RK, Sîrbulescu RF, Penson MFE, Sremac M, Engquist EN, Brauns TA, Markmann JF, Melton DA, Poznansky MC. 2019. Alginate-microencapsulation of human stem cell-derived β cells with CXCL12 prolongs their survival and function in immunocompetent mice without systemic immunosuppression. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 19(7):1930-1940. Pubmed: 30748094 DOI:10.1111/ajt.15308 Alagpulinsa DA, Cao JJL, Driscoll RK, Sîrbulescu RF, Penson MFE, Sremac M, Engquist EN, Brauns TA, Markmann JF, Melton DA, Poznansky MC. 2019. Alginate-microencapsulation of human stem cell-derived β cells with CXCL12 prolongs their survival and function in immunocompetent mice without systemic immunosuppression. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 19(7):1930-1940. Pubmed: 30748094 DOI:10.1111/ajt.15308 Pancreatic β-cell replacement by islet transplantation for the treatment of type 1 diabetes (T1D) is currently limited by donor tissue scarcity and the requirement for lifelong immunosuppression. The advent of in vitro differentiation protocols for generating functional β-like cells from human pluripotent stem cells, also referred to as SC-β cells, could eliminate these obstacles. To avoid the need for immunosuppression, alginate-microencapsulation is widely investigated as a safe path to β-cell replacement. Nonetheless, inflammatory foreign body responses leading to pericapsular fibrotic overgrowth often causes microencapsulated islet-cell death and graft failure. Here we used a novel approach to evade the pericapsular fibrotic response to alginate-microencapsulated SC-β cells; an immunomodulatory chemokine, CXCL12, was incorporated into clinical grade sodium alginate to microencapsulate SC-β cells. CXCL12 enhanced glucose-stimulated insulin secretion activity of SC-β cells and induced expression of genes associated with β-cell function in vitro. SC-β cells co-encapsulated with CXCL12 showed enhanced insulin secretion in diabetic mice and accelerated the normalization of hyperglycemia. Additionally, SC-β cells co-encapsulated with CXCL12 evaded the pericapsular fibrotic response, resulting in long-term functional competence and glycemic correction (>150 days) without systemic immunosuppression in immunocompetent C57BL/6 mice. These findings lay the groundwork for further preclinical translation of this approach into large animal models of T1D.© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons. -
Sharon N, Chawla R, Mueller J, Vanderhooft J, Whitehorn LJ, Rosenthal B, Gürtler M, Estanboulieh RR, Shvartsman D, Gifford DK, Trapnell C, Melton D. 2019. A Peninsular Structure Coordinates Asynchronous Differentiation with Morphogenesis to Generate Pancreatic Islets. Cell. 176(4):790-804.e13. Pubmed: 30661759 Sharon N, Chawla R, Mueller J, Vanderhooft J, Whitehorn LJ, Rosenthal B, Gürtler M, Estanboulieh RR, Shvartsman D, Gifford DK, Trapnell C, Melton D. 2019. A Peninsular Structure Coordinates Asynchronous Differentiation with Morphogenesis to Generate Pancreatic Islets. Cell. 176(4):790-804.e13. Pubmed: 30661759 The pancreatic islets of Langerhans regulate glucose homeostasis. The loss of insulin-producing β cells within islets results in diabetes, and islet transplantation from cadaveric donors can cure the disease. In vitro production of whole islets, not just β cells, will benefit from a better understanding of endocrine differentiation and islet morphogenesis. We used single-cell mRNA sequencing to obtain a detailed description of pancreatic islet development. Contrary to the prevailing dogma, we find islet morphology and endocrine differentiation to be directly related. As endocrine progenitors differentiate, they migrate in cohesion and form bud-like islet precursors, or "peninsulas" (literally "almost islands"). α cells, the first to develop, constitute the peninsular outer layer, and β cells form later, beneath them. This spatiotemporal collinearity leads to the typical core-mantle architecture of the mature, spherical islet. Finally, we induce peninsula-like structures in differentiating human embryonic stem cells, laying the ground for the generation of entire islets in vitro.Copyright © 2018 Elsevier Inc. All rights reserved. -
Sharon N, Vanderhooft J, Straubhaar J, Mueller J, Chawla R, Zhou Q, Engquist EN, Trapnell C, Gifford DK, Melton DA. 2019. Wnt Signaling Separates the Progenitor and Endocrine Compartments during Pancreas Development. Cell reports. 27(8):2281-2291.e5. Pubmed: 31116975 Sharon N, Vanderhooft J, Straubhaar J, Mueller J, Chawla R, Zhou Q, Engquist EN, Trapnell C, Gifford DK, Melton DA. 2019. Wnt Signaling Separates the Progenitor and Endocrine Compartments during Pancreas Development. Cell reports. 27(8):2281-2291.e5. Pubmed: 31116975 In vitro differentiation of pluripotent cells into β cells is a promising alternative to cadaveric-islet transplantation as a cure for type 1 diabetes (T1D). During the directed differentiation of human embryonic stem cells (hESCS) by exogenous factors, numerous genes that affect the differentiation process are turned on and off autonomously. Manipulating these reactions could increase the efficiency of differentiation and provide a more complete control over the final composition of cell populations. To uncover in vitro autonomous responses, we performed single-cell RNA sequencing on hESCs as they differentiate in spherical clusters. We observed that endocrine cells and their progenitors exist beside one another in separate compartments that activate distinct genetic pathways. WNT pathway inhibition in the endocrine domain of the differentiating clusters reveals a necessary role for the WNT inhibitor APC during islet formation in vivo. Accordingly, WNT inhibition in vitro causes an increase in the proportion of differentiated endocrine cells.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved. -
Tsai SL, Baselga-Garriga C, Melton DA. 2019. Blastemal progenitors modulate immune signaling during early limb regeneration. Development (Cambridge, England). 146(1). Pubmed: 30602532 Tsai SL, Baselga-Garriga C, Melton DA. 2019. Blastemal progenitors modulate immune signaling during early limb regeneration. Development (Cambridge, England). 146(1). Pubmed: 30602532 Blastema formation, a hallmark of limb regeneration, requires proliferation and migration of progenitors to the amputation plane. Although blastema formation has been well described, the transcriptional programs that drive blastemal progenitors remain unknown. We transcriptionally profiled dividing and non-dividing cells in regenerating stump tissues, as well as the wound epidermis, during early axolotl limb regeneration. Our analysis revealed unique transcriptional signatures of early dividing cells and, unexpectedly, repression of several core developmental signaling pathways in early regenerating stump tissues. We further identify an immunomodulatory role for blastemal progenitors through interleukin 8 (IL-8), a highly expressed cytokine in subpopulations of early blastemal progenitors. Ectopic expression in non-regenerating limbs induced myeloid cell recruitment, while IL-8 knockdown resulted in defective myeloid cell retention during late wound healing, delaying regeneration. Furthermore, the receptor was expressed in myeloid cells, and inhibition of CXCR-1/2 signaling during early stages of limb regeneration prevented regeneration. Altogether, our findings suggest that blastemal progenitors are active early mediators of immune support, and identify CXCR-1/2 signaling as an important immunomodulatory pathway during the initiation of regeneration.© 2019. Published by The Company of Biologists Ltd. -
Veres A, Faust AL, Bushnell HL, Engquist EN, Kenty JH, Harb G, Poh YC, Sintov E, Gürtler M, Pagliuca FW, Peterson QP, Melton DA. 2019. Charting cellular identity during human in vitro β-cell differentiation. Nature. 569(7756):368-373. Pubmed: 31068696 DOI:10.1038/s41586-019-1168-5 Veres A, Faust AL, Bushnell HL, Engquist EN, Kenty JH, Harb G, Poh YC, Sintov E, Gürtler M, Pagliuca FW, Peterson QP, Melton DA. 2019. Charting cellular identity during human in vitro β-cell differentiation. Nature. 569(7756):368-373. Pubmed: 31068696 DOI:10.1038/s41586-019-1168-5 In vitro differentiation of human stem cells can produce pancreatic β-cells; the loss of this insulin-secreting cell type underlies type 1 diabetes. Here, as a step towards understanding this differentiation process, we report the transcriptional profiling of more than 100,000 human cells undergoing in vitro β-cell differentiation, and describe the cells that emerged. We resolve populations that correspond to β-cells, α-like poly-hormonal cells, non-endocrine cells that resemble pancreatic exocrine cells and a previously unreported population that resembles enterochromaffin cells. We show that endocrine cells maintain their identity in culture in the absence of exogenous growth factors, and that changes in gene expression associated with in vivo β-cell maturation are recapitulated in vitro. We implement a scalable re-aggregation technique to deplete non-endocrine cells and identify CD49a (also known as ITGA1) as a surface marker of the β-cell population, which allows magnetic sorting to a purity of 80%. Finally, we use a high-resolution sequencing time course to characterize gene-expression dynamics during the induction of human pancreatic endocrine cells, from which we develop a lineage model of in vitro β-cell differentiation. This study provides a perspective on human stem-cell differentiation, and will guide future endeavours that focus on the differentiation of pancreatic islet cells, and their applications in regenerative medicine. 2018
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Mahmoud AI, Galdos FX, Dinan KA, Jedrychowski MP, Davis JC, Vujic A, Rachmin I, Shigley C, Pancoast JR, Lee S, Hollister-Lock J, MacGillivray CM, Gygi SP, Melton DA, Weir GC, Lee RT. 2018. Apolipoprotein E is a pancreatic extracellular factor that maintains mature β-cell gene expression. PloS one. 13(10):e0204595. Pubmed: 30303984 DOI:10.1371/journal.pone.0204595 Mahmoud AI, Galdos FX, Dinan KA, Jedrychowski MP, Davis JC, Vujic A, Rachmin I, Shigley C, Pancoast JR, Lee S, Hollister-Lock J, MacGillivray CM, Gygi SP, Melton DA, Weir GC, Lee RT. 2018. Apolipoprotein E is a pancreatic extracellular factor that maintains mature β-cell gene expression. PloS one. 13(10):e0204595. Pubmed: 30303984 DOI:10.1371/journal.pone.0204595 The in vivo microenvironment of tissues provides myriad unique signals to cells. Thus, following isolation, many cell types change in culture, often preserving some but not all of their in vivo characteristics in culture. At least some of the in vivo microenvironment may be mimicked by providing specific cues to cultured cells. Here, we show that after isolation and during maintenance in culture, adherent rat islets reduce expression of key β-cell transcription factors necessary for β-cell function and that soluble pancreatic decellularized matrix (DCM) can enhance β-cell gene expression. Following chromatographic fractionation of pancreatic DCM, we performed proteomics to identify soluble factors that can maintain β-cell stability and function. We identified Apolipoprotein E (ApoE) as an extracellular protein that significantly increased the expression of key β-cell genes. The ApoE effect on beta cells was mediated at least in part through the JAK/STAT signaling pathway. Together, these results reveal a role for ApoE as an extracellular factor that can maintain the mature β-cell gene expression profile. -
Odorico J, Markmann J, Melton D, Greenstein J, Hwa A, Nostro C, Rezania A, Oberholzer J, Pipeleers D, Yang L, Cowan C, Huangfu D, Egli D, Ben-David U, Vallier L, Grey ST, Tang Q, Roep B, Ricordi C, Naji A, Orlando G, Anderson DG, Poznansky M, Ludwig B, Tomei A, Greiner DL, Graham M, Carpenter M, Migliaccio G, D'Amour K, Hering B, Piemonti L, Berney T, Rickels M, Kay T, Adams A. 2018. Report of the Key Opinion Leaders Meeting on Stem Cell-derived Beta Cells. Transplantation. 102(8):1223-1229. Pubmed: 29781950 DOI:10.1097/TP.0000000000002217 Odorico J, Markmann J, Melton D, Greenstein J, Hwa A, Nostro C, Rezania A, Oberholzer J, Pipeleers D, Yang L, Cowan C, Huangfu D, Egli D, Ben-David U, Vallier L, Grey ST, Tang Q, Roep B, Ricordi C, Naji A, Orlando G, Anderson DG, Poznansky M, Ludwig B, Tomei A, Greiner DL, Graham M, Carpenter M, Migliaccio G, D'Amour K, Hering B, Piemonti L, Berney T, Rickels M, Kay T, Adams A. 2018. Report of the Key Opinion Leaders Meeting on Stem Cell-derived Beta Cells. Transplantation. 102(8):1223-1229. Pubmed: 29781950 DOI:10.1097/TP.0000000000002217 Beta cell replacement has the potential to restore euglycemia in patients with insulin-dependent diabetes. Although great progress has been made in establishing allogeneic islet transplantation from deceased donors as the standard of care for those with the most labile diabetes, it is also clear that the deceased donor organ supply cannot possibly treat all those who could benefit from restoration of a normal beta cell mass, especially if immunosuppression were not required. Against this background, the International Pancreas and Islet Transplant Association in collaboration with the Harvard Stem Cell Institute, the Juvenile Diabetes Research Foundation (JDRF), and the Helmsley Foundation held a 2-day Key Opinion Leaders Meeting in Boston in 2016 to bring together experts in generating and transplanting beta cells derived from stem cells. The following summary highlights current technology, recent significant breakthroughs, unmet needs and roadblocks to stem cell-derived beta cell therapies, with the aim of spurring future preclinical collaborative investigations and progress toward the clinical application of stem cell-derived beta cells. -
Zhou Q, Melton DA. 2018. Pancreas regeneration. Nature. 557(7705):351-358. Pubmed: 29769672 DOI:10.1038/s41586-018-0088-0 Zhou Q, Melton DA. 2018. Pancreas regeneration. Nature. 557(7705):351-358. Pubmed: 29769672 DOI:10.1038/s41586-018-0088-0 The pancreas is made from two distinct components: the exocrine pancreas, a reservoir of digestive enzymes, and the endocrine islets, the source of the vital metabolic hormone insulin. Human islets possess limited regenerative ability; loss of islet β-cells in diseases such as type 1 diabetes requires therapeutic intervention. The leading strategy for restoration of β-cell mass is through the generation and transplantation of new β-cells derived from human pluripotent stem cells. Other approaches include stimulating endogenous β-cell proliferation, reprogramming non-β-cells to β-like cells, and harvesting islets from genetically engineered animals. Together these approaches form a rich pipeline of therapeutic development for pancreatic regeneration. -
Ma H, Wert KJ, Shvartsman D, Melton DA, Jaenisch R. 2018. Establishment of human pluripotent stem cell-derived pancreatic β-like cells in the mouse pancreas. Proceedings of the National Academy of Sciences of the United States of America. 115(15):3924-3929. Pubmed: 29599125 DOI:10.1073/pnas.1702059115 Ma H, Wert KJ, Shvartsman D, Melton DA, Jaenisch R. 2018. Establishment of human pluripotent stem cell-derived pancreatic β-like cells in the mouse pancreas. Proceedings of the National Academy of Sciences of the United States of America. 115(15):3924-3929. Pubmed: 29599125 DOI:10.1073/pnas.1702059115 Type 1 diabetes is characterized by autoimmune destruction of β cells located in pancreatic islets. However, tractable in vivo models of human pancreatic β cells have been limited. Here, we generated xenogeneic human pancreatic β-like cells in the mouse pancreas by orthotopic transplantation of stem cell-derived β (SC-β) cells into the pancreas of neonatal mice. The engrafted β-like cells expressed β cell transcription factors and markers associated with functional maturity. Engrafted human cells recruited mouse endothelial cells, suggesting functional integration. Human insulin was detected in the blood circulation of transplanted mice for months after transplantation and increased upon glucose stimulation. In addition to β-like cells, human cells expressing markers for other endocrine pancreas cell types, acinar cells, and pancreatic ductal cells were identified in the pancreata of transplanted mice, indicating that this approach allows studying other human pancreatic cell types in the mouse pancreas. Our results demonstrate that orthotopic transplantation of human SC-β cells into neonatal mice is an experimental platform that allows the generation of mice with human pancreatic β-like cells in the endogenous niche. -
Zhou Q, Melton DA. 2018. Author Correction: Pancreas regeneration. Nature. 560(7720):E34. Pubmed: 29925956 DOI:10.1038/s41586-018-0294-9 Zhou Q, Melton DA. 2018. Author Correction: Pancreas regeneration. Nature. 560(7720):E34. Pubmed: 29925956 DOI:10.1038/s41586-018-0294-9 Change history: In this Insight Review, '1989' has been changed to '1998' in the sentence "This deep understanding of pancreatic development was put to the service of regenerative medicine in 1998, when human embryonic stem cells (hES cells) were successfully cultured and opened the door to developing methods of deriving pancreatic islets from hES cells66.". This error has been corrected online. 2016
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Vegas AJ, Veiseh O, Gürtler M, Millman JR, Pagliuca FW, Bader AR, Doloff JC, Li J, Chen M, Olejnik K, Tam HH, Jhunjhunwala S, Langan E, Aresta-Dasilva S, Gandham S, McGarrigle JJ, Bochenek MA, Hollister-Lock J, Oberholzer J, Greiner DL, Weir GC, Melton DA, Langer R, Anderson DG. 2016. Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice. Nature medicine. 22(3):306-11. Pubmed: 26808346 DOI:10.1038/nm.4030 Vegas AJ, Veiseh O, Gürtler M, Millman JR, Pagliuca FW, Bader AR, Doloff JC, Li J, Chen M, Olejnik K, Tam HH, Jhunjhunwala S, Langan E, Aresta-Dasilva S, Gandham S, McGarrigle JJ, Bochenek MA, Hollister-Lock J, Oberholzer J, Greiner DL, Weir GC, Melton DA, Langer R, Anderson DG. 2016. Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice. Nature medicine. 22(3):306-11. Pubmed: 26808346 DOI:10.1038/nm.4030 The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-β cells), which may represent an unlimited source of human cells for pancreas replacement therapy. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier. However, clinical implementation has been challenging because of host immune responses to the implant materials. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-β cells. SC-β cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells. -
Ariyachet C, Tovaglieri A, Xiang G, Lu J, Shah MS, Richmond CA, Verbeke C, Melton DA, Stanger BZ, Mooney D, Shivdasani RA, Mahony S, Xia Q, Breault DT, Zhou Q. 2016. Reprogrammed Stomach Tissue as a Renewable Source of Functional β Cells for Blood Glucose Regulation. Cell stem cell. 18(3):410-21. Pubmed: 26908146 Ariyachet C, Tovaglieri A, Xiang G, Lu J, Shah MS, Richmond CA, Verbeke C, Melton DA, Stanger BZ, Mooney D, Shivdasani RA, Mahony S, Xia Q, Breault DT, Zhou Q. 2016. Reprogrammed Stomach Tissue as a Renewable Source of Functional β Cells for Blood Glucose Regulation. Cell stem cell. 18(3):410-21. Pubmed: 26908146 The gastrointestinal (GI) epithelium is a highly regenerative tissue with the potential to provide a renewable source of insulin(+) cells after undergoing cellular reprogramming. Here, we show that cells of the antral stomach have a previously unappreciated propensity for conversion into functional insulin-secreting cells. Native antral endocrine cells share a surprising degree of transcriptional similarity with pancreatic β cells, and expression of β cell reprogramming factors in vivo converts antral cells efficiently into insulin(+) cells with close molecular and functional similarity to β cells. Induced GI insulin(+) cells can suppress hyperglycemia in a diabetic mouse model for at least 6 months and regenerate rapidly after ablation. Reprogramming of antral stomach cells assembled into bioengineered mini-organs in vitro yielded transplantable units that also suppressed hyperglycemia in diabetic mice, highlighting the potential for development of engineered stomach tissues as a renewable source of functional β cells for glycemic control.Copyright © 2016 Elsevier Inc. All rights reserved. -
Millman JR, Xie C, Van Dervort A, Gürtler M, Pagliuca FW, Melton DA. 2016. Generation of stem cell-derived β-cells from patients with type 1 diabetes. Nature communications. 7:11463. Pubmed: 27163171 DOI:10.1038/ncomms11463 Millman JR, Xie C, Van Dervort A, Gürtler M, Pagliuca FW, Melton DA. 2016. Generation of stem cell-derived β-cells from patients with type 1 diabetes. Nature communications. 7:11463. Pubmed: 27163171 DOI:10.1038/ncomms11463 We recently reported the scalable in vitro production of functional stem cell-derived β-cells (SC-β cells). Here we extend this approach to generate the first SC-β cells from type 1 diabetic patients (T1D). β-cells are destroyed during T1D disease progression, making it difficult to extensively study them in the past. These T1D SC-β cells express β-cell markers, respond to glucose both in vitro and in vivo, prevent alloxan-induced diabetes in mice and respond to anti-diabetic drugs. Furthermore, we use an in vitro disease model to demonstrate the cells respond to different forms of β-cell stress. Using these assays, we find no major differences in T1D SC-β cells compared with SC-β cells derived from non-diabetic patients. These results show that T1D SC-β cells could potentially be used for the treatment of diabetes, drug screening and the study of β-cell biology. -
Millman JR, Xie C, Van Dervort A, Gürtler M, Pagliuca FW, Melton DA. 2016. Corrigendum: Generation of stem cell-derived β-cells from patients with type 1 diabetes. Nature communications. 7:12379. Pubmed: 27487970 DOI:10.1038/ncomms12379 Millman JR, Xie C, Van Dervort A, Gürtler M, Pagliuca FW, Melton DA. 2016. Corrigendum: Generation of stem cell-derived β-cells from patients with type 1 diabetes. Nature communications. 7:12379. Pubmed: 27487970 DOI:10.1038/ncomms12379 -
Melton DA. 2016. Applied Developmental Biology: Making Human Pancreatic Beta Cells for Diabetics. Current topics in developmental biology. 117:65-73. Pubmed: 26969972 Melton DA. 2016. Applied Developmental Biology: Making Human Pancreatic Beta Cells for Diabetics. Current topics in developmental biology. 117:65-73. Pubmed: 26969972 Understanding the genes and signaling pathways that determine the differentiation and fate of a cell is a central goal of developmental biology. Using that information to gain mastery over the fates of cells presents new approaches to cell transplantation and drug discovery for human diseases including diabetes.© 2016 Elsevier Inc. All rights reserved. -
