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Ma H, Peng G, Hu Y, Lu B, Zheng Y, Wu Y, Feng W, Shi Y, Pan X, Song L, Stützer I, Liu Y, Fei J. Revealing the biological features of the axolotl pancreas as a new research model. Front Cell Dev Biol 2025; 13:1531903. [PMID: 39958891 PMCID: PMC11825805 DOI: 10.3389/fcell.2025.1531903] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2024] [Accepted: 01/07/2025] [Indexed: 02/18/2025] Open
Abstract
Introduction The pancreas plays a crucial role in digestion and blood glucose regulation. Current animal models, primarily mice and zebrafish, have limited the exploration of pancreatic biology from an evolutionary-developmental perspective. Tetrapod vertebrate axolotl (Ambystoma mexicanum) serves as a valuable model in developmental, regenerative, and evolutionary biology. However, the fundamental biology of the axolotl pancreas remains underexplored. This study aims to characterize the unique developmental, functional, and evolutionary features of the axolotl pancreas to expand the understanding of pancreatic biology. Methods We conducted morphological, histological, and transcriptomic analyses to investigate the axolotl pancreas. Pancreatic development was observed using in situ hybridization and immunostaining for key pancreatic markers. RNA sequencing was performed to profile global gene expression during larva and adult stages. And differential gene expression analysis was used to characterize the conserved and unique gene patterns in the axolotl pancreas. Functional assays, including glucose tolerance tests and insulin tolerance tests, were optimized for individual axolotls. To assess pancreatic gene function, Pdx1 mutants were generated using CRISPR/Cas9-mediated gene editing, and their effects on pancreatic morphology, endocrine cell populations, and glucose homeostasis were analyzed. Results The axolotl pancreas contains all known pancreatic cell types and develops from dorsal and ventral buds. Both of buds contribute to exocrine and endocrine glands. The dorsal bud produces the major endocrine cell types, while the ventral bud generates α and δ cells, but not β cells. Differential gene expression analysis indicated a transition in global gene expression from pancreatic cell fate commitment and the cell cycle to glucose response, hormone synthesis, and secretion, following the development progression. Notably, the adult axolotl pancreas exhibits slower metabolic activity compared to mammals, as evidenced by the results of GTT and ITT. The mutation of Pdx1 resulted in hyperglycemia and a significant reduction in pancreatic cell mass, including a complete loss of endocrine cells, although it did not lead to a lethal phenotype. Discussion This study examines the axolotl pancreas, highlighting the conservation of pancreatic development. Our study highlights the unique features of the axolotl pancreas and broadens the scope of animal models available for pancreatic evolution and disease research.
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Affiliation(s)
- Hui Ma
- Department of Pathology, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong, China
- BGI Research, Qingdao, China
- School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
| | - Guangcong Peng
- Key Laboratory of Brain, Cognition and Education Sciences, Guangdong Key Laboratory of Mental Health and Cognitive Science, Ministry of Education, Institute for Brain Research and Rehabilitation, South China Normal University, Guangzhou, China
| | - Yan Hu
- Department of Pathology, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong, China
| | - Binbin Lu
- Department of Pathology, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong, China
- The Innovation Centre of Ministry of Education for Development and Diseases, School of Medicine, South China University of Technology, Guangzhou, China
| | - Yiying Zheng
- Key Laboratory of Brain, Cognition and Education Sciences, Guangdong Key Laboratory of Mental Health and Cognitive Science, Ministry of Education, Institute for Brain Research and Rehabilitation, South China Normal University, Guangzhou, China
| | - Yingxian Wu
- Key Laboratory of Brain, Cognition and Education Sciences, Guangdong Key Laboratory of Mental Health and Cognitive Science, Ministry of Education, Institute for Brain Research and Rehabilitation, South China Normal University, Guangzhou, China
| | - Weimin Feng
- Guangdong Provincial Key Laboratory of Medical Immunology and Molecular Diagnostics, Guangdong Medical University, Dongguan, China
| | - Yu Shi
- Key Laboratory of Brain, Cognition and Education Sciences, Guangdong Key Laboratory of Mental Health and Cognitive Science, Ministry of Education, Institute for Brain Research and Rehabilitation, South China Normal University, Guangzhou, China
| | - Xiangyu Pan
- Department of Pathology, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong, China
| | - Li Song
- Department of Pathology, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong, China
| | - Ina Stützer
- Deutsche Forschungsgemeinschaft (DFG)-Center for Regenerative Therapies Dresden, Technische Universität Dresden, Dresden, Germany
| | - Yanmei Liu
- Key Laboratory of Brain, Cognition and Education Sciences, Guangdong Key Laboratory of Mental Health and Cognitive Science, Ministry of Education, Institute for Brain Research and Rehabilitation, South China Normal University, Guangzhou, China
| | - Jifeng Fei
- Department of Pathology, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong, China
- School of Basic Medical Sciences, Southern Medical University, Guangzhou, China
- The Innovation Centre of Ministry of Education for Development and Diseases, School of Medicine, South China University of Technology, Guangzhou, China
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Wokasch AS, Gannon M. CUTting through the distance: A disease-relevant long-range ONECUT1 enhancer. Cell Rep 2024; 43:114897. [PMID: 39427317 DOI: 10.1016/j.celrep.2024.114897] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2024] [Revised: 10/03/2024] [Accepted: 10/04/2024] [Indexed: 10/22/2024] Open
Abstract
In two recent issues of Cell Reports, Kaplan et al.1 and Merz et al.2 identify a distal enhancer region within the OC1 gene and demonstrate its relevance to OC1 expression, pancreatic endocrine differentiation, and diabetes susceptibility.