Baron M, Veres A, Wolock SL, Faust AL, Gaujoux R, Vetere A, Ryu JH, Wagner BK, Shen-Orr SS, Klein AM, Melton DA, Yanai I. 2016. A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-cell Population Structure. Cell Syst. 3. DOI:10.1016/j.cels.2016.08.011 Baron M, Veres A, Wolock SL, Faust AL, Gaujoux R, Vetere A, Ryu JH, Wagner BK, Shen-Orr SS, Klein AM, Melton DA, Yanai I. 2016. A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-cell Population Structure. Cell Syst. 3. DOI:10.1016/j.cels.2016.08.011 Although the function of the mammalian pancreas hinges on complex interactions of distinct cell types, gene expression profiles have primarily been described with bulk mixtures. Here we implemented a droplet-based, single-cell RNA-seq method to determine the transcriptomes of over 12,000 individual pancreatic cells from four human donors and two mouse strains. Cells could be divided into 15 clusters that matched previously characterized cell types: all endocrine cell types, including rare epsilon-cells; exocrine cell types; vascular cells; Schwann cells; quiescent and activated stellate cells; and four types of immune cells. We detected subpopulations of ductal cells with distinct expression profiles and validated their existence with immuno-histochemistry stains. Moreover, among human beta- cells, we detected heterogeneity in the regulation of genes relating to functional maturation and levels of ER stress. Finally, we deconvolved bulk gene expression samples using the single-cell data to detect disease-associated differential expression. Our dataset provides a resource for the discovery of novel cell type-specific transcription factors, signaling receptors, and medically relevant genes. Data available in BioStudies: https://www.ebi.ac.uk/biostudies/studies/S-EPMC5228327?xr=true 2015
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Ben-Zvi D, Melton DA. 2015. Modeling human nutrition using human embryonic stem cells. Cell. 161(1):12-17. Pubmed: 25815980 Ben-Zvi D, Melton DA. 2015. Modeling human nutrition using human embryonic stem cells. Cell. 161(1):12-17. Pubmed: 25815980 Nutrition presents unanswered scientific questions of high public health importance. We envision model systems composed of interacting gastrointestinal and metabolic tissues derived from human embryonic stem cells, populated by gut microbiota. The culture will be embedded in 3D scaffolds, creating a controlled experimental system that enables tissue sampling and imaging.Copyright © 2015 Elsevier Inc. All rights reserved. -
Kenty JH, Melton DA. 2015. Testing pancreatic islet function at the single cell level by calcium influx with associated marker expression. PloS one. 10(4):e0122044. Pubmed: 25853429 DOI:10.1371/journal.pone.0122044 Kenty JH, Melton DA. 2015. Testing pancreatic islet function at the single cell level by calcium influx with associated marker expression. PloS one. 10(4):e0122044. Pubmed: 25853429 DOI:10.1371/journal.pone.0122044 Studying the response of islet cells to glucose stimulation is important for understanding cell function in healthy and disease states. Most functional assays are performed on whole islets or cell populations, resulting in averaged observations and loss of information at the single cell level. We demonstrate methods to examine calcium fluxing in individual cells of intact islets in response to multiple glucose challenges. Wild-type mouse islets predominantly contained cells that responded to three (out of three) sequential high glucose challenges, whereas cells of diabetic islets (db/db or NOD) responded less frequently or not at all. Imaged islets were also immunostained for endocrine markers to associate the calcium flux profile of individual cells with gene expression. Wild-type mouse islet cells that robustly fluxed calcium expressed β cell markers (INS/NKX6.1), whereas islet cells that inversely fluxed at low glucose expressed α cell markers (GCG). Diabetic mouse islets showed a higher proportion of dysfunctional β cells that responded poorly to glucose challenges. Most of the failed calcium influx responses in β cells were observed in the second and third high glucose challenges, emphasizing the importance of multiple sequential glucose challenges for assessing the full function of islet cells. Human islet cells were also assessed and showed functional α and β cells. This approach to analyze islet responses to multiple glucose challenges in correlation with gene expression assays expands the understanding of β cell function and the diseased state. -
Chetty S, Engquist EN, Mehanna E, Lui KO, Tsankov AM, Melton DA. 2015. A Src inhibitor regulates the cell cycle of human pluripotent stem cells and improves directed differentiation. The Journal of cell biology. 210(7):1257-68. Pubmed: 26416968 DOI:10.1083/jcb.201502035 Chetty S, Engquist EN, Mehanna E, Lui KO, Tsankov AM, Melton DA. 2015. A Src inhibitor regulates the cell cycle of human pluripotent stem cells and improves directed differentiation. The Journal of cell biology. 210(7):1257-68. Pubmed: 26416968 DOI:10.1083/jcb.201502035 Driving human pluripotent stem cells (hPSCs) into specific lineages is an inefficient and challenging process. We show that a potent Src inhibitor, PP1, regulates expression of genes involved in the G1 to S phase transition of the cell cycle, activates proteins in the retinoblastoma family, and subsequently increases the differentiation propensities of hPSCs into all three germ layers. We further demonstrate that genetic suppression of Src regulates the activity of the retinoblastoma protein and enhances the differentiation potential of hPSCs across all germ layers. These positive effects extend beyond the initial germ layer specification and enable efficient differentiation at subsequent stages of differentiation.© 2015 Chetty et al. -
Ben-Zvi D, Barrandon O, Hadley S, Blum B, Peterson QP, Melton DA. 2015. Angptl4 links α-cell proliferation following glucagon receptor inhibition with adipose tissue triglyceride metabolism. Proceedings of the National Academy of Sciences of the United States of America. 112(50):15498-503. Pubmed: 26621734 DOI:10.1073/pnas.1513872112 Ben-Zvi D, Barrandon O, Hadley S, Blum B, Peterson QP, Melton DA. 2015. Angptl4 links α-cell proliferation following glucagon receptor inhibition with adipose tissue triglyceride metabolism. Proceedings of the National Academy of Sciences of the United States of America. 112(50):15498-503. Pubmed: 26621734 DOI:10.1073/pnas.1513872112 Type 2 diabetes is characterized by a reduction in insulin function and an increase in glucagon activity that together result in hyperglycemia. Glucagon receptor antagonists have been developed as drugs for diabetes; however, they often increase glucagon plasma levels and induce the proliferation of glucagon-secreting α-cells. We find that the secreted protein Angiopoietin-like 4 (Angptl4) is up-regulated via Pparγ activation in white adipose tissue and plasma following an acute treatment with a glucagon receptor antagonist. Induction of adipose angptl4 and Angptl4 supplementation promote α-cell proliferation specifically. Finally, glucagon receptor antagonist improves glycemia in diet-induced obese angptl4 knockout mice without increasing glucagon levels or α-cell proliferation, underscoring the importance of this protein. Overall, we demonstrate that triglyceride metabolism in adipose tissue regulates α-cells in the endocrine pancreas. 2014
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Pagliuca FW, Millman JR, Gürtler M, Segel M, Van Dervort A, Ryu JH, Peterson QP, Greiner D, Melton DA. 2014. Generation of functional human pancreatic β cells in vitro. Cell. 159(2):428-39. Pubmed: 25303535 Pagliuca FW, Millman JR, Gürtler M, Segel M, Van Dervort A, Ryu JH, Peterson QP, Greiner D, Melton DA. 2014. Generation of functional human pancreatic β cells in vitro. Cell. 159(2):428-39. Pubmed: 25303535 The generation of insulin-producing pancreatic β cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However, insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide β cells. Here, we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive β cells from hPSC in vitro. These stem-cell-derived β cells (SC-β) express markers found in mature β cells, flux Ca(2+) in response to glucose, package insulin into secretory granules, and secrete quantities of insulin comparable to adult β cells in response to multiple sequential glucose challenges in vitro. Furthermore, these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner, and transplantation of these cells ameliorates hyperglycemia in diabetic mice.Copyright © 2014 Elsevier Inc. All rights reserved. -
Blum B, Roose AN, Barrandon O, Maehr R, Arvanites AC, Davidow LS, Davis JC, Peterson QP, Rubin LL, Melton DA. 2014. Reversal of β cell de-differentiation by a small molecule inhibitor of the TGFβ pathway. eLife. 3:e02809. Pubmed: 25233132 DOI:10.7554/eLife.02809 Blum B, Roose AN, Barrandon O, Maehr R, Arvanites AC, Davidow LS, Davis JC, Peterson QP, Rubin LL, Melton DA. 2014. Reversal of β cell de-differentiation by a small molecule inhibitor of the TGFβ pathway. eLife. 3:e02809. Pubmed: 25233132 DOI:10.7554/eLife.02809 Dysfunction or death of pancreatic β cells underlies both types of diabetes. This functional decline begins with β cell stress and de-differentiation. Current drugs for type 2 diabetes (T2D) lower blood glucose levels but they do not directly alleviate β cell stress nor prevent, let alone reverse, β cell de-differentiation. We show here that Urocortin 3 (Ucn3), a marker for mature β cells, is down-regulated in the early stages of T2D in mice and when β cells are stressed in vitro. Using an insulin expression-coupled lineage tracer, with Ucn3 as a reporter for the mature β cell state, we screen for factors that reverse β cell de-differentiation. We find that a small molecule inhibitor of TGFβ receptor I (Alk5) protects cells from the loss of key β cell transcription factors and restores a mature β cell identity even after exposure to prolonged and severe diabetes. -
Li W, Nakanishi M, Zumsteg A, Shear M, Wright C, Melton DA, Zhou Q. 2014. In vivo reprogramming of pancreatic acinar cells to three islet endocrine subtypes. eLife. 3:e01846. Pubmed: 24714494 DOI:10.7554/eLife.01846 Li W, Nakanishi M, Zumsteg A, Shear M, Wright C, Melton DA, Zhou Q. 2014. In vivo reprogramming of pancreatic acinar cells to three islet endocrine subtypes. eLife. 3:e01846. Pubmed: 24714494 DOI:10.7554/eLife.01846 Direct lineage conversion of adult cells is a promising approach for regenerative medicine. A major challenge of lineage conversion is to generate specific cell subtypes. The pancreatic islets contain three major hormone-secreting endocrine subtypes: insulin(+) β-cells, glucagon(+) α-cells, and somatostatin(+) δ-cells. We previously reported that a combination of three transcription factors, Ngn3, Mafa, and Pdx1, directly reprograms pancreatic acinar cells to β-cells. We now show that acinar cells can be converted to δ-like and α-like cells by Ngn3 and Ngn3+Mafa respectively. Thus, three major islet endocrine subtypes can be derived by acinar reprogramming. Ngn3 promotes establishment of a generic endocrine state in acinar cells, and also promotes δ-specification in the absence of other factors. δ-specification is in turn suppressed by Mafa and Pdx1 during α- and β-cell induction. These studies identify a set of defined factors whose combinatorial actions reprogram acinar cells to distinct islet endocrine subtypes in vivo. DOI: http://dx.doi.org/10.7554/eLife.01846.001. -
Hrvatin S, O'Donnell CW, Deng F, Millman JR, Pagliuca FW, DiIorio P, Rezania A, Gifford DK, Melton DA. 2014. Differentiated human stem cells resemble fetal, not adult, β cells. Proceedings of the National Academy of Sciences of the United States of America. 111(8):3038-43. Pubmed: 24516164 DOI:10.1073/pnas.1400709111 Hrvatin S, O'Donnell CW, Deng F, Millman JR, Pagliuca FW, DiIorio P, Rezania A, Gifford DK, Melton DA. 2014. Differentiated human stem cells resemble fetal, not adult, β cells. Proceedings of the National Academy of Sciences of the United States of America. 111(8):3038-43. Pubmed: 24516164 DOI:10.1073/pnas.1400709111 Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic β cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS(+)) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS(+) cells and (ii) hPSC-derived INS(+) (hPSC-INS(+)) cells more closely resemble human fetal β cells than adult β cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS(+) cells and true β cells and provides a catalog of genes whose manipulation may convert hPSC-INS(+) cells into functional β cells. -
Hrvatin S, Deng F, O'Donnell CW, Gifford DK, Melton DA. 2014. MARIS: method for analyzing RNA following intracellular sorting. PloS one. 9(3):e89459. Pubmed: 24594682 DOI:10.1371/journal.pone.0089459 Hrvatin S, Deng F, O'Donnell CW, Gifford DK, Melton DA. 2014. MARIS: method for analyzing RNA following intracellular sorting. PloS one. 9(3):e89459. Pubmed: 24594682 DOI:10.1371/journal.pone.0089459 Transcriptional profiling is a key technique in the study of cell biology that is limited by the availability of reagents to uniquely identify specific cell types and isolate high quality RNA from them. We report a Method for Analyzing RNA following Intracellular Sorting (MARIS) that generates high quality RNA for transcriptome profiling following cellular fixation, intracellular immunofluorescent staining and FACS. MARIS can therefore be used to isolate high quality RNA from many otherwise inaccessible cell types simply based on immunofluorescent tagging of unique intracellular proteins. As proof of principle, we isolate RNA from sorted human embryonic stem cell-derived insulin-expressing cells as well as adult human β cells. MARIS is a basic molecular biology technique that could be used across several biological disciplines. 2013
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Carolan PJ, Melton DA. 2013. New findings in pancreatic and intestinal endocrine development to advance regenerative medicine. Current opinion in endocrinology, diabetes, and obesity. 20(1):1-7. Pubmed: 23249759 DOI:10.1097/MED.0b013e32835bc380 Carolan PJ, Melton DA. 2013. New findings in pancreatic and intestinal endocrine development to advance regenerative medicine. Current opinion in endocrinology, diabetes, and obesity. 20(1):1-7. Pubmed: 23249759 DOI:10.1097/MED.0b013e32835bc380 Array -
Chetty S, Pagliuca FW, Honore C, Kweudjeu A, Rezania A, Melton DA. 2013. A simple tool to improve pluripotent stem cell differentiation. Nature methods. 10(6):553-6. Pubmed: 23584186 DOI:10.1038/nmeth.2442 Chetty S, Pagliuca FW, Honore C, Kweudjeu A, Rezania A, Melton DA. 2013. A simple tool to improve pluripotent stem cell differentiation. Nature methods. 10(6):553-6. Pubmed: 23584186 DOI:10.1038/nmeth.2442 We describe a method to help overcome restrictions on the differentiation propensities of human pluripotent stem cells. Culturing pluripotent stem cells in dimethylsulfoxide (DMSO) activates the retinoblastoma protein, increases the proportion of cells in the early G1 phase of the cell cycle and, in more than 25 embryonic and induced pluripotent stem cell lines, improves directed differentiation into multiple lineages. DMSO treatment also improves differentiation into terminal cell types in several cell lines. -
Yi P, Park JS, Melton DA. 2013. Betatrophin: a hormone that controls pancreatic β cell proliferation. Cell. 153(4):747-58. Pubmed: 23623304 Yi P, Park JS, Melton DA. 2013. Betatrophin: a hormone that controls pancreatic β cell proliferation. Cell. 153(4):747-58. Pubmed: 23623304 Replenishing insulin-producing pancreatic β cell mass will benefit both type I and type II diabetics. In adults, pancreatic β cells are generated primarily by self-duplication. We report on a mouse model of insulin resistance that induces dramatic pancreatic β cell proliferation and β cell mass expansion. Using this model, we identify a hormone, betatrophin, that is primarily expressed in liver and fat. Expression of betatrophin correlates with β cell proliferation in other mouse models of insulin resistance and during gestation. Transient expression of betatrophin in mouse liver significantly and specifically promotes pancreatic β cell proliferation, expands β cell mass, and improves glucose tolerance. Thus, betatrophin treatment could augment or replace insulin injections by increasing the number of endogenous insulin-producing cells in diabetics.Copyright © 2013 Elsevier Inc. All rights reserved. -
Pagliuca FW, Melton DA. 2013. How to make a functional β-cell. Development (Cambridge, England). 140(12):2472-83. Pubmed: 23715541 DOI:10.1242/dev.093187 Pagliuca FW, Melton DA. 2013. How to make a functional β-cell. Development (Cambridge, England). 140(12):2472-83. Pubmed: 23715541 DOI:10.1242/dev.093187 Insulin-secreting pancreatic β-cells are essential regulators of mammalian metabolism. The absence of functional β-cells leads to hyperglycemia and diabetes, making patients dependent on exogenously supplied insulin. Recent insights into β-cell development, combined with the discovery of pluripotent stem cells, have led to an unprecedented opportunity to generate new β-cells for transplantation therapy and drug screening. Progress has also been made in converting terminally differentiated cell types into β-cells using transcriptional regulators identified as key players in normal development, and in identifying conditions that induce β-cell replication in vivo and in vitro. Here, we summarize what is currently known about how these strategies could be utilized to generate new β-cells and highlight how further study into the mechanisms governing later stages of differentiation and the acquisition of functional capabilities could inform this effort. -
Tajonar A, Maehr R, Hu G, Sneddon JB, Rivera-Feliciano J, Cohen DE, Elledge SJ, Melton DA. 2013. Brief report: VGLL4 is a novel regulator of survival in human embryonic stem cells. Stem cells (Dayton, Ohio). 31(12):2833-41. Pubmed: 23765749 DOI:10.1002/stem.1445 Tajonar A, Maehr R, Hu G, Sneddon JB, Rivera-Feliciano J, Cohen DE, Elledge SJ, Melton DA. 2013. Brief report: VGLL4 is a novel regulator of survival in human embryonic stem cells. Stem cells (Dayton, Ohio). 31(12):2833-41. Pubmed: 23765749 DOI:10.1002/stem.1445 Human embryonic stem cells (hESCs) are maintained in a self-renewing state by an interconnected network of mechanisms that sustain pluripotency, promote proliferation and survival, and prevent differentiation. We sought to find novel genes that could contribute to one or more of these processes using a gain-of-function screen of a large collection of human open reading frames. We identified Vestigial-like 4 (VGLL4), a cotranscriptional regulator with no previously described function in hESCs, as a positive regulator of survival in hESCs. Specifically, VGLL4 overexpression in hESCs significantly decreases cell death in response to dissociation stress. Additionally, VGLL4 overexpression enhances hESC colony formation from single cells. These effects may be attributable, in part, to a decreased activity of initiator and effector caspases observed in the context of VGLL4 overexpression. Additionally, we show an interaction between VGLL4 and the Rho/Rock pathway, previously implicated in hESC survival. This study introduces a novel gain-of-function approach for studying hESC maintenance and presents VGLL4 as a previously undescribed regulator of this process. Stem Cells 2013;31:2833-2841.© AlphaMed Press. -
Chen AE, Borowiak M, Sherwood RI, Kweudjeu A, Melton DA. 2013. Functional evaluation of ES cell-derived endodermal populations reveals differences between Nodal and Activin A-guided differentiation. Development (Cambridge, England). 140(3):675-86. Pubmed: 23293299 DOI:10.1242/dev.085431 Chen AE, Borowiak M, Sherwood RI, Kweudjeu A, Melton DA. 2013. Functional evaluation of ES cell-derived endodermal populations reveals differences between Nodal and Activin A-guided differentiation. Development (Cambridge, England). 140(3):675-86. Pubmed: 23293299 DOI:10.1242/dev.085431 Embryonic stem (ES) cells hold great promise with respect to their potential to be differentiated into desired cell types. Of interest are organs derived from the definitive endoderm, such as the pancreas and liver, and animal studies have revealed an essential role for Nodal in development of the definitive endoderm. Activin A is a related TGFβ member that acts through many of the same downstream signaling effectors as Nodal and is thought to mimic Nodal activity. Detailed characterization of ES cell-derived endodermal cell types by gene expression analysis in vitro and functional analysis in vivo reveal that, despite their similarity in gene expression, Nodal and Activin-derived endodermal cells exhibit a distinct difference in functional competence following transplantation into the developing mouse embryo. Pdx1-expressing cells arising from the respective endoderm populations exhibit extended differences in their competence to mature into insulin/c-peptide-expressing cells in vivo. Our findings underscore the importance of functional cell-type evaluation during stepwise differentiation of stem cells. 2012
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Annes JP, Ryu JH, Lam K, Carolan PJ, Utz K, Hollister-Lock J, Arvanites AC, Rubin LL, Weir G, Melton DA. 2012. Adenosine kinase inhibition selectively promotes rodent and porcine islet β-cell replication. Proceedings of the National Academy of Sciences of the United States of America. 109(10):3915-20. Pubmed: 22345561 DOI:10.1073/pnas.1201149109 Annes JP, Ryu JH, Lam K, Carolan PJ, Utz K, Hollister-Lock J, Arvanites AC, Rubin LL, Weir G, Melton DA. 2012. Adenosine kinase inhibition selectively promotes rodent and porcine islet β-cell replication. Proceedings of the National Academy of Sciences of the United States of America. 109(10):3915-20. Pubmed: 22345561 DOI:10.1073/pnas.1201149109 Diabetes is a pathological condition characterized by relative insulin deficiency, persistent hyperglycemia, and, consequently, diffuse micro- and macrovascular disease. One therapeutic strategy is to amplify insulin-secretion capacity by increasing the number of the insulin-producing β cells without triggering a generalized proliferative response. Here, we present the development of a small-molecule screening platform for the identification of molecules that increase β-cell replication. Using this platform, we identify a class of compounds [adenosine kinase inhibitors (ADK-Is)] that promote replication of primary β cells in three species (mouse, rat, and pig). Furthermore, the replication effect of ADK-Is is cell type-selective: treatment of islet cell cultures with ADK-Is increases replication of β cells but not that of α cells, PP cells, or fibroblasts. Short-term in vivo treatment with an ADK-I also increases β-cell replication but not exocrine cell or hepatocyte replication. Therefore, we propose ADK inhibition as a strategy for the treatment of diabetes. -
Blum B, Hrvatin S, Schuetz C, Bonal C, Rezania A, Melton DA. 2012. Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3. Nature biotechnology. 30(3):261-4. Pubmed: 22371083 DOI:10.1038/nbt.2141 Blum B, Hrvatin S, Schuetz C, Bonal C, Rezania A, Melton DA. 2012. Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3. Nature biotechnology. 30(3):261-4. Pubmed: 22371083 DOI:10.1038/nbt.2141 Insulin-expressing cells that have been differentiated from human pluripotent stem cells in vitro lack the glucose responsiveness characteristic of mature beta cells. Beta-cell maturation in mice was studied to find genetic markers that enable screens for factors that induce bona fide beta cells in vitro. We find that functional beta-cell maturation is marked by an increase in the glucose threshold for insulin secretion and by expression of the gene urocortin 3. -
Sneddon JB, Borowiak M, Melton DA. 2012. Self-renewal of embryonic-stem-cell-derived progenitors by organ-matched mesenchyme. Nature. 491(7426):765-8. Pubmed: 23041930 DOI:10.1038/nature11463 Sneddon JB, Borowiak M, Melton DA. 2012. Self-renewal of embryonic-stem-cell-derived progenitors by organ-matched mesenchyme. Nature. 491(7426):765-8. Pubmed: 23041930 DOI:10.1038/nature11463 One goal of regenerative medicine, to use stem cells to replace cells lost by injury or disease, depends on producing an excess of the relevant cell for study or transplantation. To this end, the stepwise differentiation of stem cells into specialized derivatives has been successful for some cell types, but a major problem remains the inefficient conversion of cells from one stage of differentiation to the next. If specialized cells are to be produced in large numbers it will be necessary to expand progenitor cells, without differentiation, at some steps of the process. Using the pancreatic lineage as a model for embryonic-stem-cell differentiation, we demonstrate that this is a solvable problem. Co-culture with organ-matched mesenchyme permits proliferation and self-renewal of progenitors, without differentiation, and enables an expansion of more than a million-fold for human endodermal cells with full retention of their developmental potential. This effect is specific both to the mesenchymal cell and to the progenitor being amplified. Progenitors that have been serially expanded on mesenchyme give rise to glucose-sensing, insulin-secreting cells when transplanted in vivo. Theoretically, the identification of stage-specific renewal signals can be incorporated into any scheme for the efficient production of large numbers of differentiated cells from stem cells and may therefore have wide application in regenerative biology. 2011
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Egli D, Chen AE, Saphier G, Ichida J, Fitzgerald C, Go KJ, Acevedo N, Patel J, Baetscher M, Kearns WG, Goland R, Leibel RL, Melton DA, Eggan K. 2011. Reprogramming within hours following nuclear transfer into mouse but not human zygotes. Nature communications. 2:488. Pubmed: 21971503 DOI:10.1038/ncomms1503 Egli D, Chen AE, Saphier G, Ichida J, Fitzgerald C, Go KJ, Acevedo N, Patel J, Baetscher M, Kearns WG, Goland R, Leibel RL, Melton DA, Eggan K. 2011. Reprogramming within hours following nuclear transfer into mouse but not human zygotes. Nature communications. 2:488. Pubmed: 21971503 DOI:10.1038/ncomms1503 Fertilized mouse zygotes can reprogram somatic cells to a pluripotent state. Human zygotes might therefore be useful for producing patient-derived pluripotent stem cells. However, logistical, legal and social considerations have limited the availability of human eggs for research. Here we show that a significant number of normal fertilized eggs (zygotes) can be obtained for reprogramming studies. Using these zygotes, we found that when the zygotic genome was replaced with that of a somatic cell, development progressed normally throughout the cleavage stages, but then arrested before the morula stage. This arrest was associated with a failure to activate transcription in the transferred somatic genome. In contrast to human zygotes, mouse zygotes reprogrammed the somatic cell genome to a pluripotent state within hours after transfer. Our results suggest that there may be a previously unappreciated barrier to successful human nuclear transfer, and that future studies could focus on the requirements for genome activation. -
Cohen DE, Melton D. 2011. Turning straw into gold: directing cell fate for regenerative medicine. Nature reviews. Genetics. 12(4):243-52. Pubmed: 21386864 DOI:10.1038/nrg2938 Cohen DE, Melton D. 2011. Turning straw into gold: directing cell fate for regenerative medicine. Nature reviews. Genetics. 12(4):243-52. Pubmed: 21386864 DOI:10.1038/nrg2938 Regenerative medicine offers the hope that cells for disease research and therapy might be created from readily available sources. To fulfil this promise, the cells available need to be converted into the desired cell types. We review two main approaches to accomplishing this goal: in vitro directed differentiation, which is used to push pluripotent stem cells, including embryonic stem cells or induced pluripotent stem cells, through steps similar to those that occur during embryonic development; and reprogramming (also known as transdifferentiation), in which a differentiated cell is converted directly into the cell of interest without proceeding through a pluripotent intermediate. We analyse the status of progress made using these strategies and highlight challenges that must be overcome to achieve the goal of cell-replacement therapy. -
Melton DA. 2011. Using stem cells to study and possibly treat type 1 diabetes. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 366(1575):2307-11. Pubmed: 21727136 DOI:10.1098/rstb.2011.0019 Melton DA. 2011. Using stem cells to study and possibly treat type 1 diabetes. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 366(1575):2307-11. Pubmed: 21727136 DOI:10.1098/rstb.2011.0019 Stem cells with the potential to form many different cell types are actively studied for their possible use in cell replacement therapies for several diseases. In addition, the differentiated derivatives of stem cells are being used as reagents to test for drugs that slow or correct disease phenotypes found in several degenerative diseases. This paper explores these approaches in the context of type 1 or juvenile diabetes, pointing to recent successes as well as the technical and theoretical challenges that lie ahead in the path to new treatments and cures. 2010
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Niakan KK, Ji H, Maehr R, Vokes SA, Rodolfa KT, Sherwood RI, Yamaki M, Dimos JT, Chen AE, Melton DA, McMahon AP, Eggan K. 2010. Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal. Genes & development. 24(3):312-26. Pubmed: 20123909 DOI:10.1101/gad.1833510 Niakan KK, Ji H, Maehr R, Vokes SA, Rodolfa KT, Sherwood RI, Yamaki M, Dimos JT, Chen AE, Melton DA, McMahon AP, Eggan K. 2010. Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal. Genes & development. 24(3):312-26. Pubmed: 20123909 DOI:10.1101/gad.1833510 In embryonic stem (ES) cells, a well-characterized transcriptional network promotes pluripotency and represses gene expression required for differentiation. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and maintain expression of pluripotency-associated transcription factors, including Oct4, Nanog, and Sox2. In contrast, forced expression of Sox17 down-regulates ES cell-associated gene expression and directly activates genes functioning in differentiation toward an extraembryonic endoderm cell fate. We show these effects of Sox17 on ES cell gene expression are mediated at least in part through a competition between Sox17 and Nanog for common DNA-binding sites. By elaborating the function of Sox17, our results provide insight into how the transcriptional network promoting ES cell self-renewal is interrupted, allowing cellular differentiation. 2009
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Chen AE, Egli D, Niakan K, Deng J, Akutsu H, Yamaki M, Cowan C, Fitz-Gerald C, Zhang K, Melton DA, Eggan K. 2009. Optimal timing of inner cell mass isolation increases the efficiency of human embryonic stem cell derivation and allows generation of sibling cell lines. Cell stem cell. 4(2):103-6. Pubmed: 19200798 DOI:10.1016/j.stem.2008.12.001 Chen AE, Egli D, Niakan K, Deng J, Akutsu H, Yamaki M, Cowan C, Fitz-Gerald C, Zhang K, Melton DA, Eggan K. 2009. Optimal timing of inner cell mass isolation increases the efficiency of human embryonic stem cell derivation and allows generation of sibling cell lines. Cell stem cell. 4(2):103-6. Pubmed: 19200798 DOI:10.1016/j.stem.2008.12.001 -
Chen S, Borowiak M, Fox JL, Maehr R, Osafune K, Davidow L, Lam K, Peng LF, Schreiber SL, Rubin LL, Melton D. 2009. A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nature chemical biology. 5(4):258-65. Pubmed: 19287398 DOI:10.1038/nchembio.154 Chen S, Borowiak M, Fox JL, Maehr R, Osafune K, Davidow L, Lam K, Peng LF, Schreiber SL, Rubin LL, Melton D. 2009. A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nature chemical biology. 5(4):258-65. Pubmed: 19287398 DOI:10.1038/nchembio.154 Stepwise differentiation from embryonic stem cells (ESCs) to functional insulin-secreting beta cells will identify key steps in beta-cell development and may yet prove useful for transplantation therapy for diabetics. An essential step in this schema is the generation of pancreatic progenitors--cells that express Pdx1 and produce all the cell types of the pancreas. High-content chemical screening identified a small molecule, (-)-indolactam V, that induces differentiation of a substantial number of Pdx1-expressing cells from human ESCs. The Pdx1-expressing cells express other pancreatic markers and contribute to endocrine, exocrine and duct cells, in vitro and in vivo. Further analyses showed that (-)-indolactam V works specifically at one stage of pancreatic development, inducing pancreatic progenitors from definitive endoderm. This study describes a chemical screening platform to investigate human ESC differentiation and demonstrates the generation of a cell population that is a key milepost on the path to making beta cells. -
Borowiak M, Melton DA. 2009. How to make beta cells?. Current opinion in cell biology. 21(6):727-32. Pubmed: 19781928 DOI:10.1016/j.ceb.2009.08.006 Borowiak M, Melton DA. 2009. How to make beta cells?. Current opinion in cell biology. 21(6):727-32. Pubmed: 19781928 DOI:10.1016/j.ceb.2009.08.006 Insulin-producing beta cells are lost or insufficient in diabetic patients, presenting the medical challenge for new beta cells. Currently, there are three strategies that offer promise. One involves the generation of beta cells de novo by directing the differentiation of either embryonic stem cells or induced pluripotent cells to the beta cell lineage. The second is based on the conversion of another terminally differentiated cell to beta cells in a process called reprogramming. The third approach is to promote the replication of existing beta cells either in vivo or in vitro. Significant progress is evident for each strategy, but it remains unclear which approach will ultimately prove successful. -
Guseh JS, Bores SA, Stanger BZ, Zhou Q, Anderson WJ, Melton DA, Rajagopal J. 2009. Notch signaling promotes airway mucous metaplasia and inhibits alveolar development. Development (Cambridge, England). 136(10):1751-9. Pubmed: 19369400 DOI:10.1242/dev.029249 Guseh JS, Bores SA, Stanger BZ, Zhou Q, Anderson WJ, Melton DA, Rajagopal J. 2009. Notch signaling promotes airway mucous metaplasia and inhibits alveolar development. Development (Cambridge, England). 136(10):1751-9. Pubmed: 19369400 DOI:10.1242/dev.029249 The airways are conduits that transport atmospheric oxygen to the distal alveolus. Normally, airway mucous cells are rare. However, diseases of the airway are often characterized by mucous metaplasia, in which there are dramatic increases in mucous cell numbers. As the Notch pathway is known to regulate cell fate in many contexts, we misexpressed the active intracellular domain of the mouse Notch1 receptor in lung epithelium. Notch misexpression resulted in an increase in mucous cells and a decrease in ciliated cells in the airway. Similarly, mouse embryonic tracheal explants and adult human airway epithelium treated with Notch agonists displayed increased mucous cell numbers and decreased ciliated cell numbers. Notch antagonists had the opposite effect. Notably, Notch antagonists blocked IL13-induced mucous metaplasia. IL13 has a well-established role as an inflammatory mediator of mucous metaplasia and functions through Stat6-mediated gene transcription. We found that Notch ligands, however, are able to cause mucous metaplasia in Stat6-null cultured trachea, thus identifying a novel pathway that stimulates mucous metaplasia. Notch signaling may therefore play an important role in airway disease and, by extension, Notch antagonists may have therapeutic value. Conversely, in the distal lung, Notch misexpression prevented the differentiation of alveolar cell types. Instead, the distal lung formed cysts composed of cells that were devoid of alveolar markers but that expressed some, but not all, markers of proximal airway epithelium. Occasional distal cystic cells appeared to differentiate into normal proximal airway cells, suggesting that ectopic Notch signaling arrests the normal differentiation of distal lung progenitors before they initiate an alveolar program. -
Maehr R, Chen S, Snitow M, Ludwig T, Yagasaki L, Goland R, Leibel RL, Melton DA. 2009. Generation of pluripotent stem cells from patients with type 1 diabetes. Proceedings of the National Academy of Sciences of the United States of America. 106(37):15768-73. Pubmed: 19720998 DOI:10.1073/pnas.0906894106 Maehr R, Chen S, Snitow M, Ludwig T, Yagasaki L, Goland R, Leibel RL, Melton DA. 2009. Generation of pluripotent stem cells from patients with type 1 diabetes. Proceedings of the National Academy of Sciences of the United States of America. 106(37):15768-73. Pubmed: 19720998 DOI:10.1073/pnas.0906894106 Type 1 diabetes (T1D) is the result of an autoimmune destruction of pancreatic beta cells. The cellular and molecular defects that cause the disease remain unknown. Pluripotent cells generated from patients with T1D would be useful for disease modeling. We show here that induced pluripotent stem (iPS) cells can be generated from patients with T1D by reprogramming their adult fibroblasts with three transcription factors (OCT4, SOX2, KLF4). T1D-specific iPS cells, termed DiPS cells, have the hallmarks of pluripotency and can be differentiated into insulin-producing cells. These results are a step toward using DiPS cells in T1D disease modeling, as well as for cell replacement therapy. -
Borowiak M, Maehr R, Chen S, Chen AE, Tang W, Fox JL, Schreiber SL, Melton DA. 2009. Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells. Cell stem cell. 4(4):348-58. Pubmed: 19341624 DOI:10.1016/j.stem.2009.01.014 Borowiak M, Maehr R, Chen S, Chen AE, Tang W, Fox JL, Schreiber SL, Melton DA. 2009. Small molecules efficiently direct endodermal differentiation of mouse and human embryonic stem cells. Cell stem cell. 4(4):348-58. Pubmed: 19341624 DOI:10.1016/j.stem.2009.01.014 An essential step for therapeutic and research applications of stem cells is the ability to differentiate them into specific cell types. Endodermal cell derivatives, including lung, liver, and pancreas, are of interest for regenerative medicine, but efforts to produce these cells have been met with only modest success. In a screen of 4000 compounds, two cell-permeable small molecules were indentified that direct differentiation of ESCs into the endodermal lineage. These compounds induce nearly 80% of ESCs to form definitive endoderm, a higher efficiency than that achieved by Activin A or Nodal, commonly used protein inducers of endoderm. The chemically induced endoderm expresses multiple endodermal markers, can participate in normal development when injected into developing embryos, and can form pancreatic progenitors. The application of small molecules to differentiate mouse and human ESCs into endoderm represents a step toward achieving a reproducible and efficient production of desired ESC derivatives. -
Brennand K, Melton D. 2009. Slow and steady is the key to beta-cell replication. Journal of cellular and molecular medicine. 13(3):472-87. Pubmed: 19379145 DOI:10.1111/j.1582-4934.2008.00635.x Brennand K, Melton D. 2009. Slow and steady is the key to beta-cell replication. Journal of cellular and molecular medicine. 13(3):472-87. Pubmed: 19379145 DOI:10.1111/j.1582-4934.2008.00635.x The beta-cells of the pancreas are responsible for insulin production and their destruction results in type I diabetes. beta-cell maintenance, growth and regenerative repair is thought to occur predominately, if not exclusively, through the replication of existing beta-cells, not via an adult stem cell. It was recently found that all beta-cells contribute equally to islet growth and maintenance. The fact that all beta-cells replicate homogeneously makes it possible to set up straightforward screens for factors that increase beta-cell replication either In vitro or in vivo. It is possible that a circulating factor may be capable of increasing beta-cell replication or that intrinsic cell cycle regulators may affect beta-cell growth. An improved understanding of the in vivo maintenance and growth of beta-cells will facilitate efforts to expand beta-cells In vitro and may lead to new treatments for diabetes. -
Sherwood RI, Chen TY, Melton DA. 2009. Transcriptional dynamics of endodermal organ formation. Developmental dynamics : an official publication of the American Association of Anatomists. 238(1):29-42. Pubmed: 19097184 DOI:10.1002/dvdy.21810 Sherwood RI, Chen TY, Melton DA. 2009. Transcriptional dynamics of endodermal organ formation. Developmental dynamics : an official publication of the American Association of Anatomists. 238(1):29-42. Pubmed: 19097184 DOI:10.1002/dvdy.21810 Although endodermal organs including the liver, pancreas, and intestine are of significant therapeutic interest, the mechanism by which the endoderm is divided into organ domains during embryogenesis is not well understood. To better understand this process, global gene expression profiling was performed on early endodermal organ domains. This global analysis was followed up by dynamic immunofluorescence analysis of key transcription factors, uncovering novel expression patterns as well as cell surface proteins that allow prospective isolation of specific endodermal organ domains. Additionally, a repressive interaction between Cdx2 and Sox2 was found to occur at the prospective stomach-intestine border, with the hepatic and pancreatic domains forming at this boundary, and Hlxb9 was revealed to have graded expression along the dorsal-ventral axis. These results contribute to understanding the mechanism of endodermal organogenesis and should assist efforts to replicate this process using pluripotent stem cells.Copyright (c) 2008 Wiley-Liss, Inc. 2008
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Huangfu D, Osafune K, Maehr R, Guo W, Eijkelenboom A, Chen S, Muhlestein W, Melton DA. 2008. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nature biotechnology. 26(11):1269-75. Pubmed: 18849973 DOI:10.1038/nbt.1502 Huangfu D, Osafune K, Maehr R, Guo W, Eijkelenboom A, Chen S, Muhlestein W, Melton DA. 2008. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nature biotechnology. 26(11):1269-75. Pubmed: 18849973 DOI:10.1038/nbt.1502 Ectopic expression of defined sets of genetic factors can reprogram somatic cells to induced pluripotent stem (iPS) cells that closely resemble embryonic stem (ES) cells. The low efficiency with which iPS cells are derived hinders studies on the molecular mechanism of reprogramming, and integration of viral transgenes, in particular the oncogenes c-Myc and Klf4, may handicap this method for human therapeutic applications. Here we report that valproic acid (VPA), a histone deacetylase inhibitor, enables reprogramming of primary human fibroblasts with only two factors, Oct4 and Sox2, without the need for the oncogenes c-Myc or Klf4. The two factor-induced human iPS cells resemble human ES cells in pluripotency, global gene expression profiles and epigenetic states. These results support the possibility of reprogramming through purely chemical means, which would make therapeutic use of reprogrammed cells safer and more practical. -
Osafune K, Caron L, Borowiak M, Martinez RJ, Fitz-Gerald CS, Sato Y, Cowan CA, Chien KR, Melton DA. 2008. Marked differences in differentiation propensity among human embryonic stem cell lines. Nature biotechnology. 26(3):313-5. Pubmed: 18278034 DOI:10.1038/nbt1383 Osafune K, Caron L, Borowiak M, Martinez RJ, Fitz-Gerald CS, Sato Y, Cowan CA, Chien KR, Melton DA. 2008. Marked differences in differentiation propensity among human embryonic stem cell lines. Nature biotechnology. 26(3):313-5. Pubmed: 18278034 DOI:10.1038/nbt1383 The differentiation potential of 17 human embryonic stem (hES) cell lines was compared. Some lines exhibit a marked propensity to differentiate into specific lineages, often with >100-fold differences in lineage-specific gene expression. For example, HUES 8 is best for pancreatic differentiation and HUES 3 for cardiomyocyte generation. These non-trivial differences in developmental potential among hES cell lines point to the importance of screening and deriving lines for lineage-specific differentiation. -
Huangfu D, Maehr R, Guo W, Eijkelenboom A, Snitow M, Chen AE, Melton DA. 2008. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nature biotechnology. 26(7):795-7. Pubmed: 18568017 DOI:10.1038/nbt1418 Huangfu D, Maehr R, Guo W, Eijkelenboom A, Snitow M, Chen AE, Melton DA. 2008. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nature biotechnology. 26(7):795-7. Pubmed: 18568017 DOI:10.1038/nbt1418 Reprogramming of mouse and human somatic cells can be achieved by ectopic expression of transcription factors, but with low efficiencies. We report that DNA methyltransferase and histone deacetylase (HDAC) inhibitors improve reprogramming efficiency. In particular, valproic acid (VPA), an HDAC inhibitor, improves reprogramming efficiency by more than 100-fold, using Oct4-GFP as a reporter. VPA also enables efficient induction of pluripotent stem cells without introduction of the oncogene c-Myc. -
Anderson WJ, Zhou Q, Alcalde V, Kaneko OF, Blank LJ, Sherwood RI, Guseh JS, Rajagopal J, Melton DA. 2008. Genetic targeting of the endoderm with claudin-6CreER. Developmental dynamics : an official publication of the American Association of Anatomists. 237(2):504-12. Pubmed: 18213590 DOI:10.1002/dvdy.21437 Anderson WJ, Zhou Q, Alcalde V, Kaneko OF, Blank LJ, Sherwood RI, Guseh JS, Rajagopal J, Melton DA. 2008. Genetic targeting of the endoderm with claudin-6CreER. Developmental dynamics : an official publication of the American Association of Anatomists. 237(2):504-12. Pubmed: 18213590 DOI:10.1002/dvdy.21437 A full description of the ontogeny of the beta cell would guide efforts to generate beta cells from embryonic stem cells (ESCs). The first step requires an understanding of definitive endoderm: the genes and signals responsible for its specification, proliferation, and patterning. This report describes a global marker of definitive endoderm, Claudin-6 (Cldn6). We report its expression in early development with particular attention to definitive endoderm derivatives. To create a genetic system to drive gene expression throughout the definitive endoderm with both spatial and temporal control, we target the endogenous locus with an inducible Cre recombinase (Cre-ER(T2)) cassette. Cldn6 null mice are viable and fertile with no obvious phenotypic abnormalities. We also report a lineage analysis of the fate of Cldn6-expressing embryonic cells, which is relevant to the development of the pancreas, lung, and liver. -