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Affiliation(s)
- Anthony S Wokasch
- Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA
| | - Maureen Gannon
- Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA; Department of Veterans Affairs Tennessee Valley, Nashville, TN, USA; Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA; Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA.
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Kaplan SJ, Wong W, Yan J, Pulecio J, Cho HS, Li Q, Zhao J, Leslie-Iyer J, Kazakov J, Murphy D, Luo R, Dey KK, Apostolou E, Leslie CS, Huangfu D. CRISPR screening uncovers a long-range enhancer for ONECUT1 in pancreatic differentiation and links a diabetes risk variant. Cell Rep 2024; 43:114640. [PMID: 39163202 PMCID: PMC11406439 DOI: 10.1016/j.celrep.2024.114640] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2024] [Revised: 07/01/2024] [Accepted: 07/31/2024] [Indexed: 08/22/2024] Open
Abstract
Functional enhancer annotation is critical for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants. However, unbiased enhancer discovery in disease-relevant contexts remains challenging. To identify enhancers pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers identified, we focused on an enhancer we named ONECUT1e-664kb, ∼664 kb from the ONECUT1 promoter. Previous studies have linked ONECUT1 coding mutations to pancreatic hypoplasia and neonatal diabetes. We found that homozygous deletion of ONECUT1e-664kb in hPSCs leads to a near-complete loss of ONECUT1 expression and impaired pancreatic differentiation. ONECUT1e-664kb contains a type 2 diabetes-associated variant (rs528350911) disrupting a GATA motif. Introducing the risk variant into hPSCs reduced binding of key pancreatic transcription factors (GATA4, GATA6, and FOXA2), supporting its causal role in diabetes. This work highlights the utility of unbiased enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.
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Affiliation(s)
- Samuel Joseph Kaplan
- Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medical College, New York, NY 10065, USA; Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Wilfred Wong
- Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medical College, New York, NY 10065, USA; Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Jielin Yan
- Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Julian Pulecio
- Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Hyein S Cho
- Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Qianzi Li
- Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medical College, New York, NY 10065, USA; Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Jiahui Zhao
- Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medical College, New York, NY 10065, USA
| | - Jayanti Leslie-Iyer
- Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Jonathan Kazakov
- Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Dylan Murphy
- Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medical College, New York, NY 10065, USA
| | - Renhe Luo
- Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Kushal K Dey
- Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Effie Apostolou
- Meyer Cancer Center, Division of Neuro-Oncology, Department of Neurology, Sandra and Edward Meyer Cancer Center, New York-Presbyterian Hospital/Weill Cornell Medicine, New York, NY 10065, USA
| | - Christina S Leslie
- Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
| | - Danwei Huangfu
- Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
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Yang Y, Wang J, Wan J, Cheng Q, Cheng Z, Zhou X, Wang O, Shi K, Wang L, Wang B, Zhu X, Chen J, Feng D, Liu Y, Jahan-Mihan Y, Haddock AN, Edenfield BH, Peng G, Hohenstein JD, McCabe CE, O'Brien DR, Wang C, Ilyas SI, Jiang L, Torbenson MS, Wang H, Nakhleh RE, Shi X, Wang Y, Bi Y, Gores GJ, Patel T, Ji B. PTEN deficiency induces an extrahepatic cholangitis-cholangiocarcinoma continuum via aurora kinase A in mice. J Hepatol 2024; 81:120-134. [PMID: 38428643 PMCID: PMC11259013 DOI: 10.1016/j.jhep.2024.02.018] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/31/2023] [Revised: 02/09/2024] [Accepted: 02/18/2024] [Indexed: 03/03/2024]
Abstract
BACKGROUND & AIMS The PTEN-AKT pathway is frequently altered in extrahepatic cholangiocarcinoma (eCCA). We aimed to evaluate the role of PTEN in the pathogenesis of eCCA and identify novel therapeutic targets for this disease. METHODS The Pten gene was genetically deleted using the Cre-loxp system in biliary epithelial cells. The pathologies were evaluated both macroscopically and histologically. The characteristics were further analyzed by immunohistochemistry, reverse-transcription PCR, cell culture, and RNA sequencing. Some features were compared to those in human eCCA samples. Further mechanistic studies utilized the conditional knockout of Trp53 and Aurora kinase A (Aurka) genes. We also tested the effectiveness of an Aurka inhibitor. RESULTS We observed that genetic deletion of the Pten gene in the extrahepatic biliary epithelium and peri-ductal glands initiated sclerosing cholangitis-like lesions in mice, resulting in enlarged and distorted extrahepatic bile ducts in mice as early as 1 month after birth. Histologically, these lesions exhibited increased epithelial proliferation, inflammatory cell infiltration, and fibrosis. With aging, the lesions progressed from low-grade dysplasia to invasive carcinoma. Trp53 inactivation further accelerated disease progression, potentially by downregulating senescence. Further mechanistic studies showed that both human and mouse eCCA showed high expression of AURKA. Notably, the genetic deletion of Aurka completely eliminated Pten deficiency-induced extrahepatic bile duct lesions. Furthermore, pharmacological inhibition of Aurka alleviated disease progression. CONCLUSIONS Pten deficiency in extrahepatic cholangiocytes and peribiliary glands led to a cholangitis-to-cholangiocarcinoma continuum that was dependent on Aurka. These findings offer new insights into preventive and therapeutic interventions for extrahepatic CCA. IMPACT AND IMPLICATIONS The aberrant PTEN-PI3K-AKT signaling pathway is commonly observed in human extrahepatic cholangiocarcinoma (eCCA), a disease with a poor prognosis. In our study, we developed a mouse model mimicking cholangitis to eCCA progression by conditionally deleting the Pten gene via Pdx1-Cre in epithelial cells and peribiliary glands of the extrahepatic biliary duct. The conditional Pten deletion in these cells led to cholangitis, which gradually advanced to dysplasia, ultimately resulting in eCCA. The loss of Pten heightened Akt signaling, cell proliferation, inflammation, fibrosis, DNA damage, epigenetic signaling, epithelial-mesenchymal transition, cell dysplasia, and cellular senescence. Genetic deletion or pharmacological inhibition of Aurka successfully halted disease progression. This model will be valuable for testing novel therapies and unraveling the mechanisms of eCCA tumorigenesis.
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Affiliation(s)
- Yan Yang
- Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA; Department of Medical Oncology, The First Affiliated Hospital of Bengbu Medical University, Bengbu, Anhui, China
| | - Jiale Wang
- Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA
| | - Jianhua Wan
- Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA
| | - Qianqian Cheng
- Department of Medical Oncology, The First Affiliated Hospital of Bengbu Medical University, Bengbu, Anhui, China
| | - Zenong Cheng
- Department of Pathology, The First Affiliated Hospital of Bengbu Medical University, Bengbu, Anhui, China
| | - Xueli Zhou
- Department of Medical Oncology, The First Affiliated Hospital of Bengbu Medical University, Bengbu, Anhui, China
| | - Oliver Wang
- Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA
| | - Kelvin Shi
- Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA
| | - Lingxiang Wang
- Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA
| | - Bin Wang
- Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA
| | - Xiaohui Zhu
- Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA
| | - Jiaxiang Chen
- Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA
| | - Dongfeng Feng
- Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA
| | - Yang Liu
- Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA
| | | | - Ashley N Haddock
- Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA
| | | | - Guang Peng
- Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | | | - Chantal E McCabe
- Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota, USA
| | - Daniel R O'Brien
- Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota, USA
| | - Chen Wang
- Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota, USA
| | - Sumera I Ilyas
- Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, USA
| | - Liuyan Jiang
- Department of Laboratory Medicine and Pathology, Mayo Clinic, Jacksonville, Florida, USA
| | - Michael S Torbenson
- Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA
| | - Huamin Wang
- Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Raouf E Nakhleh
- Department of Laboratory Medicine and Pathology, Mayo Clinic, Jacksonville, Florida, USA
| | - Xuemei Shi
- Greenwood Genetic Center, Greenwood, South Carolina, USA
| | - Ying Wang
- Department of Cardiovascular Medicine, Mayo Clinic, Rochester, Minnesota, USA
| | - Yan Bi
- Department of Gastroenterology and Hepatology, Mayo Clinic, Jacksonville, Florida, USA
| | - Gregory J Gores
- Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, USA
| | - Tushar Patel
- Department of Transplantation, Mayo Clinic, Jacksonville, Florida, USA
| | - Baoan Ji
- Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, USA.