Rajagopal J, Carroll TJ, Guseh JS, Bores SA, Blank LJ, Anderson WJ, Yu J, Zhou Q, McMahon AP, Melton DA. 2008. Wnt7b stimulates embryonic lung growth by coordinately increasing the replication of epithelium and mesenchyme. Development (Cambridge, England). 135(9):1625-34. Pubmed: 18367557 DOI:10.1242/dev.015495 Rajagopal J, Carroll TJ, Guseh JS, Bores SA, Blank LJ, Anderson WJ, Yu J, Zhou Q, McMahon AP, Melton DA. 2008. Wnt7b stimulates embryonic lung growth by coordinately increasing the replication of epithelium and mesenchyme. Development (Cambridge, England). 135(9):1625-34. Pubmed: 18367557 DOI:10.1242/dev.015495 The effects of Wnt7b on lung development were examined using a conditional Wnt7b-null mouse. Wnt7b-null lungs are markedly hypoplastic, yet display largely normal patterning and cell differentiation. In contrast to findings in prior hypomorphic Wnt7b models, we find decreased replication of both developing epithelium and mesenchyme, without abnormalities of vascular smooth muscle development. We further demonstrate that Wnt7b signals to neighboring cells to activate both autocrine and paracrine canonical Wnt signaling cascades. In contrast to results from hypomorphic models, we show that Wnt7b modulates several important signaling pathways in the lung. Together, these cascades result in the coordinated proliferation of adjacent epithelial and mesenchymal cells to stimulate organ growth with few alterations in differentiation and patterning. -
Zhou Q, Melton DA. 2008. Pathways to new beta cells. Cold Spring Harbor symposia on quantitative biology. 73:175-81. Pubmed: 19478324 DOI:10.1101/sqb.2008.73.002 Zhou Q, Melton DA. 2008. Pathways to new beta cells. Cold Spring Harbor symposia on quantitative biology. 73:175-81. Pubmed: 19478324 DOI:10.1101/sqb.2008.73.002 Diabetes is a leading health problem of the world and its prevalence continues to rise. With Type I diabetes, and in some patients with Type II, the lack of insulin can be counterbalanced by providing new beta (insulin-producing) cells. For Type I diabetes, treating the autoimmune attack remains a serious challenge. Several strategies to produce new beta cells have been proposed. These include differentiation from embryonic stem cells, proliferation of existing adult beta cells, derivation from putative adult progenitors/stem cells, and reprogramming of non-beta cells to beta cells. Each of these strategies has distinct merits and risks, and they are at different stages of understanding and development. In particular, the approach based on differentiation from embryonic stem cells has had strong support and in recent years has made notable progress. Nevertheless, significant hurdles remain to transform the current research into future therapies. To expedite this transformation, we believe particular emphasis should be placed on overcoming key knowledge gaps in beta-cell biology, developing strategies that produce patient-specific beta cells, and carefully addressing potential treatment-related complications or limitations. 2007
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Greenwood AL, Li S, Jones K, Melton DA. 2007. Notch signaling reveals developmental plasticity of Pax4(+) pancreatic endocrine progenitors and shunts them to a duct fate. Mechanisms of development. 124(2):97-107. Pubmed: 17196797 Greenwood AL, Li S, Jones K, Melton DA. 2007. Notch signaling reveals developmental plasticity of Pax4(+) pancreatic endocrine progenitors and shunts them to a duct fate. Mechanisms of development. 124(2):97-107. Pubmed: 17196797 Relatively little is known about the developmental signals that specify the types and numbers of pancreatic cells. Previous studies suggested that Notch signaling in the pancreas inhibits differentiation and promotes the maintenance of progenitor cells, but it remains unclear whether Notch also controls cell fate choices as it does in other tissues. To study the impact of Notch in progenitors of the beta cell lineage, we generated mice that express Cre-recombinase under control of the Pax4 promoter. Lineage analysis of Pax4(+) cells demonstrates they are specified endocrine progenitors that contribute equally to four islet cell fates, contrary to expectations raised by the dispensable role of Pax4 in the specification of the alpha and PP subtypes. In addition, we show that activation of Notch in Pax4(+) progenitors inhibits their differentiation into alpha and beta endocrine cells and shunts them instead toward a duct fate. These observations reveal an unappreciated degree of developmental plasticity among early endocrine progenitors and raise the possibility that a bipotent duct-endocrine progenitor exists during development. Furthermore, the redirection of Pax4(+) cells from alpha and beta endocrine fates toward a duct cell type suggests a positive role for Notch signaling in duct specification and is consistent with the more widely defined role for Notch in cell fate determination. -
Brennand K, Huangfu D, Melton D. 2007. All beta cells contribute equally to islet growth and maintenance. PLoS biology. 5(7):e163. Pubmed: 17535113 Brennand K, Huangfu D, Melton D. 2007. All beta cells contribute equally to islet growth and maintenance. PLoS biology. 5(7):e163. Pubmed: 17535113 In healthy adult mice, the beta cell population is not maintained by stem cells but instead by the replication of differentiated beta cells. It is not known, however, whether all beta cells contribute equally to growth and maintenance, as it may be that some cells replicate while others do not. Understanding precisely which cells are responsible for beta cell replication will inform attempts to expand beta cells in vitro, a potential source for cell replacement therapy to treat diabetes. Two experiments were performed to address this issue. First, the level of fluorescence generated by a pulse of histone 2B-green fluorescent protein (H2BGFP) expression was followed over time to determine how this marker is diluted with cell division; a uniform loss of label across the entire beta cell population was observed. Second, clonal analysis of dividing beta cells was completed; all clones were of comparable size. These results support the conclusion that the beta cell pool is homogeneous with respect to replicative capacity and suggest that all beta cells are candidates for in vitro expansion. Given similar observations in the hepatocyte population, we speculate that for tissues lacking an adult stem cell, they are replenished equally by replication of all differentiated cells. -
Sherwood RI, Jitianu C, Cleaver O, Shaywitz DA, Lamenzo JO, Chen AE, Golub TR, Melton DA. 2007. Prospective isolation and global gene expression analysis of definitive and visceral endoderm. Developmental biology. 304(2):541-55. Pubmed: 17328885 Sherwood RI, Jitianu C, Cleaver O, Shaywitz DA, Lamenzo JO, Chen AE, Golub TR, Melton DA. 2007. Prospective isolation and global gene expression analysis of definitive and visceral endoderm. Developmental biology. 304(2):541-55. Pubmed: 17328885 In spite of the therapeutic importance of endoderm derivatives such as the pancreas, liver, lung, and intestine, there are few molecular markers specific for early endoderm. In order to identify endoderm-specific genes as well as to define transcriptional differences between definitive and visceral endoderm, we performed microarray analysis on E8.25 definitive and visceral endoderm. We have developed an early endoderm gene expression signature, and clarified the transcriptional similarities and differences between definitive and visceral endoderm. Additionally, we have developed methods for flow cytometric isolation of definitive and visceral endoderm. These results shed light on the mechanism of endoderm formation and should facilitate investigation of endoderm formation from embryonic stem cells. -
Zhou Q, Law AC, Rajagopal J, Anderson WJ, Gray PA, Melton DA. 2007. A multipotent progenitor domain guides pancreatic organogenesis. Developmental cell. 13(1):103-14. Pubmed: 17609113 Zhou Q, Law AC, Rajagopal J, Anderson WJ, Gray PA, Melton DA. 2007. A multipotent progenitor domain guides pancreatic organogenesis. Developmental cell. 13(1):103-14. Pubmed: 17609113 The mammalian pancreas is constructed during embryogenesis by multipotent progenitors, the identity and function of which remain poorly understood. We performed genome-wide transcription factor expression analysis of the developing pancreas to identify gene expression domains that may represent distinct progenitor cell populations. Five discrete domains were discovered. Genetic lineage-tracing experiments demonstrate that one specific domain, located at the tip of the branching pancreatic tree, contains multipotent progenitors that produce exocrine, endocrine, and duct cells in vivo. These multipotent progenitors are Pdx1(+)Ptf1a(+)cMyc(High)Cpa1(+) and negative for differentiated lineage markers. The outgrowth of multipotent tip cells leaves behind differentiated progeny that form the trunk of the branches. These findings define a multipotent compartment within the developing pancreas and suggest a model of how branching is coordinated with cell type specification. In addition, this comprehensive analysis of >1,100 transcription factors identified genes that are likely to control critical decisions in pancreas development and disease. -
Stanger BZ, Tanaka AJ, Melton DA. 2007. Organ size is limited by the number of embryonic progenitor cells in the pancreas but not the liver. Nature. 445(7130):886-91. Pubmed: 17259975 Stanger BZ, Tanaka AJ, Melton DA. 2007. Organ size is limited by the number of embryonic progenitor cells in the pancreas but not the liver. Nature. 445(7130):886-91. Pubmed: 17259975 The determinants of vertebrate organ size are poorly understood, but the process is thought to depend heavily on growth factors and other environmental cues. In the blood and central nervous system, for example, organ mass is determined primarily by growth-factor-regulated cell proliferation and apoptosis to achieve a final target size. Here, we report that the size of the mouse pancreas is constrained by an intrinsic programme established early in development, one that is essentially not subject to growth compensation. Specifically, final pancreas size is limited by the size of the progenitor cell pool that is set aside in the developing pancreatic bud. By contrast, the size of the liver is not constrained by reductions in the progenitor cell pool. These findings show that progenitor cell number, independently of regulation by growth factors, can be a key determinant of organ size. 2006
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Akutsu H, Cowan CA, Melton D. 2006. Human embryonic stem cells. Methods in enzymology. 418:78-92. Pubmed: 17141030 Akutsu H, Cowan CA, Melton D. 2006. Human embryonic stem cells. Methods in enzymology. 418:78-92. Pubmed: 17141030 Human embryonic stem cells hold great promise in furthering our treatment of disease and increasing our understanding of early development. This chapter describes protocols for the derivation and maintenance of human embryonic stem cells. In addition, it summarizes briefly several alternative methods for the culture of human embryonic stem cells. Thus, this chapter provides a good starting point for researchers interested in harnessing the potential of human embryonic stem cells. -
Nikolova G, Jabs N, Konstantinova I, Domogatskaya A, Tryggvason K, Sorokin L, Fässler R, Gu G, Gerber HP, Ferrara N, Melton DA, Lammert E. 2006. The vascular basement membrane: a niche for insulin gene expression and Beta cell proliferation. Developmental cell. 10(3):397-405. Pubmed: 16516842 Nikolova G, Jabs N, Konstantinova I, Domogatskaya A, Tryggvason K, Sorokin L, Fässler R, Gu G, Gerber HP, Ferrara N, Melton DA, Lammert E. 2006. The vascular basement membrane: a niche for insulin gene expression and Beta cell proliferation. Developmental cell. 10(3):397-405. Pubmed: 16516842 Endocrine pancreatic beta cells require endothelial signals for their differentiation and function. However, the molecular basis for such signals remains unknown. Here, we show that beta cells, in contrast to the exocrine pancreatic cells, do not form a basement membrane. Instead, by using VEGF-A, they attract endothelial cells, which form capillaries with a vascular basement membrane next to the beta cells. We have identified laminins, among other vascular basement membrane proteins, as endothelial signals, which promote insulin gene expression and proliferation in beta cells. We further demonstrate that beta1-integrin is required for the beta cell response to the laminins. The proposed mechanism explains why beta cells must interact with endothelial cells, and it may apply to other cellular processes in which endothelial signals are required. -
Melton DA. 2006. Reversal of type 1 diabetes in mice. The New England journal of medicine. 355(1):89-90. Pubmed: 16823003 Melton DA. 2006. Reversal of type 1 diabetes in mice. The New England journal of medicine. 355(1):89-90. Pubmed: 16823003 2005
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Cowan CA, Atienza J, Melton DA, Eggan K. 2005. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science (New York, N.Y.). 309(5739):1369-73. Pubmed: 16123299 Cowan CA, Atienza J, Melton DA, Eggan K. 2005. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science (New York, N.Y.). 309(5739):1369-73. Pubmed: 16123299 We have explored the use of embryonic stem cells as an alternative to oocytes for reprogramming human somatic nuclei. Human embryonic stem (hES) cells were fused with human fibroblasts, resulting in hybrid cells that maintain a stable tetraploid DNA content and have morphology, growth rate, and antigen expression patterns characteristic of hES cells. Differentiation of hybrid cells in vitro and in vivo yielded cell types from each embryonic germ layer. Analysis of genome-wide transcriptional activity, reporter gene activation, allele-specific gene expression, and DNA methylation showed that the somatic genome was reprogrammed to an embryonic state. These results establish that hES cells can reprogram the transcriptional state of somatic nuclei and provide a system for investigating the underlying mechanisms. -
Murtaugh LC, Law AC, Dor Y, Melton DA. 2005. Beta-catenin is essential for pancreatic acinar but not islet development. Development (Cambridge, England). 132(21):4663-74. Pubmed: 16192304 Murtaugh LC, Law AC, Dor Y, Melton DA. 2005. Beta-catenin is essential for pancreatic acinar but not islet development. Development (Cambridge, England). 132(21):4663-74. Pubmed: 16192304 Despite our increasingly sophisticated understanding of transcriptional regulation in pancreas development, we know relatively little about the extrinsic signaling pathways involved in this process. We show here that the early pancreatic epithelium exhibits a specific enrichment in unphosphorylated beta-catenin protein, a hallmark of activation of the canonical Wnt signaling pathway. To determine if this pathway is functionally required for normal pancreas development, we have specifically deleted the beta-catenin gene in these cells. Pancreata developing without beta-catenin are hypoplastic, although their early progenitors appear normal and exhibit no premature differentiation or death. Surprisingly, and in marked contrast to its role in the intestine, loss of beta-catenin does not significantly perturb islet endocrine cell mass or function. The major defect of the beta-catenin-deficient pancreas is an almost complete lack of acinar cells, which normally comprise the majority of the organ. beta-Catenin appears to be cell-autonomously required for the specification of acinar cells, rather than for their survival or maintenance, as deletion of beta-catenin specifically in differentiated acinar cells has no effect. Thus, our data are consistent with a crucial role for canonical Wnt signals in acinar lineage specification and differentiation. -
Stanger BZ, Stiles B, Lauwers GY, Bardeesy N, Mendoza M, Wang Y, Greenwood A, Cheng KH, McLaughlin M, Brown D, Depinho RA, Wu H, Melton DA, Dor Y. 2005. Pten constrains centroacinar cell expansion and malignant transformation in the pancreas. Cancer cell. 8(3):185-95. Pubmed: 16169464 Stanger BZ, Stiles B, Lauwers GY, Bardeesy N, Mendoza M, Wang Y, Greenwood A, Cheng KH, McLaughlin M, Brown D, Depinho RA, Wu H, Melton DA, Dor Y. 2005. Pten constrains centroacinar cell expansion and malignant transformation in the pancreas. Cancer cell. 8(3):185-95. Pubmed: 16169464 To determine the role of the phosphatidylinositol 3-kinase (PI3-K) pathway in pancreas development, we generated a pancreas-specific knockout of Pten, a negative regulator of PI3-K signaling. Knockout mice display progressive replacement of the acinar pancreas with highly proliferative ductal structures that contain abundant mucins and express Pdx1 and Hes1, two markers of pancreatic progenitor cells. Moreover, a fraction of these mice develop ductal malignancy. We provide evidence that ductal metaplasia results from the expansion of centroacinar cells rather than transdifferentiation of acinar cells. These results indicate that Pten actively maintains the balance between different cell types in the adult pancreas and that misregulation of the PI3-K pathway in centroacinar cells may contribute to the initiation of pancreatic carcinoma in vivo. -
Stanger BZ, Datar R, Murtaugh LC, Melton DA. 2005. Direct regulation of intestinal fate by Notch. Proceedings of the National Academy of Sciences of the United States of America. 102(35):12443-8. Pubmed: 16107537 Stanger BZ, Datar R, Murtaugh LC, Melton DA. 2005. Direct regulation of intestinal fate by Notch. Proceedings of the National Academy of Sciences of the United States of America. 102(35):12443-8. Pubmed: 16107537 The signals that maintain the proper balance between adult intestinal cell types are poorly understood. Loss-of-function studies have implicated the Notch pathway in the regulation of intestinal fate during development. However, it is unknown whether Notch has a role in maintaining the balance of different cell types in the adult intestine and whether it acts reversibly. To determine whether Notch has a direct effect on intestinal development and adult intestinal cell turnover, we have used a gain-of-function approach to activate Notch. Ectopic Notch signaling in adult intestinal progenitor cells leads to a bias against secretory fates, whereas ectopic Notch activation in the embryonic foregut results in reversible defects in villus morphogenesis and loss of the proliferative progenitor compartment. We conclude that Notch regulates adult intestinal development by controlling the balance between secretory and absorptive cell types. In the embryo, Notch activation perturbs morphogenesis, possibly through effects on stem or progenitor cells. 2004
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Gu G, Wells JM, Dombkowski D, Preffer F, Aronow B, Melton DA. 2004. Global expression analysis of gene regulatory pathways during endocrine pancreatic development. Development (Cambridge, England). 131(1):165-79. Pubmed: 14660441 Gu G, Wells JM, Dombkowski D, Preffer F, Aronow B, Melton DA. 2004. Global expression analysis of gene regulatory pathways during endocrine pancreatic development. Development (Cambridge, England). 131(1):165-79. Pubmed: 14660441 To define genetic pathways that regulate development of the endocrine pancreas, we generated transcriptional profiles of enriched cells isolated from four biologically significant stages of endocrine pancreas development: endoderm before pancreas specification, early pancreatic progenitor cells, endocrine progenitor cells and adult islets of Langerhans. These analyses implicate new signaling pathways in endocrine pancreas development, and identified sets of known and novel genes that are temporally regulated, as well as genes that spatially define developing endocrine cells from their neighbors. The differential expression of several genes from each time point was verified by RT-PCR and in situ hybridization. Moreover, we present preliminary functional evidence suggesting that one transcription factor encoding gene (Myt1), which was identified in our screen, is expressed in endocrine progenitors and may regulate alpha, beta and delta cell development. In addition to identifying new genes that regulate endocrine cell fate, this global gene expression analysis has uncovered informative biological trends that occur during endocrine differentiation. -
Melton DA, Daley GQ, Jennings CG. 2004. Altered nuclear transfer in stem-cell research - a flawed proposal. The New England journal of medicine. 351(27):2791-2. Pubmed: 15625329 Melton DA, Daley GQ, Jennings CG. 2004. Altered nuclear transfer in stem-cell research - a flawed proposal. The New England journal of medicine. 351(27):2791-2. Pubmed: 15625329 -
Dor Y, Melton DA. 2004. How important are adult stem cells for tissue maintenance?. Cell cycle (Georgetown, Tex.). 3(9):1104-6. Pubmed: 15326371 Dor Y, Melton DA. 2004. How important are adult stem cells for tissue maintenance?. Cell cycle (Georgetown, Tex.). 3(9):1104-6. Pubmed: 15326371 Most tissues undergo significant expansion during postnatal life, as well as a lifelong cellular turnover to compensate for cell loss. What is the identity of cells that give rise to newly differentiated cells? This fundamental yet understudied question is now attracting the attention of biologists and clinicians, triggered by reports that essentially every tissue can be replenished and repaired by multipotent adult stem cells. The identification of the "cell of origin" for a given tissue is essential for understanding its dynamics during adult life, and may have important therapeutic implications for both degenerative and neoplastic diseases. In this commentary, we briefly outline classic and current views on the question of the cell of origin. We also describe a general method that we have recently developed for addressing this issue, and its first application for the study of pancreatic beta cells. -
Dor Y, Brown J, Martinez OI, Melton DA. 2004. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature. 429(6987):41-6. Pubmed: 15129273 Dor Y, Brown J, Martinez OI, Melton DA. 2004. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature. 429(6987):41-6. Pubmed: 15129273 How tissues generate and maintain the correct number of cells is a fundamental problem in biology. In principle, tissue turnover can occur by the differentiation of stem cells, as is well documented for blood, skin and intestine, or by the duplication of existing differentiated cells. Recent work on adult stem cells has highlighted their potential contribution to organ maintenance and repair. However, the extent to which stem cells actually participate in these processes in vivo is not clear. Here we introduce a method for genetic lineage tracing to determine the contribution of stem cells to a tissue of interest. We focus on pancreatic beta-cells, whose postnatal origins remain controversial. Our analysis shows that pre-existing beta-cells, rather than pluripotent stem cells, are the major source of new beta-cells during adult life and after pancreatectomy in mice. These results suggest that terminally differentiated beta-cells retain a significant proliferative capacity in vivo and cast doubt on the idea that adult stem cells have a significant role in beta-cell replenishment. -
Cowan CA, Klimanskaya I, McMahon J, Atienza J, Witmyer J, Zucker JP, Wang S, Morton CC, McMahon AP, Powers D, Melton DA. 2004. Derivation of embryonic stem-cell lines from human blastocysts. The New England journal of medicine. 350(13):1353-6. Pubmed: 14999088 Cowan CA, Klimanskaya I, McMahon J, Atienza J, Witmyer J, Zucker JP, Wang S, Morton CC, McMahon AP, Powers D, Melton DA. 2004. Derivation of embryonic stem-cell lines from human blastocysts. The New England journal of medicine. 350(13):1353-6. Pubmed: 14999088 -