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Kaplan SJ, Wong W, Yan J, Pulecio J, Cho HS, Li Q, Zhao J, Leslie-Iyer J, Kazakov J, Murphy D, Luo R, Dey KK, Apostolou E, Leslie CS, Huangfu D. CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.04.26.591412. [PMID: 38746154 PMCID: PMC11092487 DOI: 10.1101/2024.04.26.591412] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/16/2024]
Abstract
Functional enhancer annotation is a valuable first step for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants for investigation. However, unbiased enhancer discovery in physiologically relevant contexts remains a major challenge. To discover regulatory elements pertinent to diabetes, we conducted a CRISPR interference screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers uncovered, we focused on a long-range enhancer ∼664 kb from the ONECUT1 promoter, since coding mutations in ONECUT1 cause pancreatic hypoplasia and neonatal diabetes. Homozygous enhancer deletion in hPSCs was associated with a near-complete loss of ONECUT1 gene expression and compromised pancreatic differentiation. This enhancer contains a confidently fine-mapped type 2 diabetes associated variant (rs528350911) which disrupts a GATA motif. Introduction of the risk variant into hPSCs revealed substantially reduced binding of key pancreatic transcription factors (GATA4, GATA6 and FOXA2) on the edited allele, accompanied by a slight reduction of ONECUT1 transcription, supporting a causal role for this risk variant in metabolic disease. This work expands our knowledge about transcriptional regulation in pancreatic development through the characterization of a long-range enhancer and highlights the utility of enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.
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Sanchez JG, Rankin S, Paul E, McCauley HA, Kechele DO, Enriquez JR, Jones NH, Greeley SAW, Letourneau-Frieberg L, Zorn AM, Krishnamurthy M, Wells JM. RFX6 regulates human intestinal patterning and function upstream of PDX1. Development 2024; 151:dev202529. [PMID: 38587174 PMCID: PMC11128285 DOI: 10.1242/dev.202529] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2023] [Accepted: 03/12/2024] [Indexed: 04/09/2024]
Abstract
The gastrointestinal (GI) tract is complex and consists of multiple organs with unique functions. Rare gene variants can cause congenital malformations of the human GI tract, although the molecular basis of these has been poorly studied. We identified a patient with compound-heterozygous variants in RFX6 presenting with duodenal malrotation and atresia, implicating RFX6 in development of the proximal intestine. To identify how mutations in RFX6 impact intestinal patterning and function, we derived induced pluripotent stem cells from this patient to generate human intestinal organoids (HIOs). We identified that the duodenal HIOs and human tissues had mixed regional identity, with gastric and ileal features. CRISPR-mediated correction of RFX6 restored duodenal identity. We then used gain- and loss-of-function and transcriptomic approaches in HIOs and Xenopus embryos to identify that PDX1 is a downstream transcriptional target of RFX6 required for duodenal development. However, RFX6 had additional PDX1-independent transcriptional targets involving multiple components of signaling pathways that are required for establishing early regional identity in the GI tract. In summary, we have identified RFX6 as a key regulator in intestinal patterning that acts by regulating transcriptional and signaling pathways.
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Affiliation(s)
- J Guillermo Sanchez
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
- Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
| | - Scott Rankin
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
- Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
| | - Emily Paul
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
- Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
| | - Heather A McCauley
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
- Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
- Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA
| | - Daniel O Kechele
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
- Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
| | - Jacob R Enriquez
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
- Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
| | - Nana-Hawa Jones
- Division of Endocrinology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Siri A W Greeley
- Department of Medicine, University of Chicago, Chicago, IL 60637, USA
- Department of Pediatrics, University of Chicago, Chicago, IL 60637, USA
| | | | - Aaron M Zorn
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
- Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
| | - Mansa Krishnamurthy
- Division of Endocrinology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - James M Wells
- Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
- Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Cincinnati OH 45229, USA
- Division of Endocrinology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
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Siwan D, Nandave M, Gilhotra R, Almalki WH, Gupta G, Gautam RK. Unlocking β-cell restoration: The crucial role of PDX1 in diabetes therapy. Pathol Res Pract 2024; 254:155131. [PMID: 38309018 DOI: 10.1016/j.prp.2024.155131] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/01/2023] [Revised: 01/08/2024] [Accepted: 01/10/2024] [Indexed: 02/05/2024]
Abstract
Diabetes has been a significant healthcare problem worldwide for a considerable period. The primary objective of diabetic treatment plans is to control the symptoms associated with the pathology. To effectively combat diabetes, it is crucial to comprehend the disease's etiology, essential factors, and the relevant processes involving β-cells. The development of the pancreas, maturation, and maintenance of β-cells, and their role in regular insulin function are all regulated by PDX1. Therefore, understanding the regulation of PDX1 and its interactions with signaling pathways involved in β-cell differentiation and proliferation are crucial elements of alternative diabetes treatment strategies. The present review aims to explore the protective role of PDX1 in β-cell proliferation through signaling pathways. The main keywords chosen for this review include "PDX1 for β-cell mass," "β-cell proliferation," "β-cell restoration via PDX1," and "mechanism of PDX1 in β-cells." A comprehensive literature search was conducted using various internet search engines, such as PubMed, Science Direct, and other publication databases. We summarize several approaches to generating β-cells from alternative cell sources, employing PDX1 under various modified growth conditions and different transcriptional factors. Our analysis highlights the unique potential of PDX1 as a promising target in molecular and cell-based therapies for diabetes.
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Affiliation(s)
- Deepali Siwan
- Department of Pharmacology, Delhi Pharmaceutical Sciences and Research University (DPSRU), New Delhi 110017, India
| | - Mukesh Nandave
- Department of Pharmacology, Delhi Pharmaceutical Sciences and Research University (DPSRU), New Delhi 110017, India.
| | - Ritu Gilhotra
- School of Pharmacy, Suresh Gyan Vihar University, Mahal Road, Jagatpura, Jaipur, India
| | - Waleed Hassan Almalki
- Department of Pharmacology, College of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia
| | - Gaurav Gupta
- Center for Global Health Research, Saveetha Medical College, Saveetha Institute of Medical and Technical Sciences, Saveetha University, India; School of Pharmacy, Graphic Era Hill University, Dehradun 248007, India; Centre of Medical and Bio-allied Health Sciences Research, Ajman University, Ajman, Ajman, 346, United Arab Emirates
| | - Rupesh K Gautam
- Department of Pharmacology, Indore Institute of Pharmacy, IIST Campus, Opposite IIM Indore, Rau-Pithampur Road, Indore 453331, Madhya Pradesh, India
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8
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Weidemann BJ, Marcheva B, Kobayashi M, Omura C, Newman MV, Kobayashi Y, Waldeck NJ, Perelis M, Lantier L, McGuinness OP, Ramsey KM, Stein RW, Bass J. Repression of latent NF-κB enhancers by PDX1 regulates β cell functional heterogeneity. Cell Metab 2024; 36:90-102.e7. [PMID: 38171340 PMCID: PMC10793877 DOI: 10.1016/j.cmet.2023.11.018] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/07/2022] [Revised: 07/17/2023] [Accepted: 11/30/2023] [Indexed: 01/05/2024]
Abstract
Interactions between lineage-determining and activity-dependent transcription factors determine single-cell identity and function within multicellular tissues through incompletely known mechanisms. By assembling a single-cell atlas of chromatin state within human islets, we identified β cell subtypes governed by either high or low activity of the lineage-determining factor pancreatic duodenal homeobox-1 (PDX1). β cells with reduced PDX1 activity displayed increased chromatin accessibility at latent nuclear factor κB (NF-κB) enhancers. Pdx1 hypomorphic mice exhibited de-repression of NF-κB and impaired glucose tolerance at night. Three-dimensional analyses in tandem with chromatin immunoprecipitation (ChIP) sequencing revealed that PDX1 silences NF-κB at circadian and inflammatory enhancers through long-range chromatin contacts involving SIN3A. Conversely, Bmal1 ablation in β cells disrupted genome-wide PDX1 and NF-κB DNA binding. Finally, antagonizing the interleukin (IL)-1β receptor, an NF-κB target, improved insulin secretion in Pdx1 hypomorphic islets. Our studies reveal functional subtypes of single β cells defined by a gradient in PDX1 activity and identify NF-κB as a target for insulinotropic therapy.
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Affiliation(s)
- Benjamin J Weidemann
- Department of Medicine, Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Biliana Marcheva
- Department of Medicine, Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Mikoto Kobayashi
- Department of Medicine, Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Chiaki Omura
- Department of Medicine, Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Marsha V Newman
- Department of Medicine, Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Yumiko Kobayashi
- Department of Medicine, Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Nathan J Waldeck
- Department of Medicine, Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Mark Perelis
- Department of Medicine, Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Ionis Pharmaceuticals, Carlsbad, CA 92010, USA
| | - Louise Lantier
- Vanderbilt-NIH Mouse Metabolic Phenotyping Center, Nashville, TN 37232, USA; Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA
| | - Owen P McGuinness
- Vanderbilt-NIH Mouse Metabolic Phenotyping Center, Nashville, TN 37232, USA; Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA
| | - Kathryn Moynihan Ramsey
- Department of Medicine, Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Roland W Stein
- Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA
| | - Joseph Bass
- Department of Medicine, Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
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9
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Wong YF, Kumar Y, Proks M, Herrera JAR, Rothová MM, Monteiro RS, Pozzi S, Jennings RE, Hanley NA, Bickmore WA, Brickman JM. Expansion of ventral foregut is linked to changes in the enhancer landscape for organ-specific differentiation. Nat Cell Biol 2023; 25:481-492. [PMID: 36690849 PMCID: PMC10014581 DOI: 10.1038/s41556-022-01075-8] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2022] [Accepted: 12/14/2022] [Indexed: 01/24/2023]
Abstract
Cell proliferation is fundamental for almost all stages of development and differentiation that require an increase in cell number. Although cell cycle phase has been associated with differentiation, the actual process of proliferation has not been considered as having a specific role. Here we exploit human embryonic stem cell-derived endodermal progenitors that we find are an in vitro model for the ventral foregut. These cells exhibit expansion-dependent increases in differentiation efficiency to pancreatic progenitors that are linked to organ-specific enhancer priming at the level of chromatin accessibility and the decommissioning of lineage-inappropriate enhancers. Our findings suggest that cell proliferation in embryonic development is about more than tissue expansion; it is required to ensure equilibration of gene regulatory networks allowing cells to become primed for future differentiation. Expansion of lineage-specific intermediates may therefore be an important step in achieving high-fidelity in vitro differentiation.