Annerén C, Cowan CA, Melton DA. 2004. The Src family of tyrosine kinases is important for embryonic stem cell self-renewal. The Journal of biological chemistry. 279(30):31590-8. Pubmed: 15148312 Annerén C, Cowan CA, Melton DA. 2004. The Src family of tyrosine kinases is important for embryonic stem cell self-renewal. The Journal of biological chemistry. 279(30):31590-8. Pubmed: 15148312 cYes, a member of the Src family of non-receptor tyrosine kinases, is highly expressed in mouse and human embryonic stem (ES) cells. We demonstrate that cYes kinase activity is regulated by leukemia inhibitory factor (LIF) and serum and is down-regulated when cells differentiate. Moreover, selective chemical inhibition of Src family kinases decreases growth and expression of stem cell genes that mark the undifferentiated state, including Oct3/4, alkaline phosphatase, fibroblast growth factor 4, and Nanog. A synergistic effect on differentiation is observed when ES cells are cultured with an Src family inhibitor and low levels of retinoic acid. Src family kinase inhibition does not interfere with LIF-induced JAK/STAT3 (Janus-associated tyrosine kinases/signal transducer and activator of transcription 3) or p42/p44 MAPK (mitogen-activated protein kinase) phosphorylation. Together the results suggest that the activation of the Src family is important for maintaining mouse and human ES in an undifferentiated state and may represent a third, independent pathway, downstream of LIF in mouse ES cells. 2003
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Murtaugh LC, Stanger BZ, Kwan KM, Melton DA. 2003. Notch signaling controls multiple steps of pancreatic differentiation. Proceedings of the National Academy of Sciences of the United States of America. 100(25):14920-5. Pubmed: 14657333 Murtaugh LC, Stanger BZ, Kwan KM, Melton DA. 2003. Notch signaling controls multiple steps of pancreatic differentiation. Proceedings of the National Academy of Sciences of the United States of America. 100(25):14920-5. Pubmed: 14657333 Multiple cell types of the pancreas appear asynchronously during embryogenesis, which requires that pancreatic progenitor cell potential changes over time. Loss-of-function studies have shown that Notch signaling modulates the differentiation of these progenitors, but it remains unclear how and when the Notch pathway acts. We established a modular transgenic system to heritably activate mouse Notch1 in multiple types of progenitors and differentiated cells. We find that misexpression of activated Notch in Pdx1-expressing progenitor cells prevents differentiation of both exocrine and endocrine lineages. Progenitors remain trapped in an undifferentiated state even if Notch activation occurs long after the pancreas has been specified. Furthermore, endocrine differentiation is associated with escape from this activity, because Ngn3-expressing endocrine precursors are susceptible to Notch inhibition, whereas fully differentiated endocrine cells are resistant. -
Gu G, Brown JR, Melton DA. 2003. Direct lineage tracing reveals the ontogeny of pancreatic cell fates during mouse embryogenesis. Mechanisms of development. 120(1):35-43. Pubmed: 12490294 Gu G, Brown JR, Melton DA. 2003. Direct lineage tracing reveals the ontogeny of pancreatic cell fates during mouse embryogenesis. Mechanisms of development. 120(1):35-43. Pubmed: 12490294 Lineage tracing follows the progeny of labeled cells through development. This technique identifies precursors of mature cell types in vivo and describes the cell fate restriction steps they undergo in temporal order. In the mouse pancreas, direct cell lineage tracing reveals that Pdx1- expressing progenitors in the early embryo give rise to all pancreatic cells. The progenitors for the mature pancreatic ducts separate from the endocrine/exocrine tissues before E12.5. Expression of Ngn3 and pancreatic polypeptide marks endocrine cell lineages during early embryogenesis, and these cells behave as transient progenitors rather than stem cells. In adults, Ngn3 is expressed within the endocrine islets, and the NGN3+ cells seem to contribute to pancreatic islet renewal. These results indicate the stage at which each progenitor population is restricted to a particular fate and provide markers for isolating progenitors to study their growth, differentiation, and the genes necessary for their development. -
Lammert E, Cleaver O, Melton D. 2003. Role of endothelial cells in early pancreas and liver development. Mechanisms of development. 120(1):59-64. Pubmed: 12490296 Lammert E, Cleaver O, Melton D. 2003. Role of endothelial cells in early pancreas and liver development. Mechanisms of development. 120(1):59-64. Pubmed: 12490296 Liver and pancreas initially develop by budding from the embryonic endoderm. The formation of these organs coincides with the appearance of endothelial cells (ECs) adjacent to the endoderm. ECs either develop in situ in organs, or are recruited by organs and are induced to form blood vessels. Recent reports on liver and pancreas have now shown that ECs also induce essential steps in organ formation such as morphogenesis and cell differentiation. This review summarizes reports on EC signaling during organogenesis and cell differentiation. -
Kumar M, Jordan N, Melton D, Grapin-Botton A. 2003. Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate. Developmental biology. 259(1):109-22. Pubmed: 12812792 Kumar M, Jordan N, Melton D, Grapin-Botton A. 2003. Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate. Developmental biology. 259(1):109-22. Pubmed: 12812792 During embryonic development, organs arise along the gut tube as a series of buds in a stereotyped anterior-posterior (A-P) pattern. Using chick-quail chimeras and in vitro tissue recombination, we studied the interactions governing the induction and maintenance of endodermal organ identify focusing on the pancreas. Though several permissive signals in pancreatic development have been previously identified, here we provide evidence that lateral plate mesoderm sends instructive signals to the endoderm, signals that induce expression of the pancreatic genes Pdx1, p48, Nkx6.1, glucagon, and insulin. Moreover, this instructive signal directs cells to form ectopic insulin-positive islet-like clusters in endoderm that would otherwise form more rostral organs. Once generated, endocrine cells no longer require interaction with mesoderm, but nonendocrine cells continue to require permissive signals from the mesoderm. Stimulation of activin, BMP, or retinoic acid signaling is sufficient to induce Pdx1 expression in endoderm anterior to the pancreas. Lateral plate mesoderm appears to pattern the endoderm in a posterior-dominant fashion as first noted in the patterning of the neural tube at the same embryonic stage. These findings argue for a central role of the mesoderm in coordinating the A-P pattern of all three primary germ layers. -
Kumar M, Melton D. 2003. Pancreas specification: a budding question. Current opinion in genetics & development. 13(4):401-7. Pubmed: 12888014 Kumar M, Melton D. 2003. Pancreas specification: a budding question. Current opinion in genetics & development. 13(4):401-7. Pubmed: 12888014 Much recent investigation has been carried out into the mechanisms by which the pancreas is specified from the early endoderm. Recent advances have highlighted important roles for retinoic acid and bone morphogenetic protein signalling in patterning the endoderm at late gastrulation. Subsequently, interactions with the endothelium of the aorta in the dorsal pancreas domain and lateral plate mesoderm in the ventral pancreas domain are the source of essential pancreas-inductive signals. Additionally, the transcription factor Ptf1a has been demonstrated to have a previously unappreciated role in distinguishing pancreas from surrounding duodenal fates. -
Cleaver O, Melton DA. 2003. Endothelial signaling during development. Nature medicine. 9(6):661-8. Pubmed: 12778164 Cleaver O, Melton DA. 2003. Endothelial signaling during development. Nature medicine. 9(6):661-8. Pubmed: 12778164 Blood vessels perfuse all tissues in the body and mediate vital metabolic exchange between tissues and blood. Increasing evidence, however, points to a direct role for paracrine signaling between blood vessel cells and surrounding target organ cells, during embryonic development and cell differentiation. Understanding the nature of this signaling and its heterogeneity, both in the embryo and in adult tissues, may not only provide insights into mechanisms for normal developmental cell fate decisions, but could also lead to novel targeted therapeutic approaches for a variety of diseases such as heart disease, diabetes or cancer. -
Murtaugh LC, Melton DA. 2003. Genes, signals, and lineages in pancreas development. Annual review of cell and developmental biology. 19:71-89. Pubmed: 14570564 Murtaugh LC, Melton DA. 2003. Genes, signals, and lineages in pancreas development. Annual review of cell and developmental biology. 19:71-89. Pubmed: 14570564 Type I diabetes results from the autoimmune-mediated destruction of pancreatic beta cells, which regulate blood sugar levels by secretion of insulin. Recent clinical data suggest that the disease could be cured if an adequate supply of new beta-cells were available, and one goal of pancreatic developmental biology is to understand how endogenous beta-cells are made, with the hope of making them exogenously. Much is now known about the transcriptional regulation of pancreatic organ specification, growth, and lineage allocation; less is known about intercellular signals that regulate this process, but candidates continue to emerge. Additional insights, often contradicting older models, have come from the application of new lineage-tracing techniques. Altogether, these studies also shed light on the still-elusive pancreatic stem cell, which may participate in normal organ maintenance as well as recovery from injury. A rigorous proof of the existence of such a cell, whether in vivo or in vitro, would offer real hope for the prospect of controlled beta-cell generation in a clinical setting. -
Rajagopal J, Anderson WJ, Kume S, Martinez OI, Melton DA. 2003. Insulin staining of ES cell progeny from insulin uptake. Science (New York, N.Y.). 299(5605):363. Pubmed: 12532008 Rajagopal J, Anderson WJ, Kume S, Martinez OI, Melton DA. 2003. Insulin staining of ES cell progeny from insulin uptake. Science (New York, N.Y.). 299(5605):363. Pubmed: 12532008 -
Chiang MK, Melton DA. 2003. Single-cell transcript analysis of pancreas development. Developmental cell. 4(3):383-93. Pubmed: 12636919 Chiang MK, Melton DA. 2003. Single-cell transcript analysis of pancreas development. Developmental cell. 4(3):383-93. Pubmed: 12636919 DNA microarray analysis was combined with a modified single-cell PCR procedure to study gene expression profiles of single cells at different stages of pancreatic development. This method identifies distinct cell types at embryonic day 10.5, a stage when the pancreatic epithelium is morphologically uniform. Some cells express unexpected combinations of genes, and these expression patterns provide new insights into pancreas development. Following on these findings, we use PCR products from different cell types to identify novel pancreatic genes, some of which mark subtypes of developing pancreatic cells. By integrating these data with previous genetic and biochemical studies, we propose a pathway for pancreatic cell development. This form of single-cell transcriptional analysis can be applied to any developmental process or tissue to characterize distinct cell types. 2002
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Fishman MP, Melton DA. 2002. Pancreatic lineage analysis using a retroviral vector in embryonic mice demonstrates a common progenitor for endocrine and exocrine cells. The International journal of developmental biology. 46(2):201-7. Pubmed: 11934148 Fishman MP, Melton DA. 2002. Pancreatic lineage analysis using a retroviral vector in embryonic mice demonstrates a common progenitor for endocrine and exocrine cells. The International journal of developmental biology. 46(2):201-7. Pubmed: 11934148 The origin of pancreatic endocrine cells is unknown. Some studies have suggested that there is a common pancreatic progenitor which gives rise to both endocrine and exocrine cells, while others have suggested separate endocrine and exocrine lineages. Previous conclusions have been based on indirect data, such as the co-expression of molecular markers. We directly assessed the relationship between endocrine and exocrine cells during development using a lineage tracer. A replication-incompetent retrovirus was used to introduce the reporter gene alkaline phosphatase into single cells in explants of mouse embryonic pancreas. After a week in culture, the subsequent fate of the infected cells could then be determined. The results show that a common pancreatic progenitor cell exists, which gives rise to both endocrine and exocrine cells. -
Gu G, Dubauskaite J, Melton DA. 2002. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development (Cambridge, England). 129(10):2447-57. Pubmed: 11973276 Gu G, Dubauskaite J, Melton DA. 2002. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development (Cambridge, England). 129(10):2447-57. Pubmed: 11973276 The location and lineage of cells that give rise to endocrine islets during embryogenesis has not been established nor has the origin or identity of adult islet stem cells. We have employed an inducible Cre-ER(TM)-LoxP system to indelibly mark the progeny of cells expressing either Ngn3 or Pdx1 at different stages of development. The results provide direct evidence that NGN3+ cells are islet progenitors during embryogenesis and in adult mice. In addition, we find that cells expressing Pdx1 give rise to all three types of pancreatic tissue: exocrine, endocrine and duct. Furthermore, exocrine and endocrine cells are derived from Pdx1-expressing progenitors throughout embryogenesis. By contrast, the pancreatic duct arises from PDX1+ progenitors that are set aside around embryonic day 10.5 (E9.5-E11.5). These findings suggest that lineages for exocrine, endocrine islet and duct progenitors are committed at mid-gestation. -
Ramalho-Santos M, Yoon S, Matsuzaki Y, Mulligan RC, Melton DA. 2002. "Stemness": transcriptional profiling of embryonic and adult stem cells. Science (New York, N.Y.). 298(5593):597-600. Pubmed: 12228720 Ramalho-Santos M, Yoon S, Matsuzaki Y, Mulligan RC, Melton DA. 2002. "Stemness": transcriptional profiling of embryonic and adult stem cells. Science (New York, N.Y.). 298(5593):597-600. Pubmed: 12228720 The transcriptional profiles of mouse embryonic, neural, and hematopoietic stem cells were compared to define a genetic program for stem cells. A total of 216 genes are enriched in all three types of stem cells, and several of these genes are clustered in the genome. When compared to differentiated cell types, stem cells express a significantly higher number of genes (represented by expressed sequence tags) whose functions are unknown. Embryonic and neural stem cells have many similarities at the transcriptional level. These results provide a foundation for a more detailed understanding of stem cell biology. 2001
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Lammert E, Cleaver O, Melton D. 2001. Induction of pancreatic differentiation by signals from blood vessels. Science (New York, N.Y.). 294(5542):564-7. Pubmed: 11577200 Lammert E, Cleaver O, Melton D. 2001. Induction of pancreatic differentiation by signals from blood vessels. Science (New York, N.Y.). 294(5542):564-7. Pubmed: 11577200 Blood vessels supply developing organs with metabolic sustenance. Here, we demonstrate a role for blood vessels as a source of developmental signals during pancreatic organogenesis. In vitro experiments with embryonic mouse tissues demonstrate that blood vessel endothelium induces insulin expression in isolated endoderm. Removal of the dorsal aorta in Xenopus laevis embryos results in the failure of insulin expression in vivo. Furthermore, using transgenic mice, we show that ectopic vascularization in the posterior foregut leads to ectopic insulin expression and islet hyperplasia. These results indicate that vessels not only provide metabolic sustenance, but also provide inductive signals for organ development. -
Grapin-Botton A, Majithia AR, Melton DA. 2001. Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes. Genes & development. 15(4):444-54. Pubmed: 11230152 Grapin-Botton A, Majithia AR, Melton DA. 2001. Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes. Genes & development. 15(4):444-54. Pubmed: 11230152 The mechanisms by which the epithelium of the digestive tract and its associated glands are specified are largely unknown. One clue is that several transcription factors are expressed in specific regions of the endoderm prior to and during organogenesis. Pdx-1, for example, is expressed in the duodenum and pancreas and Pdx-1 inactivation results in an arrest of pancreatic development after buds formation. Similarly, ngn3 is transiently expressed in the developing pancreas and a knockout results in the absence of endocrine cells. This paper focuses on the question of whether these and other transcription factors, known to be necessary for pancreatic development, are also sufficient to drive a program of pancreatic organogenesis. Using in ovo electroporation of chick embryos, we show that ectopic expression of Pdx-1 or ngn3 causes cells to bud out of the epithelium like pancreatic progenitors. The Pdx-1-expressing cells extinguish markers for other nonpancreatic regions of the endoderm and initiate, but do not complete, pancreatic cytodifferentiation. Ectopic expression of ngn3 is sufficient to turn endodermal cells of any region into endocrine cells that form islets expressing glucagon and somatostatin in the mesenchyme. The results suggest that simple gene combinations could be used in stem cells to achieve specific endodermal tissue differentiation. 2000
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Hebrok M, Kim SK, St Jacques B, McMahon AP, Melton DA. 2000. Regulation of pancreas development by hedgehog signaling. Development (Cambridge, England). 127(22):4905-13. Pubmed: 11044404 Hebrok M, Kim SK, St Jacques B, McMahon AP, Melton DA. 2000. Regulation of pancreas development by hedgehog signaling. Development (Cambridge, England). 127(22):4905-13. Pubmed: 11044404 Pancreas organogenesis is regulated by the interaction of distinct signaling pathways that promote or restrict morphogenesis and cell differentiation. Previous work has shown that activin, a TGF(beta+) signaling molecule, permits pancreas development by repressing expression of Sonic hedgehog (Shh), a member of the hedgehog family of signaling molecules that antagonize pancreas development. Here we show that Indian hedgehog (Ihh), another hedgehog family member, and Patched 1 (Ptc1), a receptor and negative regulator of hedgehog activity, are expressed in pancreatic tissue. Targeted inactivation of Ihh in mice allows ectopic branching of ventral pancreatic tissue resulting in an annulus that encircles the duodenum, a phenotype frequently observed in humans suffering from a rare disorder known as annular pancreas. Shh(-)(/)(-) and Shh(-)(/)(-) Ihh(+/)(-) mutants have a threefold increase in pancreas mass, and a fourfold increase in pancreatic endocrine cell numbers. In contrast, mutations in Ptc1 reduce pancreas gene expression and impair glucose homeostasis. Thus, islet cell, pancreatic mass and pancreatic morphogenesis are regulated by hedgehog signaling molecules expressed within and adjacent to the embryonic pancreas. Defects in hedgehog signaling may lead to congenital pancreatic malformations and glucose intolerance. -
Kelly OG, Melton DA. 2000. Development of the pancreas in Xenopus laevis. Developmental dynamics : an official publication of the American Association of Anatomists. 218(4):615-27. Pubmed: 10906780 Kelly OG, Melton DA. 2000. Development of the pancreas in Xenopus laevis. Developmental dynamics : an official publication of the American Association of Anatomists. 218(4):615-27. Pubmed: 10906780 Xenopus embryos have several experimental advantages for studying development. Although these advantages have traditionally been used to elucidate mechanisms of early development, they can also be exploited to investigate issues later in development such as organogenesis. We have begun to study pancreatic organogenesis in Xenopus. Using histological and molecular marker analysis, we characterized the anatomy of the developing pancreas in Xenopus embryos from the time of initial pancreatic rudiment formation to the time when the tadpole starts to feed. We examined the expression of various endocrine hormones, exocrine gene products, and pancreatic transcription factors. Interestingly, the endocrine hormone insulin has restricted expression in the dorsal pancreas. Investigation of pancreatic specification during gastrulation demonstrates that insulin expression is regionalized along the dorsoventral axis early in development.Copyright 2000 Wiley-Liss, Inc. -
Lammert E, Brown J, Melton DA. 2000. Notch gene expression during pancreatic organogenesis. Mechanisms of development. 94(1-2):199-203. Pubmed: 10842072 Lammert E, Brown J, Melton DA. 2000. Notch gene expression during pancreatic organogenesis. Mechanisms of development. 94(1-2):199-203. Pubmed: 10842072 Notch receptors are involved in regulating the balance between cell differentiation and stem cell proliferation during the development of numerous tissues (Artavanis-Tsakonas, S., Matsuno, K., Fortini, M. E., 1995. Notch signaling. Science 268, 225-232). Here the expression of all four vertebrate Notch genes, their ligands, and some down-stream targets is analyzed during mouse pancreatic organogenesis. Notch 1 is the first Notch gene expressed in the pancreatic epithelium, and coexpression with HES 1 suggests that the Notch 1 pathway is activated. Notch 2 expression follows later when pancreatic buds branch and is restricted to embryonic ducts, believed to be the source for endocrine and exocrine stem cells. Notch 3 and Notch 4 are expressed in pancreatic mesenchyme and later in endothelial cells. Together these descriptive data comprise a framework for understanding the cellular basis for Notch function during pancreatic development. -
Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N. 2000. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America. 97(21):11307-12. Pubmed: 11027332 Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N. 2000. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America. 97(21):11307-12. Pubmed: 11027332 Human embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of in vitro fertilized human blastocysts. We examined the potential of eight growth factors [basic fibroblast growth factor (bFGF), transforming growth factor beta1 (TGF-beta1), activin-A, bone morphogenic protein 4 (BMP-4), hepatocyte growth factor (HGF), epidermal growth factor (EGF), beta nerve growth factor (betaNGF), and retinoic acid] to direct the differentiation of human ES-derived cells in vitro. We show that human ES cells that have initiated development as aggregates (embryoid bodies) express a receptor for each of these factors, and that their effects are evident by differentiation into cells with different epithelial or mesenchymal morphologies. Differentiation of the cells was assayed by expression of 24 cell-specific molecular markers that cover all embryonic germ layers and 11 different tissues. Each growth factor has a unique effect that may result from directed differentiation and/or cell selection, and we can divide the overall effects of the factors into three categories: growth factors (Activin-A and TGFbeta1) that mainly induce mesodermal cells; factors (retinoic acid, EGF, BMP-4, and bFGF) that activate ectodermal and mesodermal markers; and factors (NGF and HGF) that allow differentiation into the three embryonic germ layers, including endoderm. None of the growth factors directs differentiation exclusively to one cell type. This analysis sets the stage for directing differentiation of human ES cells in culture and indicates that multiple human cell types may be enriched in vitro by specific factors. -