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Affiliation(s)
- Yan Fung Wong
- Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), University of Copenhagen, Copenhagen, Denmark
| | - Yatendra Kumar
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK
| | - Martin Proks
- Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), University of Copenhagen, Copenhagen, Denmark
| | - Jose Alejandro Romero Herrera
- Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), University of Copenhagen, Copenhagen, Denmark
- Center for Health Data Science, University of Copenhagen, Copenhagen, Denmark
| | - Michaela Mrugala Rothová
- Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), University of Copenhagen, Copenhagen, Denmark
| | - Rita S Monteiro
- Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), University of Copenhagen, Copenhagen, Denmark
| | - Sara Pozzi
- Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), University of Copenhagen, Copenhagen, Denmark
| | - Rachel E Jennings
- Faculty of Biology, Medicine & Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK
| | - Neil A Hanley
- Faculty of Biology, Medicine & Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK
| | - Wendy A Bickmore
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, UK.
| | - Joshua M Brickman
- Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), University of Copenhagen, Copenhagen, Denmark.
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10
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Ebrahim N, Shakirova K, Dashinimaev E. PDX1 is the cornerstone of pancreatic β-cell functions and identity. Front Mol Biosci 2022; 9:1091757. [PMID: 36589234 PMCID: PMC9798421 DOI: 10.3389/fmolb.2022.1091757] [Citation(s) in RCA: 28] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2022] [Accepted: 12/01/2022] [Indexed: 12/23/2022] Open
Abstract
Diabetes has been a worldwide healthcare problem for many years. Current methods of treating diabetes are still largely directed at symptoms, aiming to control the manifestations of the pathology. This creates an overall need to find alternative measures that can impact on the causes of the disease, reverse diabetes, or make it more manageable. Understanding the role of key players in the pathogenesis of diabetes and the related β-cell functions is of great importance in combating diabetes. PDX1 is a master regulator in pancreas organogenesis, the maturation and identity preservation of β-cells, and of their role in normal insulin function. Mutations in the PDX1 gene are correlated with many pancreatic dysfunctions, including pancreatic agenesis (homozygous mutation) and MODY4 (heterozygous mutation), while in other types of diabetes, PDX1 expression is reduced. Therefore, alternative approaches to treat diabetes largely depend on knowledge of PDX1 regulation, its interaction with other transcription factors, and its role in obtaining β-cells through differentiation and transdifferentiation protocols. In this article, we review the basic functions of PDX1 and its regulation by genetic and epigenetic factors. Lastly, we summarize different variations of the differentiation protocols used to obtain β-cells from alternative cell sources, using PDX1 alone or in combination with various transcription factors and modified culture conditions. This review shows the unique position of PDX1 as a potential target in the genetic and cellular treatment of diabetes.
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Affiliation(s)
- Nour Ebrahim
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Moscow, Russia,Moscow Institute of Physics and Technology (State University), Dolgoprudny, Russia
| | - Ksenia Shakirova
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Moscow, Russia
| | - Erdem Dashinimaev
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Moscow, Russia,Moscow Institute of Physics and Technology (State University), Dolgoprudny, Russia,*Correspondence: Erdem Dashinimaev,
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11
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Duque M, Amorim JP, Bessa J. Ptf1a function and transcriptional cis-regulation, a cornerstone in vertebrate pancreas development. FEBS J 2022; 289:5121-5136. [PMID: 34125483 PMCID: PMC9545688 DOI: 10.1111/febs.16075] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2020] [Revised: 04/23/2021] [Accepted: 06/14/2021] [Indexed: 12/11/2022]
Abstract
Vertebrate pancreas organogenesis is a stepwise process regulated by a complex network of signaling and transcriptional events, progressively steering the early endoderm toward pancreatic fate. Many crucial players of this process have been identified, including signaling pathways, cis-regulatory elements, and transcription factors (TFs). Pancreas-associated transcription factor 1a (PTF1A) is one such TF, crucial for pancreas development. PTF1A mutations result in dramatic pancreatic phenotypes associated with severe complications, such as neonatal diabetes and impaired food digestion due to exocrine pancreatic insufficiency. Here, we present a brief overview of vertebrate pancreas development, centered on Ptf1a function and transcriptional regulation, covering similarities and divergences in three broadly studied organisms: human, mouse and zebrafish.