Wells JM, Melton DA. 2000. Early mouse endoderm is patterned by soluble factors from adjacent germ layers. Development (Cambridge, England). 127(8):1563-72. Pubmed: 10725233 Wells JM, Melton DA. 2000. Early mouse endoderm is patterned by soluble factors from adjacent germ layers. Development (Cambridge, England). 127(8):1563-72. Pubmed: 10725233 Endoderm that forms the respiratory and digestive tracts is a sheet of approximately 500-1000 cells around the distal cup of an E7.5 mouse embryo. Within 2 days, endoderm folds into a primitive gut tube from which numerous organs will bud. To characterize the signals involved in the developmental specification of this early endoderm, we have employed an in vitro assay using germ layer explants and show that adjacent germ layers provide soluble, temporally specific signals that induce organ-specific gene expression in endoderm. Furthermore, we show that FGF4 expressed in primitive streak-mesoderm can induce the differentiation of endoderm in a concentration-dependent manner. We conclude that the differentiation of gastrulation-stage endoderm is directed by adjacent mesoderm and ectoderm, one of the earliest reported patterning events in formation of the vertebrate gut tube. -
Grapin-Botton A, Melton DA. 2000. Endoderm development: from patterning to organogenesis. Trends in genetics : TIG. 16(3):124-30. Pubmed: 10689353 Grapin-Botton A, Melton DA. 2000. Endoderm development: from patterning to organogenesis. Trends in genetics : TIG. 16(3):124-30. Pubmed: 10689353 Although the ectoderm and mesoderm have been the focus of intensive work in the recent era of studies on the molecular control of vertebrate development, the endoderm has received less attention. Because signaling must occur between germ layers in order to achieve a properly organized body, our understanding of the coordinated development of all organs requires a more thorough consideration of the endoderm and its derivatives. This review focuses on present knowledge and perspectives concerning endoderm patterning and organogenesis. Some of the classical embryology of the endoderm is discussed and the progress and deficiencies in cellular and molecular studies are noted. -
Ramalho-Santos M, Melton DA, McMahon AP. 2000. Hedgehog signals regulate multiple aspects of gastrointestinal development. Development (Cambridge, England). 127(12):2763-72. Pubmed: 10821773 Ramalho-Santos M, Melton DA, McMahon AP. 2000. Hedgehog signals regulate multiple aspects of gastrointestinal development. Development (Cambridge, England). 127(12):2763-72. Pubmed: 10821773 The gastrointestinal tract develops from the embryonic gut, which is composed of an endodermally derived epithelium surrounded by cells of mesodermal origin. Cell signaling between these two tissue layers appears to play a critical role in coordinating patterning and organogenesis of the gut and its derivatives. We have assessed the function of Sonic hedgehog and Indian hedgehog genes, which encode members of the Hedgehog family of cell signals. Both are expressed in gut endoderm, whereas target genes are expressed in discrete layers in the mesenchyme. It was unclear whether functional redundancy between the two genes would preclude a genetic analysis of the roles of Hedgehog signaling in the mouse gut. We show here that the mouse gut has both common and separate requirements for Sonic hedgehog and Indian hedgehog. Both Sonic hedgehog and Indian hedgehog mutant mice show reduced smooth muscle, gut malrotation and annular pancreas. Sonic hedgehog mutants display intestinal transformation of the stomach, duodenal stenosis (obstruction), abnormal innervation of the gut and imperforate anus. Indian hedgehog mutants show reduced epithelial stem cell proliferation and differentiation, together with features typical of Hirschsprung's disease (aganglionic colon). These results show that Hedgehog signals are essential for organogenesis of the mammalian gastrointestinal tract and suggest that mutations in members of this signaling pathway may be involved in human gastrointestinal malformations. 1999
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Wells JM, Melton DA. 1999. Vertebrate endoderm development. Annual review of cell and developmental biology. 15:393-410. Pubmed: 10611967 Wells JM, Melton DA. 1999. Vertebrate endoderm development. Annual review of cell and developmental biology. 15:393-410. Pubmed: 10611967 Endoderm, one of the three principal germ layers, contributes to all organs of the alimentary tract. For simplicity, this review divides formation of endodermal organs into four fundamental steps: (a) formation of endoderm during gastrulation, (b) morphogenesis of a gut tube from a sheet of cells, (c) budding of organ domains from the tube, and (d) differentiation of organ-specific cell types within the growing buds. We discuss possible mechanisms that regulate how undifferentiated endoderm becomes specified into a myriad of cell types that populate the respiratory and gastrointestinal tracts. -
Hebrok M, Kim SK, Melton DA. 1999. Screening for novel pancreatic genes expressed during embryogenesis. Diabetes. 48(8):1550-6. Pubmed: 10426372 Hebrok M, Kim SK, Melton DA. 1999. Screening for novel pancreatic genes expressed during embryogenesis. Diabetes. 48(8):1550-6. Pubmed: 10426372 We have combined suppressive subtractive hybridization with in situ hybridization to identify genes expressed at early stages of pancreas development. By using polymerase chain reaction amplification and subtractive hybridization, this protocol for screening can be applied when the amount of RNA is limited. Seven genes expressed in or adjacent to the pancreas anlage were isolated, three of which show similarity to known genes. The expression pattern and sequence information indicate that some of the genes could govern pancreas development. 1998
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Hebrok M, Kim SK, Melton DA. 1998. Notochord repression of endodermal Sonic hedgehog permits pancreas development. Genes & development. 12(11):1705-13. Pubmed: 9620856 Hebrok M, Kim SK, Melton DA. 1998. Notochord repression of endodermal Sonic hedgehog permits pancreas development. Genes & development. 12(11):1705-13. Pubmed: 9620856 Notochord signals to the endoderm are required for development of the chick dorsal pancreas. Sonic hedgehog (SHH) is normally absent from pancreatic endoderm, and we provide evidence that notochord, in contrast to its effects on adjacent neuroectoderm where SHH expression is induced, represses SHH expression in adjacent nascent pancreatic endoderm. We identify activin-betaB and FGF2 as notochord factors that can repress endodermal SHH and thereby permit expression of pancreas genes including Pdx1 and insulin. Endoderm treatment with antibodies that block hedgehog activity also results in pancreatic gene expression. Prevention of SHH expression in prepancreatic dorsal endoderm by intercellular signals, like activin and FGF, may be critical for permitting early steps of chick pancreatic development. -
Joseph EM, Melton DA. 1998. Mutant Vg1 ligands disrupt endoderm and mesoderm formation in Xenopus embryos. Development (Cambridge, England). 125(14):2677-85. Pubmed: 9636082 Joseph EM, Melton DA. 1998. Mutant Vg1 ligands disrupt endoderm and mesoderm formation in Xenopus embryos. Development (Cambridge, England). 125(14):2677-85. Pubmed: 9636082 The Xenopus Vg1 gene, a TGFbeta superfamily member, is expressed as a maternal mRNA localized to prospective endoderm, and mature Vg1 protein can induce both endodermal and mesodermal markers in embryonic cells. Most previous work on embryonic inducers, including activin, BMPs and Vg1, has relied on ectopic expression to assay for gene function. Here we employ a mutant ligand approach to block Vg1 signaling in developing embryos. The results indicate that Vg1 expression is essential for normal endodermal development and the induction of dorsal mesoderm in vivo. -
Henry GL, Melton DA. 1998. Mixer, a homeobox gene required for endoderm development. Science (New York, N.Y.). 281(5373):91-6. Pubmed: 9651252 Henry GL, Melton DA. 1998. Mixer, a homeobox gene required for endoderm development. Science (New York, N.Y.). 281(5373):91-6. Pubmed: 9651252 An expression cloning strategy in Xenopus laevis was used to isolate a homeobox-containing gene, Mixer, that can cause embryonic cells to form endoderm. Mixer transcripts are found specifically in the prospective endoderm of gastrula, which coincides with the time and place that endodermal cells become histologically distinct and irreversibly determined. Loss-of-function studies with a dominant inhibitory mutant demonstrate that Mixer activity is required for endoderm development. In particular, the expression of Sox17alpha and Sox17beta, two previously identified endodermal determinants, require Mixer function. Together, these data suggest that Mixer is an embryonic transcription factor involved in specifying the endodermal germ layer. -
Kim SK, Melton DA. 1998. Pancreas development is promoted by cyclopamine, a hedgehog signaling inhibitor. Proceedings of the National Academy of Sciences of the United States of America. 95(22):13036-41. Pubmed: 9789036 Kim SK, Melton DA. 1998. Pancreas development is promoted by cyclopamine, a hedgehog signaling inhibitor. Proceedings of the National Academy of Sciences of the United States of America. 95(22):13036-41. Pubmed: 9789036 Exposure to cyclopamine, a steroid alkaloid that blocks Sonic hedgehog (Shh) signaling, promotes pancreatic expansion in embryonic chicks. Heterotopic development of pancreatic endocrine and exocrine structures occurs in regions adjacent to the pancreas including stomach and duodenum, and insulin-producing islets in the pancreas are enlarged. The homeodomain transcription factor PDX1, required for pancreas development, is expressed broadly in the posterior foregut but pancreas development normally initiates only in a restricted region of PDX1-expressing posterior foregut where endodermal Shh expression is repressed. The results suggests that cyclopamine expands the endodermal region where Shh signaling does not occur, resulting in pancreatic differentiation in a larger region of PDX1-expressing foregut endoderm. Cyclopamine reveals the capacity of a broad region of the posterior embryonic foregut to form pancreatic cells and provides a means for expanding embryonic pancreas development. 1997
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Kim SK, Hebrok M, Melton DA. 1997. Notochord to endoderm signaling is required for pancreas development. Development (Cambridge, England). 124(21):4243-52. Pubmed: 9334273 Kim SK, Hebrok M, Melton DA. 1997. Notochord to endoderm signaling is required for pancreas development. Development (Cambridge, England). 124(21):4243-52. Pubmed: 9334273 The role of the notochord in inducing and patterning adjacent neural and mesodermal tissues is well established. We provide evidence that the notochord is also required for one of the earliest known steps in the development of the pancreas, an endodermally derived organ. At a developmental stage in chick embryos when the notochord touches the endoderm, removal of notochord eliminates subsequent expression of several markers of dorsal pancreas bud development, including insulin, glucagon and carboxypeptidase A. Pancreatic gene expression can be initiated and maintained in prepancreatic chick endoderm grown in vitro with notochord. Non-pancreatic endoderm, however, does not express pancreatic genes when recombined with the same notochord. The results suggest that the notochord provides a permissive signal to endoderm to specify pancreatic fate in a stepwise manner. -
Joseph EM, Melton DA. 1997. Xnr4: a Xenopus nodal-related gene expressed in the Spemann organizer. Developmental biology. 184(2):367-72. Pubmed: 9133442 Joseph EM, Melton DA. 1997. Xnr4: a Xenopus nodal-related gene expressed in the Spemann organizer. Developmental biology. 184(2):367-72. Pubmed: 9133442 We have isolated a novel TGF beta-like gene from Xenopus which is highly related to mouse nodal The gene, Xnr4, is expressed at the gastrula stage in the Spemann organizer and at later stages in the notochord and neural tube. Ectopic expression of Xnr4 can induce and dorsalize mesoderm. These studies suggest that Xnr4, along with other nodal-related genes (Xnr1-3), may participate in mesodermal patterning and possibly neural development. -
Kim SK, Hebrok M, Melton DA. 1997. Pancreas development in the chick embryo. Cold Spring Harbor symposia on quantitative biology. 62:377-83. Pubmed: 9598372 Kim SK, Hebrok M, Melton DA. 1997. Pancreas development in the chick embryo. Cold Spring Harbor symposia on quantitative biology. 62:377-83. Pubmed: 9598372 1996
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Klein PS, Melton DA. 1996. A molecular mechanism for the effect of lithium on development. Proceedings of the National Academy of Sciences of the United States of America. 93(16):8455-9. Pubmed: 8710892 Klein PS, Melton DA. 1996. A molecular mechanism for the effect of lithium on development. Proceedings of the National Academy of Sciences of the United States of America. 93(16):8455-9. Pubmed: 8710892 Lithium, one of the most effective drugs for the treatment of bipolar (manic-depressive) disorder, also has dramatic effects on morphogenesis in the early development of numerous organisms. How lithium exerts these diverse effects is unclear, but the favored hypothesis is that lithium acts through inhibition of inositol monophosphatase (IMPase). We show here that complete inhibition of IMPase has no effect on the morphogenesis of Xenopus embryos and present a different hypothesis to explain the broad action of lithium. Our results suggest that lithium acts through inhibition of glycogen synthase kinase-3 beta (GSK-3 beta), which regulates cell fate determination in diverse organisms including Dictyostelium, Drosophila, and Xenopus. Lithium potently inhibits GSK-3 beta activity (Ki = 2 mM), but is not a general inhibitor of other protein kinases. In support of this hypothesis, lithium treatment phenocopies loss of GSK-3 beta function in Xenopus and Dictyostelium. These observations help explain the effect of lithium on cell-fate determination and could provide insights into the pathogenesis and treatment of bipolar disorder. -
Reilly KM, Melton DA. 1996. Short-range signaling by candidate morphogens of the TGF beta family and evidence for a relay mechanism of induction. Cell. 86(5):743-54. Pubmed: 8797821 Reilly KM, Melton DA. 1996. Short-range signaling by candidate morphogens of the TGF beta family and evidence for a relay mechanism of induction. Cell. 86(5):743-54. Pubmed: 8797821 The specification and patterning of cell fates by a morphogen gradient is a unifying theme of developmental biology, yet little evidence exists for the presence of gradients in vivo or to show how such putative gradients form. Vg1 and activin are candidate morphogens involved in Xenopus mesoderm induction. This study suggests that these TGF beta family members act on adjacent cells but do not travel through the intact extracellular space to induce distant cells directly. Moreover, we present evidence for the presence of secondary inducing signals that could be involved in relaying signals to distant cells. These results suggest that if a localized cellular source of an inducer acts to pattern mesodermal cells at a distance in Xenopus embryos, it does so by a relay mechanism. -
Henry GL, Brivanlou IH, Kessler DS, Hemmati-Brivanlou A, Melton DA. 1996. TGF-beta signals and a pattern in Xenopus laevis endodermal development. Development (Cambridge, England). 122(3):1007-15. Pubmed: 8631246 Henry GL, Brivanlou IH, Kessler DS, Hemmati-Brivanlou A, Melton DA. 1996. TGF-beta signals and a pattern in Xenopus laevis endodermal development. Development (Cambridge, England). 122(3):1007-15. Pubmed: 8631246 We have analyzed two gene products expressed in the early endoderm of Xenopus laevis: Xlhbox-8, a pancreas-specific transcription factor and intestinal fatty acid binding protein (IFABP), a marker of small intestinal epithelium. Expression of the pancreas marker relies on cell signaling mediated by both the TGF-beta and FGF classes of secreted peptide growth factors, whereas, expression of the more posterior small intestinal marker does not. Endodermal explants devoid of mesoderm express both markers in a regionalized manner. Cortical rotation is required for the expression of the more anterior marker, Xlhbox-8, but not for the small intestinal marker, IFABP. These findings suggest that endodermal patterning is dependent, in part, on the same events and signals known to play important roles in mesodermal development. Furthermore, inhibition of TGF-beta signaling in the endoderm leads to ectopic expression of both mesodermal and ectodermal markers, suggesting the TGF-beta signaling may play a general role in the segregation of the three embryonic germ layers. -
Graff JM, Bansal A, Melton DA. 1996. Xenopus Mad proteins transduce distinct subsets of signals for the TGF beta superfamily. Cell. 85(4):479-87. Pubmed: 8653784 Graff JM, Bansal A, Melton DA. 1996. Xenopus Mad proteins transduce distinct subsets of signals for the TGF beta superfamily. Cell. 85(4):479-87. Pubmed: 8653784 Xenopus cDNAs homologous to the Drosophila Mad gene and C. elegans CEM genes have been cloned and functionally analyzed by microinjection into frog embryos. The results show that these genes (Xmad) encode intracellular proteins that act downstream of TGF beta superfamily ligands. Most interesting is the fact that different Xmad proteins produce distinct biological responses. Xmad1 produces ventral mesoderm, apparently transducing a signal for BMP2 and BMP4, whereas Xmad2 induces dorsal mesoderm like Vg1, activin, and nodal. These results suggest that an individual Xmad protein waits poised in the cytoplasm for instruction from a distinct subset of TGF beta ligands and then conveys specific information to the nucleus. -
Dohrmann CE, Kessler DS, Melton DA. 1996. Induction of axial mesoderm by zDVR-1, the zebrafish orthologue of Xenopus Vg1. Developmental biology. 175(1):108-17. Pubmed: 8608857 Dohrmann CE, Kessler DS, Melton DA. 1996. Induction of axial mesoderm by zDVR-1, the zebrafish orthologue of Xenopus Vg1. Developmental biology. 175(1):108-17. Pubmed: 8608857 The zebrafish DVR-1 (zDVR-1) gene, like Xenopus Vg1, is present maternally as an unprocessed precursor protein which is distributed ubiquitously along the future dorsoventral axis. Also, like Vg1, overexpression of zDVR-1 in zebrafish directs synthesis of more precursor, but no processed protein. However, the native zDVR-1 precursor is processed to mature protein when expressed in Xenopus. Like processed Vg1, mature zDVR-1 is a potent inducer of axial mesoderm. The parallels in expression pattern, apparent regulation of protein processing, and mesoderm-inducing activity support the hypothesis that localized protein processing controls production of a dorsal mesoderm inducer in these two species. Furthermore, using mutant mRNAs, we show that cleavage site sequences of the precursor protein are important in regulating protein processing. -
Newfeld SJ, Chartoff EH, Graff JM, Melton DA, Gelbart WM. 1996. Mothers against dpp encodes a conserved cytoplasmic protein required in DPP/TGF-beta responsive cells. Development (Cambridge, England). 122(7):2099-108. Pubmed: 8681791 Newfeld SJ, Chartoff EH, Graff JM, Melton DA, Gelbart WM. 1996. Mothers against dpp encodes a conserved cytoplasmic protein required in DPP/TGF-beta responsive cells. Development (Cambridge, England). 122(7):2099-108. Pubmed: 8681791 The proteins necessary for signal transduction in cells responding to ligands of the TGF-beta family are largely unknown. We have previously identified Mad (Mothers against dpp), a gene that interacts with the TGF-beta family member encoded by decapentaplegic (dpp) in Drosophila. Assay of Mad's role in the DPP-dependent events of embryonic midgut development demonstrates that Mad is required for any response of the visceral mesoderm or endoderm to DPP signals from the visceral mesoderm. Replacement of the normal DPP promoter with a heterologous (hsp70) promoter fails to restore DPP-dependent responses in Mad mutant midguts. Experiments utilizing Mad transgenes regulated by tissue-specific promoters show that MAD is required specifically in cells responding to DPP. Immunohistochemical studies localize MAD to the cytoplasm in all tissues examined. Experiments in Xenopus embryos demonstrate that Drosophila MAD can function in the signaling pathway of BMP-4, a vertebrate homolog of dpp. Based on these results, we propose that Mad is a highly conserved and essential element of the DPP signal transduction pathway. 1995
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Kessler DS, Melton DA. 1995. Induction of dorsal mesoderm by soluble, mature Vg1 protein. Development (Cambridge, England). 121(7):2155-64. Pubmed: 7635059 Kessler DS, Melton DA. 1995. Induction of dorsal mesoderm by soluble, mature Vg1 protein. Development (Cambridge, England). 121(7):2155-64. Pubmed: 7635059 Mesoderm induction during Xenopus development has been extensively studied, and two members of the transforming growth factor-beta family, activin beta B and Vg1, have emerged as candidates for a natural inducer of dorsal mesoderm. Heretofore, analysis of Vg1 activity has relied on injection of hybrid Vg1 mRNAs, which have not been shown to direct efficient secretion of ligand and, therefore, the mechanism of mesoderm induction by processed Vg1 protein is unclear. This report describes injection of Xenopus oocytes with a chimeric activin-Vg1 mRNA, encoding the pro-region of activin beta B fused to the mature region of Vg1, resulting in the processing and secretion of mature Vg1. Treatment of animal pole explants with mature Vg1 protein resulted in differentiation of dorsal, but not ventral, mesodermal tissues and dose-dependent activation of both dorsal and ventrolateral mesodermal markers. At high doses, mature Vg1 induced formation of 'embryoids' with a rudimentary axial pattern, head structures including eyes and a functional neuromuscular system. Furthermore, truncated forms of the activin and FGF receptors, which block mesoderm induction in the intact embryo, fully inhibited mature Vg1 activity. To examine the mechanism of inhibition, we have performed receptor-binding assays with radiolabeled Vg1. Finally, follistatin, a specific inhibitor of activin beta B which is shown not to block endogenous dorsal mesoderm induction, failed to inhibit Vg1. The results support a role for endogenous Vg1 in dorsal mesoderm induction during Xenopus development. -