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Affiliation(s)
- Marta Duque
- Instituto de Biologia Molecular e Celular (IBMC)Universidade do PortoPortugal
- Instituto de Investigação e Inovação em Saúde (i3S)Universidade do PortoPortugal
- Doctoral program in Molecular and Cell Biology (MCbiology)Instituto de Ciências Biomédicas Abel Salazar (ICBAS)Universidade do PortoPortugal
| | - João Pedro Amorim
- Instituto de Biologia Molecular e Celular (IBMC)Universidade do PortoPortugal
- Instituto de Investigação e Inovação em Saúde (i3S)Universidade do PortoPortugal
- Doctoral program in Molecular and Cell Biology (MCbiology)Instituto de Ciências Biomédicas Abel Salazar (ICBAS)Universidade do PortoPortugal
| | - José Bessa
- Instituto de Biologia Molecular e Celular (IBMC)Universidade do PortoPortugal
- Instituto de Investigação e Inovação em Saúde (i3S)Universidade do PortoPortugal
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12
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Bordeira-Carriço R, Teixeira J, Duque M, Galhardo M, Ribeiro D, Acemel RD, Firbas PN, Tena JJ, Eufrásio A, Marques J, Ferreira FJ, Freitas T, Carneiro F, Goméz-Skarmeta JL, Bessa J. Multidimensional chromatin profiling of zebrafish pancreas to uncover and investigate disease-relevant enhancers. Nat Commun 2022; 13:1945. [PMID: 35410466 PMCID: PMC9001708 DOI: 10.1038/s41467-022-29551-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2020] [Accepted: 03/17/2022] [Indexed: 11/26/2022] Open
Abstract
The pancreas is a central organ for human diseases. Most alleles uncovered by genome-wide association studies of pancreatic dysfunction traits overlap with non-coding sequences of DNA. Many contain epigenetic marks of cis-regulatory elements active in pancreatic cells, suggesting that alterations in these sequences contribute to pancreatic diseases. Animal models greatly help to understand the role of non-coding alterations in disease. However, interspecies identification of equivalent cis-regulatory elements faces fundamental challenges, including lack of sequence conservation. Here we combine epigenetic assays with reporter assays in zebrafish and human pancreatic cells to identify interspecies functionally equivalent cis-regulatory elements, regardless of sequence conservation. Among other potential disease-relevant enhancers, we identify a zebrafish ptf1a distal-enhancer whose deletion causes pancreatic agenesis, a phenotype previously found to be induced by mutations in a distal-enhancer of PTF1A in humans, further supporting the causality of this condition in vivo. This approach helps to uncover interspecies functionally equivalent cis-regulatory elements and their potential role in human disease. Alterations in cis-regulatory elements (CREs) can contribute to pancreatic diseases. Here the authors combine chromatin profiling and interaction points with in vivo reporter assays in zebrafish to uncover functionally equivalent human CREs, helping to predict disease-relevant enhancers.
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13
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Transcriptional control of pancreatic β-cell identity and plasticity during the pathogenesis of type 2 diabetes. J Genet Genomics 2022; 49:316-328. [DOI: 10.1016/j.jgg.2022.03.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2021] [Revised: 02/23/2022] [Accepted: 03/06/2022] [Indexed: 11/21/2022]
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14
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Sáenz JB. Follow the Metaplasia: Characteristics and Oncogenic Implications of Metaplasia's Pattern of Spread Throughout the Stomach. Front Cell Dev Biol 2021; 9:741574. [PMID: 34869328 PMCID: PMC8633114 DOI: 10.3389/fcell.2021.741574] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2021] [Accepted: 10/29/2021] [Indexed: 12/12/2022] Open
Abstract
The human stomach functions as both a digestive and innate immune organ. Its main product, acid, rapidly breaks down ingested products and equally serves as a highly effective microbial filter. The gastric epithelium has evolved mechanisms to appropriately handle the myriad of injurious substances, both exogenous and endogenous, to maintain the epithelial barrier and restore homeostasis. The most significant chronic insult that the stomach must face is Helicobacter pylori (Hp), a stomach-adapted bacterium that can colonize the stomach and induce chronic inflammatory and pre-neoplastic changes. The progression from chronic inflammation to dysplasia relies on the decades-long interplay between this oncobacterium and its gastric host. This review summarizes the functional and molecular regionalization of the stomach at homeostasis and details how chronic inflammation can lead to characteristic alterations in these developmental demarcations, both at the topographic and glandular levels. More importantly, this review illustrates our current understanding of the epithelial mechanisms that underlie the pre-malignant gastric landscape, how Hp adapts to and exploits these changes, and the clinical implications of identifying these changes in order to stratify patients at risk of developing gastric cancer, a leading cause of cancer-related deaths worldwide.