Kelly OG, Melton DA. 1995. Induction and patterning of the vertebrate nervous system. Trends in genetics : TIG. 11(7):273-8. Pubmed: 7482777 Kelly OG, Melton DA. 1995. Induction and patterning of the vertebrate nervous system. Trends in genetics : TIG. 11(7):273-8. Pubmed: 7482777 The formation of the vertebrate central nervous system depends on a series of inductive interactions. Ventral ectoderm gives rise to epidermis (skin), whereas dorsal ectoderm forms neurectoderm, which is regionalized along the anteroposterior axis in response to inductive signals from dorsal mesoderm. Specific neural cell types are established along the dorsoventral axis of the neural tube by inductive signals from the dorsal mesoderm and prospective epidermis. We review the induction and patterning of the nervous system, with an emphasis on molecules that might play a role in these interactions. -
Klein PS, Melton DA. 1995. Translational control of activin in Xenopus laevis embryos. Developmental genetics. 17(1):55-64. Pubmed: 7554495 Klein PS, Melton DA. 1995. Translational control of activin in Xenopus laevis embryos. Developmental genetics. 17(1):55-64. Pubmed: 7554495 Activin is a potent mesoderm inducing factor present in embryos of Xenopus laevis. Recent evidence has implicated activin in the inhibition of neural development in addition to the well-established induction of mesoderm in ectodermal explants. These diverse effects are critically dependent on the concentration of activin yet little is known about the mechanisms regulating the level of activin in the embryo. We report that the 3' untranslated region (3' UTR) of activin beta B mRNA inhibits the translation of activin in embryos. Micro-injection of activin mRNA from which the 3' UTR has been deleted is 8-10-fold more potent in inducing mesoderm than mRNA containing the 3' UTR. Truncation of the 3' UTR also leads to a marked enhancement of activin protein levels in embryos but has no effect when the truncated mRNA is translated in vitro. The 3' UTR also confers translational inhibition on a heterologous mRNA. These data show that a maternal factor(s) present in X. laevis regulates the translation of injected activin beta B mRNA. This factor(s) could be responsible for regulating the levels of endogenous activin beta B protein during mesoderm induction and the specification of ectodermal derivatives such as neural and epidermal tissues. 1994
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Hemmati-Brivanlou A, Melton DA. 1994. Inhibition of activin receptor signaling promotes neuralization in Xenopus. Cell. 77(2):273-81. Pubmed: 8168134 Hemmati-Brivanlou A, Melton DA. 1994. Inhibition of activin receptor signaling promotes neuralization in Xenopus. Cell. 77(2):273-81. Pubmed: 8168134 Expression of a truncated activin type II receptor, which blocks signaling by activin, neuralizes explants of embryonic cells that would otherwise become epidermal cells. This neuralization is direct and does not require the presence of mesoderm. The induced neural tissue expresses general molecular markers of the central nervous system as well as an array of neural markers along the anteroposterior axis. In the context of the whole embryo, expression of this truncated activin receptor diverts prospective ectoderm and endoderm to a neural fate. We propose that inhibition of the activin type II receptor signaling causes the cells of Xenopus embryos to adopt a neural fate. These results, along with previous experiments performed in Drosophila, suggest that the formation of the nervous system in vertebrates and invertebrates occurs by a common strategy. -
Klein PS, Melton DA. 1994. Hormonal regulation of embryogenesis: the formation of mesoderm in Xenopus laevis. Endocrine reviews. 15(3):326-41. Pubmed: 8076585 Klein PS, Melton DA. 1994. Hormonal regulation of embryogenesis: the formation of mesoderm in Xenopus laevis. Endocrine reviews. 15(3):326-41. Pubmed: 8076585 -
Hemmati-Brivanlou A, Kelly OG, Melton DA. 1994. Follistatin, an antagonist of activin, is expressed in the Spemann organizer and displays direct neuralizing activity. Cell. 77(2):283-95. Pubmed: 8168135 Hemmati-Brivanlou A, Kelly OG, Melton DA. 1994. Follistatin, an antagonist of activin, is expressed in the Spemann organizer and displays direct neuralizing activity. Cell. 77(2):283-95. Pubmed: 8168135 In the accompanying paper, we show that the expression of a dominant negative activin receptor can convert prospective ectoderm into neural tissue, which suggests that activin is an inhibitor of neuralization. Here we report the isolation and characterization of an activin antagonist, follistatin, that can induce neural tissue directly in vivo. Follistatin RNA is localized in the Spemann organizer and notochord, tissues known to be potent neural inducers. We demonstrate that follistatin RNA and protein are able to block the activity of activin in embryonic explants. Furthermore, we show that follistatin RNA directly neuralizes ectodermal explants in the absence of detectable mesoderm. Thus, follistatin is present at the correct time and location to play a role in neural induction in vivo. -
Kessler DS, Melton DA. 1994. Vertebrate embryonic induction: mesodermal and neural patterning. Science (New York, N.Y.). 266(5185):596-604. Pubmed: 7939714 Kessler DS, Melton DA. 1994. Vertebrate embryonic induction: mesodermal and neural patterning. Science (New York, N.Y.). 266(5185):596-604. Pubmed: 7939714 Within the fertilized egg lies the information necessary to generate a diversity of cell types in the precise pattern of tissues and organs that comprises the vertebrate body. Seminal embryological experiments established the importance of induction, or cell interactions, in the formation of embryonic tissues and provided a foundation for molecular studies. In recent years, secreted gene products capable of inducing or patterning embryonic tissues have been identified. Despite these advances, embryologists remain challenged by fundamental questions: What are the endogenous inducing molecules? How is the action of an inducer spatially and temporally restricted? How does a limited group of inducers give rise to diversity of tissues? In this review, the focus is on the induction and patterning of mesodermal and neural tissues in the frog Xenopus laevis, with an emphasis on families of secreted molecules that appear to underlie inductive events throughout vertebrate embryogenesis. -
Graff JM, Thies RS, Song JJ, Celeste AJ, Melton DA. 1994. Studies with a Xenopus BMP receptor suggest that ventral mesoderm-inducing signals override dorsal signals in vivo. Cell. 79(1):169-79. Pubmed: 7522972 Graff JM, Thies RS, Song JJ, Celeste AJ, Melton DA. 1994. Studies with a Xenopus BMP receptor suggest that ventral mesoderm-inducing signals override dorsal signals in vivo. Cell. 79(1):169-79. Pubmed: 7522972 We report the isolation of a Xenopus BMP receptor that is expressed maternally in the appropriate location to play a role in mesoderm induction. This receptor binds both BMP-2 and BMP-4 with high affinity. A truncated form of this BMP receptor specifically blocks BMP-4 signaling. Expression of this truncated BMP receptor during embryogenesis converts ventral mesoderm to dorsal mesoderm. Contrary to the popularly held view that ventral is the ground state for all mesoderm, our results suggest that formation of ventral mesoderm requires an active signal and that, in the absence of this ventral signal, dorsal mesoderm is formed. -
Klein PS, Melton DA. 1994. Induction of mesoderm in Xenopus laevis embryos by translation initiation factor 4E. Science (New York, N.Y.). 265(5173):803-6. Pubmed: 8047887 Klein PS, Melton DA. 1994. Induction of mesoderm in Xenopus laevis embryos by translation initiation factor 4E. Science (New York, N.Y.). 265(5173):803-6. Pubmed: 8047887 The microinjection of messenger RNA encoding the eukaryotic translation initiation factor 4E (eIF-4E) into early embryos of Xenopus laevis leads to the induction of mesoderm in ectodermal explants. This induction occurs without a stimulation of overall protein synthesis and is blocked by the co-expression of a dominant negative mutant of the proto-oncogene ras or a truncated activin type II receptor. Although other translation factors have been studied in vertebrate and invertebrate embryos, none have been shown to play a direct role in development. The results here suggest a mechanism for relaying and amplifying signals for mesoderm induction. -
Wilson PA, Melton DA. 1994. Mesodermal patterning by an inducer gradient depends on secondary cell-cell communication. Current biology : CB. 4(8):676-86. Pubmed: 7953553 Wilson PA, Melton DA. 1994. Mesodermal patterning by an inducer gradient depends on secondary cell-cell communication. Current biology : CB. 4(8):676-86. Pubmed: 7953553 Array 1993
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Dohrmann CE, Hemmati-Brivanlou A, Thomsen GH, Fields A, Woolf TM, Melton DA. 1993. Expression of activin mRNA during early development in Xenopus laevis. Developmental biology. 157(2):474-83. Pubmed: 8500654 Dohrmann CE, Hemmati-Brivanlou A, Thomsen GH, Fields A, Woolf TM, Melton DA. 1993. Expression of activin mRNA during early development in Xenopus laevis. Developmental biology. 157(2):474-83. Pubmed: 8500654 Activins are members of the transforming growth factor-beta superfamily, a class of peptide growth factors that can regulate the growth and differentiation of a variety of cell types. In mesoderm induction assays, activins A and B were shown to be very potent inducers and it was only recently demonstrated that they are crucial for initial mesoderm induction in Xenopus embryos. To determine the source of activin protein for initial mesoderm induction and to investigate whether activins may play further roles in embryonic development we have examined the localization of the mRNAs encoding the activin beta A and beta B subunits in Xenopus embryos. Activin beta A and beta B mRNAs are found in the follicle cells surrounding oocytes but not in oocytes themselves or fertilized eggs. During embryogenesis activin mRNA is first detected after the midblastula transition and expression increases as development proceeds. Activin beta B mRNA is homogeneously distributed during blastula and early gastrula stages but restricted to the dorso-anterior region in neurula stage embryos. At the early tailbud stage activin expression becomes confined to the brain, eye analgen, visceral pouches, otic vesicles, and the anterior notochord. -
Thomsen GH, Melton DA. 1993. Processed Vg1 protein is an axial mesoderm inducer in Xenopus. Cell. 74(3):433-41. Pubmed: 8348610 Thomsen GH, Melton DA. 1993. Processed Vg1 protein is an axial mesoderm inducer in Xenopus. Cell. 74(3):433-41. Pubmed: 8348610 Vg1 is a TGF beta-related growth factor encoded by a maternal mRNA localized to vegetal blastomeres in Xenopus embryos. Vg1 precursor protein is abundant in vegetal cells, but the processed mature form has not been readily detected and no activity has been demonstrated for the putative Vg1 mature protein. We have engineered a BMP2-Vg1 fusion (BVg1) that promotes formation of mature Vg1 protein in vivo. Injection of BVg1 mRNA induces dorsal mesoderm in animal cap cells, and BVg1 expression in ultraviolet-ventralized embryos fully restores a normal dorsal axis. Blastomeres expressing BVg1 act as a Nieuwkoop center, the region that induces the Spemann organizer. our results lead us to suggest that localized posttranslational processing of Vg1 precursor protein on the future dorsal side of the embryo is a key step in generating dorsal mesoderm and the body axis in Xenopus. -
Ku M, Melton DA. 1993. Xwnt-11: a maternally expressed Xenopus wnt gene. Development (Cambridge, England). 119(4):1161-73. Pubmed: 8306880 Ku M, Melton DA. 1993. Xwnt-11: a maternally expressed Xenopus wnt gene. Development (Cambridge, England). 119(4):1161-73. Pubmed: 8306880 We have isolated and characterized a novel Xenopus wnt gene, Xwnt-11, whose expression pattern and overexpression phenotype suggest that it may be important for dorsal-ventral axis formation. Xwnt-11 mRNA is present during oogenesis and embryonic development through swimming tadpole stages. Xwnt-11 mRNA is ubiquitous in early oocytes and is localized during mid-oogenesis. By late oocyte stages, Xwnt-11 mRNA is localized to the vegetal cortex, with some mRNA in the vegetal cytoplasm. After egg maturation, Xwnt-11 mRNA is released from the vegetal cortex and is found in the vegetal cytoplasm. This early pattern of Xwnt-11 mRNA localization is similar to another vegetally localized maternal mRNA, Vg1 (D. A. Melton (1987) Nature 328, 80-82). In the late blastula, Xwnt-11 mRNA is found at high levels in the dorsal marginal zone. As gastrulation proceeds, Xwnt-11 mRNA appears in the lateral and ventral marginal zone and, during tadpole stages, it is found in the somites and first branchial arch. Injection of Xwnt-11 mRNA into UV-ventralized embryos can substantially rescue the UV defect by inducing the formation of dorsal tissues. The rescued embryos develop somitic muscle and neural tube; however, they lack notochord and anterior head structures. 1992
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Jessell TM, Melton DA. 1992. Diffusible factors in vertebrate embryonic induction. Cell. 68(2):257-70. Pubmed: 1310256 Jessell TM, Melton DA. 1992. Diffusible factors in vertebrate embryonic induction. Cell. 68(2):257-70. Pubmed: 1310256 -
Woolf TM, Melton DA, Jennings CG. 1992. Specificity of antisense oligonucleotides in vivo. Proceedings of the National Academy of Sciences of the United States of America. 89(16):7305-9. Pubmed: 1380154 Woolf TM, Melton DA, Jennings CG. 1992. Specificity of antisense oligonucleotides in vivo. Proceedings of the National Academy of Sciences of the United States of America. 89(16):7305-9. Pubmed: 1380154 Antisense oligonucleotides are widely used as inhibitors of gene expression in cultured cells and have been proposed as potential therapeutic agents, but it is not known to what extent they are specific for their intended target RNAs. Statistical considerations indicate that if oligonucleotides can form hybrids with mRNA molecules in vivo by means of short or imperfect regions of complementarity, then the specificity of oligonucleotides as antisense reagents will be greatly compromised. We have used Xenopus oocytes as a model system in which to investigate the potential specificity of antisense oligonucleotides in vivo. We injected perfect and partially matched antisense oligonucleotides into oocytes and measured the resulting degradation of the target RNA in each case. On the basis of the extent to which antisense oligonucleotides can cause cleavage of RNAs at imperfectly matched target sites, we conclude that in this system it is probably not possible to obtain specific cleavage of an intended target RNA without also causing at least the partial destruction of many nontargeted RNAs. -
Mowry KL, Melton DA. 1992. Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes. Science (New York, N.Y.). 255(5047):991-4. Pubmed: 1546297 Mowry KL, Melton DA. 1992. Vegetal messenger RNA localization directed by a 340-nt RNA sequence element in Xenopus oocytes. Science (New York, N.Y.). 255(5047):991-4. Pubmed: 1546297 Contained within a single cell, the fertilized egg, is information that will ultimately specify the entire organism. During early embryonic cleavages, cells acquire distinct fates and their differences in developmental potential might be explained by localization of informational molecules in the egg. The mechanisms by which Vg1 RNA, a maternal mRNA, is translocated to the vegetal pole of Xenopus oocytes may indicate how developmental signals are localized. Data presented here show that a 340-nucleotide localization signal present in the 3' untranslated region of Vg1 RNA is sufficient to direct RNA localization to the vegetal pole. -
Hemmati-Brivanlou A, Melton DA. 1992. A truncated activin receptor inhibits mesoderm induction and formation of axial structures in Xenopus embryos. Nature. 359(6396):609-14. Pubmed: 1328888 Hemmati-Brivanlou A, Melton DA. 1992. A truncated activin receptor inhibits mesoderm induction and formation of axial structures in Xenopus embryos. Nature. 359(6396):609-14. Pubmed: 1328888 Activins can induce mesoderm in embryonic explants and have been proposed as the natural inducer in Xenopus. A mutant activin receptor that inhibits activin signalling is used to show that activin is required for the induction of mesoderm in vivo and the patterning of the embryonic body plan. Blocking the activin signal transduction pathway also reveals autonomous induction of a neural marker and unmasks a relationship between activin and fibroblast growth factor. -
Hemmati-Brivanlou A, Wright DA, Melton DA. 1992. Embryonic expression and functional analysis of a Xenopus activin receptor. Developmental dynamics : an official publication of the American Association of Anatomists. 194(1):1-11. Pubmed: 1384808 Hemmati-Brivanlou A, Wright DA, Melton DA. 1992. Embryonic expression and functional analysis of a Xenopus activin receptor. Developmental dynamics : an official publication of the American Association of Anatomists. 194(1):1-11. Pubmed: 1384808 We report the isolation and characterization of a Xenopus activin receptor (XAR1). The amino acid sequence of this protein shows extensive homology with a murine activin receptor. The mRNA is expressed maternally and is ubiquitously distributed during the early stages of embryogenesis. Consistent with a possible role in mesoderm induction and patterning, interference with the normal expression of the receptor by overexpression in the early embryo results in the formation of ectopic dorsal axial structures. During neurulation the XAR1 mRNA is expressed predominantly in the presumptive brain and spinal cord, suggesting an additional function for XAR1 in neurogenesis. -
Sokol SY, Melton DA. 1992. Interaction of Wnt and activin in dorsal mesoderm induction in Xenopus. Developmental biology. 154(2):348-55. Pubmed: 1426642 Sokol SY, Melton DA. 1992. Interaction of Wnt and activin in dorsal mesoderm induction in Xenopus. Developmental biology. 154(2):348-55. Pubmed: 1426642 Both the activin and Wnt families of peptide growth factors are capable of inducing dorsal mesoderm in Xenopus embryos. Presumptive ventral ectoderm cells isolated from embryos injected with Xwnt8 mRNA were cultured in the presence of activin A to study the possible interactions between these two classes of signaling proteins. We find that overexpression of Xwnt8 RNA alters the response of ventral ectoderm to activin such that ventral explants differentiate dorsoanterior structures including notochord and eyes. This response is similar to the response of dorsal ectoderm to activin alone. When embryos are irradiated with uv light to inhibit dorsal axis formation, ectodermal explants differentiate notochord when they are induced by a combination of both signaling factors, but not when cells receive only one inducing signal (activin or Xwnt8). This result is further supported by the observation that goosecoid (gsc) mRNA, an early marker for dorsal mesoderm, is expressed in these explants only when they are injected with Xwnt8 mRNA followed by exposure to activin. Early morphogenetic movements of the induced cells and activation of muscle-specific actin and Brachyury (Xbra) genes also reveal a cooperation of activin A and Xwnt8 in mesoderm induction. 1991
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Sokol S, Christian JL, Moon RT, Melton DA. 1991. Injected Wnt RNA induces a complete body axis in Xenopus embryos. Cell. 67(4):741-52. Pubmed: 1834344 Sokol S, Christian JL, Moon RT, Melton DA. 1991. Injected Wnt RNA induces a complete body axis in Xenopus embryos. Cell. 67(4):741-52. Pubmed: 1834344 Studies in Xenopus have shown that growth factors of the TGF beta and Wnt oncogene families can mimic aspects of dorsal axis formation. Here we directly compare the inductive properties of two Wnt proteins by injecting synthetic mRNA into developing embryos. The results show that Wnt-1 and Xwnt-8 can induce a new and complete dorsal axis and can rescue the development of axis-deficient, UV-irradiated embryos. In contrast, activin mRNA injection induces only a partial dorsal axis that lacks anterior structures. These studies demonstrate that the mechanism of Wnt-induced axis duplication results from the creation of an independent Spemann organizer. The relationship between the properties of the endogenous dorsal inducer and the effects of Wnts and activins is discussed. -
Melton DA. 1991. Pattern formation during animal development. Science (New York, N.Y.). 252(5003):234-41. Pubmed: 1672778 Melton DA. 1991. Pattern formation during animal development. Science (New York, N.Y.). 252(5003):234-41. Pubmed: 1672778 At the beginning of this century, embryologists defined the central problems of developmental biology that remain today. These questions include how differentiated cells arise and form tissues and organs and how pattern is generated. In short, how does an egg give rise to an adult? In recent years, the application of molecular biology to embryological problems has led to significant advances and recast old problems in molecular and cellular terms. Although not necessarily comprehensive, this idiosyncratic review is intended to highlight selected findings and indicate where there are important gaps in our knowledge for those less than familiar with developmental biology. -
Sokol S, Melton DA. 1991. Pre-existent pattern in Xenopus animal pole cells revealed by induction with activin. Nature. 351(6325):409-11. Pubmed: 2034291 Sokol S, Melton DA. 1991. Pre-existent pattern in Xenopus animal pole cells revealed by induction with activin. Nature. 351(6325):409-11. Pubmed: 2034291 Activin, a peptide growth factor related to tumour growth factor-beta, has been implicated in early inductive interactions in vertebrates and can induce Xenopus blastula ectodermal explants to develop a rudimentary axial pattern with anteroposterior and dorsoventral polarity. Here we demonstrate that prospective dorsal and ventral regions of the ectoderm respond differently to the same concentration of activin. Thus, activin does not seem to endow ectodermal cells with polarity but rather reveals a pre-existent pattern. Our results suggest that patterning of mesoderm is determined not only by a localized inducer, but also by the differential competence of cells in the responding tissue. -
Vize PD, Melton DA, Hemmati-Brivanlou A, Harland RM. 1991. Assays for gene function in developing Xenopus embryos. Methods in cell biology. 36:367-87. Pubmed: 1811145 Vize PD, Melton DA, Hemmati-Brivanlou A, Harland RM. 1991. Assays for gene function in developing Xenopus embryos. Methods in cell biology. 36:367-87. Pubmed: 1811145 -
O'Keefe HP, Melton DA, Ferreiro B, Kintner C. 1991. In situ hyridization. Methods in cell biology. 36:443-63. Pubmed: 1811146 O'Keefe HP, Melton DA, Ferreiro B, Kintner C. 1991. In situ hyridization. Methods in cell biology. 36:443-63. Pubmed: 1811146 1990
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Woolf TM, Jennings CG, Rebagliati M, Melton DA. 1990. The stability, toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos. Nucleic acids research. 18(7):1763-9. Pubmed: 1692405 Woolf TM, Jennings CG, Rebagliati M, Melton DA. 1990. The stability, toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos. Nucleic acids research. 18(7):1763-9. Pubmed: 1692405 The properties of antisense phosphorothioate and unmodified oligodeoxynucleotides have been studied in Xenopus oocytes and embryos. We find that phosphorothioates, like unmodified oligodeoxynucleotides, can degrade Vg1 mRNA in oocytes via an endogenous RNase H-like activity. In oocytes, phosphorothioate oligodeoxynucleotides are more stable than unmodified oligodeoxynucleotides and are more effective in degrading Vg1 mRNA. In embryos, neither unmodified nor phosphorothioate deoxyoligonucleotides were effective in degrading Vg1 message at sub-toxic doses. -
Yisraeli JK, Sokol S, Melton DA. 1990. A two-step model for the localization of maternal mRNA in Xenopus oocytes: involvement of microtubules and microfilaments in the translocation and anchoring of Vg1 mRNA. Development (Cambridge, England). 108(2):289-98. Pubmed: 2351071 Yisraeli JK, Sokol S, Melton DA. 1990. A two-step model for the localization of maternal mRNA in Xenopus oocytes: involvement of microtubules and microfilaments in the translocation and anchoring of Vg1 mRNA. Development (Cambridge, England). 108(2):289-98. Pubmed: 2351071 In an effort to understand how polarity is established in Xenopus oocytes, we have analyzed the process of localization of the maternal mRNA, Vg1. In fully grown oocytes, Vg1 mRNA is tightly localized at the vegetal cortex. Biochemical fractionation shows that the mRNA is preferentially associated with a detergent-insoluble subcellular fraction. The use of cytoskeletal inhibitors suggests that (1) microtubules are involved in the translocation of the message to the vegetal hemisphere and (2) microfilaments are important for the anchoring of the message at the cortex. Furthermore, immunohistochemistry reveals that a cytoplasmic microtubule array exists during translocation. These results suggest a role for the cytoskeleton in localizing information in the oocyte. -
Ruiz i Altaba A, Melton DA. 1990. Axial patterning and the establishment of polarity in the frog embryo. Trends in genetics : TIG. 6(2):57-64. Pubmed: 2186530 Ruiz i Altaba A, Melton DA. 1990. Axial patterning and the establishment of polarity in the frog embryo. Trends in genetics : TIG. 6(2):57-64. Pubmed: 2186530 We review the early development of the frog Xenopus laevis, concentrating on events that lead to the establishment of the body plan. Recent molecular and embryological experiments suggest that peptide growth factors and homeobox genes interact to specify cell fates and pattern during early development of this vertebrate. -
Whitman M, Melton DA. 1990. Signal transduction during mesoderm induction in Xenopus. Journal of reproduction and fertility. Supplement. 42:249-54. Pubmed: 2077128 Whitman M, Melton DA. 1990. Signal transduction during mesoderm induction in Xenopus. Journal of reproduction and fertility. Supplement. 42:249-54. Pubmed: 2077128 -
Sokol S, Wong GG, Melton DA. 1990. A mouse macrophage factor induces head structures and organizes a body axis in Xenopus. Science (New York, N.Y.). 249(4968):561-4. Pubmed: 2382134 Sokol S, Wong GG, Melton DA. 1990. A mouse macrophage factor induces head structures and organizes a body axis in Xenopus. Science (New York, N.Y.). 249(4968):561-4. Pubmed: 2382134 Soluble peptide factors have been implicated as the agents responsible for embryonic inductions in vertebrates. Here, a protein (PIF) secreted by a mouse macrophage cell line is shown to change the developmental fate of Xenopus embryonic cells. Exposure to PIF causes presumptive ectodermal explants to form anterior neural and mesodermal tissues, including brain and eye, instead of ciliated epidermis. In addition, the induced tissues are organized into a rudimentary embryonic axis. These results suggest that PIF or a closely related molecule is involved in inducing anterior structures and organizing the frog body plan. -
Thomsen G, Woolf T, Whitman M, Sokol S, Vaughan J, Vale W, Melton DA. 1990. Activins are expressed early in Xenopus embryogenesis and can induce axial mesoderm and anterior structures. Cell. 63(3):485-93. Pubmed: 2225062 Thomsen G, Woolf T, Whitman M, Sokol S, Vaughan J, Vale W, Melton DA. 1990. Activins are expressed early in Xenopus embryogenesis and can induce axial mesoderm and anterior structures. Cell. 63(3):485-93. Pubmed: 2225062 We show that mammalian and Xenopus activins induce dorsal axial mesoderm and anterior structures in explants of Xenopus blastula cells that would otherwise form epidermis. The induced explants of animal cap cells can form notochord, muscle, neural tissue, and eyes all arranged in a rudimentary axial pattern. Activin A shares inductive properties and antigenic determinants with PIF, an inducing factor recently isolated from mouse macrophage culture supernatants. Genes encoding Xenopus activin beta A and beta B chains were cloned. Activin beta B transcripts are first detected in Xenopus blastula, whereas activin beta A transcripts do not appear until the late gastrula stage. Recombinant Xenopus activin beta B protein induces mesodermal and neural tissues similar to those induced by mammalian activin A and PIF. Furthermore, ectopic expression of Xenopus activin beta B produces a second body axis in embryos injected with synthetic mRNA. Our results suggest that early induction and axial patterning are accomplished by endogenous activin B, not activin A, in Xenopus. 1989
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Tannahill D, Melton DA. 1989. Localized synthesis of the Vg1 protein during early Xenopus development. Development (Cambridge, England). 106(4):775-85. Pubmed: 2562668 Tannahill D, Melton DA. 1989. Localized synthesis of the Vg1 protein during early Xenopus development. Development (Cambridge, England). 106(4):775-85. Pubmed: 2562668 The Xenopus Vg1 gene encodes a maternal mRNA that is localized to the vegetal hemisphere of both oocytes and embryos and encodes a protein related to the TGF-beta family of small secreted growth factors. We have raised antibodies to recombinant Vg1 protein and used them to show that Vg1 protein is first detected in stage IV oocytes and reaches maximal levels in stage VI oocytes and eggs. During embryogenesis, Vg1 protein is synthesized until the gastrula stage. The embryonically synthesized Vg1 protein is present only in vegetal cells of an early blastula. We find that Vg1 protein is glycosylated and associated with membranes in the early embryo. Our results also suggest that a small proportion of the full-length Vg1 protein is cleaved to give a small peptide of M(r) = approximately 17 x 10(3). These results support the proposal that the Vg1 protein is an endogenous growth-factor-like molecule involved in mesoderm induction within the amphibian embryo. -
Ruiz i Altaba A, Melton DA. 1989. Bimodal and graded expression of the Xenopus homeobox gene Xhox3 during embryonic development. Development (Cambridge, England). 106(1):173-83. Pubmed: 2576399 Ruiz i Altaba A, Melton DA. 1989. Bimodal and graded expression of the Xenopus homeobox gene Xhox3 during embryonic development. Development (Cambridge, England). 106(1):173-83. Pubmed: 2576399 A Xenopus laevis homeobox gene, Xhox3, has been isolated using the homeobox of the Drosophila pair-rule gene even skipped as a hybridization probe. Xhox3 is first transcribed at the midblastula transition; RNA levels peak at the early neurula stage and decrease thereafter. During the early period of Xhox3 expression, the gastrula and neurula stages, transcripts are found in a graded fashion along the anteroposterior (A-P) axis in the mesoderm and are most concentrated at the posterior pole. In the late period of expression, the tailbud and tadpole stage, transcripts are concentrated at the two ends of the embryo: in the anterior nervous system and posterior tail bud. Analysis of Xhox3 expression in experimentally perturbed embryos shows that different A-P fates in the mesoderm are correlated with different levels of Xhox3 expression. Based on these results and those with other frog homeobox genes, we propose a role for homeobox genes in the patterning of the A-P embryonic axis. -
Krieg PA, Varnum SM, Wormington WM, Melton DA. 1989. The mRNA encoding elongation factor 1-alpha (EF-1 alpha) is a major transcript at the midblastula transition in Xenopus. Developmental biology. 133(1):93-100. Pubmed: 2707491 Krieg PA, Varnum SM, Wormington WM, Melton DA. 1989. The mRNA encoding elongation factor 1-alpha (EF-1 alpha) is a major transcript at the midblastula transition in Xenopus. Developmental biology. 133(1):93-100. Pubmed: 2707491 A Xenopus laevis gastrula cDNA library has been screened in order to identify sequences that are expressed early in development. We find that the mRNA encoding translation elongation factor 1-alpha (EF-1 alpha) is synthesized in very large amounts in the early embryo. Transcription of EF-1 alpha mRNA commences at the midblastula transition (MBT), and new EF-1 alpha protein is synthesized very soon after this, as determined by the association of EF-1 alpha mRNA with polysomes. The nucleotide sequence of a full-length EF-1 alpha cDNA clone and the deduced amino acid sequence of Xenopus EF-1 alpha protein are presented. -
Ruiz i Altaba A, Melton DA. 1989. Involvement of the Xenopus homeobox gene Xhox3 in pattern formation along the anterior-posterior axis. Cell. 57(2):317-26. Pubmed: 2564813 Ruiz i Altaba A, Melton DA. 1989. Involvement of the Xenopus homeobox gene Xhox3 in pattern formation along the anterior-posterior axis. Cell. 57(2):317-26. Pubmed: 2564813 The Xenopus homeobox gene Xhox3 shows a graded expression in the axial mesoderm, with the highest concentration in the posterior end of frog gastrula and neurula embryos. To investigate the function of the Xhox3 gene, synthetic Xhox3 mRNA was injected into different regions of developing embryos. In particular, Xhox3 was supplied in excess to anterior cells, which normally have the lowest levels of Xhox3 RNA. The results show that injection of Xhox3, but not control, mRNA into prospective anterior regions of developing embryos produces a series of graded axial defects. The injected embryos gastrulate normally but fail to form anterior (head) structures. Our findings suggest that Xhox3 is involved in establishing anterior-posterior cell identities during pattern formation of the axial mesoderm in early embryonic development. -
Ruiz i Altaba A, Melton DA. 1989. Interaction between peptide growth factors and homoeobox genes in the establishment of antero-posterior polarity in frog embryos. Nature. 341(6237):33-8. Pubmed: 2570357 Ruiz i Altaba A, Melton DA. 1989. Interaction between peptide growth factors and homoeobox genes in the establishment of antero-posterior polarity in frog embryos. Nature. 341(6237):33-8. Pubmed: 2570357 The expression of the Xenopus homoeobox gene xhox3 is an early response to mesoderm induction by peptide growth factors and the level of xhox3 expression marks the antero-posterior character of the induced mesoderm. Different peptide growth factors specify different antero-posterior mesodermal cell fates as seen by the level of xhox3 expression and the capacity to induce specific secondary neural/epidermal structures. These factors and homoeobox genes thus form part of the mechanism necessary for establishing antero-posterior polarity in the frog embryo. -
Whitman M, Melton DA. 1989. Growth factors in early embryogenesis. Annual review of cell biology. 5:93-117. Pubmed: 2688710 Whitman M, Melton DA. 1989. Growth factors in early embryogenesis. Annual review of cell biology. 5:93-117. Pubmed: 2688710 -
Yisraeli JK, Sokol S, Melton DA. 1989. The process of localizing a maternal messenger RNA in Xenopus oocytes. Development (Cambridge, England). 107 Suppl:31-6. Pubmed: 2636138 Yisraeli JK, Sokol S, Melton DA. 1989. The process of localizing a maternal messenger RNA in Xenopus oocytes. Development (Cambridge, England). 107 Suppl:31-6. Pubmed: 2636138 The maternal mRNA Vg1 is localized to the vegetal pole during oogenesis in Xenopus. We have cultured oocytes in vitro to begin to understand how this localization occurs. Endogenous Vg1 mRNA undergoes localization when oocytes are cultured in vitro, and synthetic Vg1 mRNA injected into such oocytes is localized in the same fashion. Vg1 mRNA is associated with a detergent-insoluble fraction from homogenized oocytes, suggesting a possible cytoskeletal association. The use of cytoskeletal inhibitors reveals a two-step process for localizing Vg1 mRNA. Microtubule inhibitors such as nocodazole and colchicine inhibit the localization of Vg1 mRNA in late stage III/early stage IV oocytes, but have no effect on Vg1 mRNA once it is localized. The microfilament inhibitor cytochalasin B, however, has little effect on the translocation of Vg1 mRNA in middle-stage oocytes but causes a release of the message in late-stage oocytes. We propose a model for the localization of Vg1 mRNA in which translocation of the message to the vegetal cortex is achieved via cytoplasmic microtubules and the anchoring of the message at the cortex involves cortical microfilaments. -
Whitman M, Melton DA. 1989. Induction of mesoderm by a viral oncogene in early Xenopus embryos. Science (New York, N.Y.). 244(4906):803-6. Pubmed: 2658054 Whitman M, Melton DA. 1989. Induction of mesoderm by a viral oncogene in early Xenopus embryos. Science (New York, N.Y.). 244(4906):803-6. Pubmed: 2658054 During frog embryogenesis, mesoderm is specified in the equatorial region of the early embryo by a signal from the vegetal hemisphere. Prospective ectodermal cells dissected from the animal hemisphere can be respecified to form mesodermal tissues by recombination with vegetal tissue or by treatment with any of several polypeptide growth factors or growth factor-like molecules. Together with the discovery that several developmental mutations in Drosophila are in genes with significant homology to mammalian mitogens and oncogenes, these observations suggest that early developmental signals may use similar transduction pathways to mitogenic signals characterized in cultured mammalian cells. Whether mesoderm can be induced by activation of intracellular signal transduction pathways implicated in mitogenesis and oncogenesis has been investigated with the viral oncogene polyoma middle T. Microinjection of middle T messenger RNA into early embryos results in the respecification of isolated prospective ectodermal tissue to form characteristic mesodermal structures. Middle T in frog blastomeres appears to associate with cellular activities similar to those observed in polyoma-transformed mouse cells, and transformation-defective middle T mutants fail to induce mesoderm. These results suggest that early inductive signals and mitogenic and oncogenic stimuli may share common signal transduction pathways. -
Melton DA, Ruiz i Altaba A, Yisraeli J, Sokol S. 1989. Localization of mRNA and axis formation during Xenopus embryogenesis. Ciba Foundation symposium. 144:16-29; discussion 29-36, 92-8. Pubmed: 2673675 Melton DA, Ruiz i Altaba A, Yisraeli J, Sokol S. 1989. Localization of mRNA and axis formation during Xenopus embryogenesis. Ciba Foundation symposium. 144:16-29; discussion 29-36, 92-8. Pubmed: 2673675 This paper summarizes our recent work concerned with the developmental polarity of the frog egg and the patterning of the embryonic body plan. In two separate projects, we are studying genes involved in setting up basic embryonic axes. One of these genes, Vg1, codes for a maternal mRNA that is localized in the frog egg. The Vg1 gene is used in studies on the induction of mesoderm and as a probe to understand how the polarity of an egg is established. A second gene, Xhox3, contains a homeodomain and is differentially expressed in the axial mesoderm. Our studies suggest that this homeobox gene is critically involved in setting up different positional values along the anteroposterior axis. 1988
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Yisraeli JK, Melton DA. 1988. The material mRNA Vg1 is correctly localized following injection into Xenopus oocytes. Nature. 336(6199):592-5. Pubmed: 3200307 Yisraeli JK, Melton DA. 1988. The material mRNA Vg1 is correctly localized following injection into Xenopus oocytes. Nature. 336(6199):592-5. Pubmed: 3200307 The animal and vegetal ends of Xenopus oocytes have distinctly different developmental fates. At the molecular level, several maternal mRNAs have been isolated that are localized to either the animal or vegetal hemisphere. One of these mRNAs, Vg1, is distributed homogeneously throughout the cytoplasm of early-stage oocytes and gets localized during oogenesis to a tight shell at the vegetal cortex of middle and late-stage oocytes. We have used an in vitro culture system to demonstrate that exogeneous Vg1 mRNA injected into middle-stage, but not late-stage, oocytes gets localized in a similar fashion to the endogenous message. Furthermore, translation of Vg1 mRNA is not required for the localization of the message itself. These results show that the information necessary to interpret the animal-vegetal polarity in oocytes is present in the naked mRNA transcript. -
Harvey RP, Melton DA. 1988. Microinjection of synthetic Xhox-1A homeobox mRNA disrupts somite formation in developing Xenopus embryos. Cell. 53(5):687-97. Pubmed: 2897242 Harvey RP, Melton DA. 1988. Microinjection of synthetic Xhox-1A homeobox mRNA disrupts somite formation in developing Xenopus embryos. Cell. 53(5):687-97. Pubmed: 2897242 The structural similarity between Drosophila and vertebrate homeobox genes begs the question of whether the vertebrate gene products affect cell fate and pattern formation. To study the function of the Xenopus homeobox protein, Xhox-1A, we microinjected fertilized Xenopus eggs with an excess of synthetic Xhox-RNA and assayed for effects on development. The predominant phenotype is a disturbance in somite formation. When embryos are injected with Xhox-1A mRNA, but not with control mRNAs, morphogenesis of somites occurs chaotically and individual segments are lost. Histological staining, in situ hybridization, and immunohistochemistry indicate that the disorganized somitic tissue has differentiated into muscle cells. Overall, these results suggest that correct regulation of the Xhox-1A gene may be important for the normal development of the segmented somite pattern in early embryos. Moreover, the inferred role of Xhox-1A in somite formation indicates that there may be molecular parallels between mechanisms of segmentation in flies and vertebrates. 1987
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Kintner CR, Melton DA. 1987. Expression of Xenopus N-CAM RNA in ectoderm is an early response to neural induction. Development (Cambridge, England). 99(3):311-25. Pubmed: 2443340 Kintner CR, Melton DA. 1987. Expression of Xenopus N-CAM RNA in ectoderm is an early response to neural induction. Development (Cambridge, England). 99(3):311-25. Pubmed: 2443340 We have isolated Xenopus laevis N-CAM cDNA clones and used these to study the expression of N-CAM RNA during neural induction. The results show that the first marked increase in N-CAM RNA levels occurs during gastrulation when mesoderm comes in contact with ectoderm and induces neural development. In situ hybridization results show that the early expression of N-CAM RNA is localized to the neural plate and its later expression is confined to the neural tube. Induction experiments with explanted germ layers show that N-CAM RNA is not expressed in ectoderm unless there is contact with inducing tissue. Together these results suggest an approach to studying how ectoderm is committed to form neural rather than epidermal tissue. Specifically, the data suggest that neural commitment is marked and perhaps mediated by the transcriptional activation of genes, like N-CAM, in the neural ectoderm. -
Ruiz i Altaba A, Perry-O'Keefe H, Melton DA. 1987. Xfin: an embryonic gene encoding a multifingered protein in Xenopus. The EMBO journal. 6(10):3065-70. Pubmed: 2826129 Ruiz i Altaba A, Perry-O'Keefe H, Melton DA. 1987. Xfin: an embryonic gene encoding a multifingered protein in Xenopus. The EMBO journal. 6(10):3065-70. Pubmed: 2826129 The Xenopus laevis genome was screened for putative DNA-binding gene products by using the 'finger' region of the Drosophila gene Krüppel as a probe. The one gene detected, named Xfin, codes for a protein with 37 finger domains that comprise nearly 90% of the protein. In the light of studies by Rhodes and Klug (Cell, 46, 123-132, 1986), these data suggest that the Xfin protein has the capacity to bind an unusually large stretch (185 bases) of DNA. The Xfin gene is expressed as a maternal and zygotic mRNA that undergoes extensive polyadenylation changes during early development. The Xfin mRNA expression pattern and the potential DNA binding activity of the protein point to the possibility that the Xfin gene may have a role in controlling gene activity during early embryonic development. -
Weeks DL, Melton DA. 1987. A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF-beta. Cell. 51(5):861-7. Pubmed: 3479264 Weeks DL, Melton DA. 1987. A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF-beta. Cell. 51(5):861-7. Pubmed: 3479264 We report that Vg1, a maternal mRNA localized to the vegetal hemisphere of frog eggs, encodes a member of the transforming growth factor-beta (TGF-beta) family of proteins. Furthermore, we show that Vg1 mRNA is distributed to presumptive endodermal cells after fertilization. Previous studies had shown that the vegetal end of a frog egg produces a signal that induces the overlying animal pole cells to form mesodermal tissue. More recently it has been shown that fibroblast growth factor (FGF) and TGF-beta can participate in the induction of muscle. Together, these results lead us to propose that the formation of mesoderm during frog development is specified by the products of localized maternal mRNAs, including Vg1. -
Sherwood RI, Maehr R, Mazzoni EO, Melton DA. Wnt signaling specifies and patterns intestinal endoderm. Mechanisms of development. 128(7-10):387-400. Pubmed: 21854845 DOI:10.1016/j.mod.2011.07.005 Sherwood RI, Maehr R, Mazzoni EO, Melton DA. Wnt signaling specifies and patterns intestinal endoderm. Mechanisms of development. 128(7-10):387-400. Pubmed: 21854845 DOI:10.1016/j.mod.2011.07.005 Wnt signaling has been implicated in many developmental processes, but its role in early endoderm development is not well understood. Wnt signaling is active in posterior endoderm as early as E7.5. Genetic and chemical activation show that the Wnt pathway acts directly on endoderm to induce the intestinal master regulator Cdx2, shifting global gene away from anterior endoderm and toward a posterior, intestinal program. In a mouse embryonic stem cell differentiation platform that yields pure populations of definitive endoderm, Wnt signaling induces intestinal gene expression in all cells. We have identified a set of genes specific to the anterior small intestine, posterior small intestine, and large intestine during early development, and show that Wnt, through Cdx2, activates large intestinal gene expression at high doses and small intestinal gene expression at lower doses. These findings shed light on the mechanism of embryonic intestinal induction and provide a method to manipulate intestinal development from embryonic stem cells.Copyright © 2011 Elsevier Ireland Ltd. All rights reserved. -
Melton D. Signals for tissue induction and organ formation in vertebrate embryos. Harvey lectures. 93:49-64. Pubmed: 10941418 Melton D. Signals for tissue induction and organ formation in vertebrate embryos. Harvey lectures. 93:49-64. Pubmed: 10941418