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Affiliation(s)
- José B Sáenz
- Division of Gastroenterology, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, United States
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15
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Fukaishi T, Nakagawa Y, Fukunaka A, Sato T, Hara A, Nakao K, Saito M, Kohno K, Miyatsuka T, Tamaki M, Matsuhisa M, Matsuoka TA, Yamada T, Watada H, Fujitani Y. Characterisation of Ppy-lineage cells clarifies the functional heterogeneity of pancreatic beta cells in mice. Diabetologia 2021; 64:2803-2816. [PMID: 34498099 PMCID: PMC8563568 DOI: 10.1007/s00125-021-05560-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/13/2021] [Accepted: 06/28/2021] [Indexed: 12/16/2022]
Abstract
AIMS/HYPOTHESIS Pancreatic polypeptide (PP) cells, which secrete PP (encoded by the Ppy gene), are a minor population of pancreatic endocrine cells. Although it has been reported that the loss of beta cell identity might be associated with beta-to-PP cell-fate conversion, at present, little is known regarding the characteristics of Ppy-lineage cells. METHODS We used Ppy-Cre driver mice and a PP-specific monoclonal antibody to investigate the association between Ppy-lineage cells and beta cells. The molecular profiles of endocrine cells were investigated by single-cell transcriptome analysis and the glucose responsiveness of beta cells was assessed by Ca2+ imaging. Diabetic conditions were experimentally induced in mice by either streptozotocin or diphtheria toxin. RESULTS Ppy-lineage cells were found to contribute to the four major types of endocrine cells, including beta cells. Ppy-lineage beta cells are a minor subpopulation, accounting for 12-15% of total beta cells, and are mostly (81.2%) localised at the islet periphery. Unbiased single-cell analysis with a Ppy-lineage tracer demonstrated that beta cells are composed of seven clusters, which are categorised into two groups (i.e. Ppy-lineage and non-Ppy-lineage beta cells). These subpopulations of beta cells demonstrated distinct characteristics regarding their functionality and gene expression profiles. Ppy-lineage beta cells had a reduced glucose-stimulated Ca2+ signalling response and were increased in number in experimental diabetes models. CONCLUSIONS/INTERPRETATION Our results indicate that an unexpected degree of beta cell heterogeneity is defined by Ppy gene activation, providing valuable insight into the homeostatic regulation of pancreatic islets and future therapeutic strategies against diabetes. DATA AVAILABILITY The single-cell RNA sequence (scRNA-seq) analysis datasets generated in this study have been deposited in the Gene Expression Omnibus (GEO) under the accession number GSE166164 ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE166164 ).
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Affiliation(s)
- Takahiro Fukaishi
- Laboratory of Developmental Biology & Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Gunma, Japan
- Department of Molecular Endocrinology and Metabolism, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| | - Yuko Nakagawa
- Laboratory of Developmental Biology & Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Gunma, Japan
| | - Ayako Fukunaka
- Laboratory of Developmental Biology & Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Gunma, Japan
| | - Takashi Sato
- Laboratory of Developmental Biology & Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Gunma, Japan
| | - Akemi Hara
- Department of Metabolism & Endocrinology, Juntendo University Graduate School of Medicine, Tokyo, Japan
- Center for Therapeutic Innovations in Diabetes, Juntendo University Graduate School of Medicine, Tokyo, Japan
- Department of Physiology, Faculty of Medicine, Saitama Medical University, Saitama, Japan
| | - Keiko Nakao
- Department of Physiology, Faculty of Medicine, Saitama Medical University, Saitama, Japan
| | - Michiko Saito
- Institute for Research Initiatives, Nara Institute of Science and Technology (NAIST), Nara, Japan
- Bio-science Research Center, Kyoto Pharmaceutical University, Kyoto, Japan
| | - Kenji Kohno
- Institute for Research Initiatives, Nara Institute of Science and Technology (NAIST), Nara, Japan
| | - Takeshi Miyatsuka
- Department of Metabolism & Endocrinology, Juntendo University Graduate School of Medicine, Tokyo, Japan
- Center for Therapeutic Innovations in Diabetes, Juntendo University Graduate School of Medicine, Tokyo, Japan
| | - Motoyuki Tamaki
- Diabetes Therapeutics and Research Center, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan
| | - Munehide Matsuhisa
- Diabetes Therapeutics and Research Center, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan
| | - Taka-Aki Matsuoka
- The First Department of Medicine, Wakayama Medical University, Wakayama, Japan
| | - Tetsuya Yamada
- Department of Molecular Endocrinology and Metabolism, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| | - Hirotaka Watada
- Department of Metabolism & Endocrinology, Juntendo University Graduate School of Medicine, Tokyo, Japan
- Center for Therapeutic Innovations in Diabetes, Juntendo University Graduate School of Medicine, Tokyo, Japan
- Center for Identification of Diabetic Therapeutic Targets, Juntendo University Graduate School of Medicine, Tokyo, Japan
- Sportology Center, Juntendo University Graduate School of Medicine, Tokyo, Japan
| | - Yoshio Fujitani
- Laboratory of Developmental Biology & Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Gunma, Japan.
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16
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Nagaya M, Hasegawa K, Watanabe M, Nakano K, Okamoto K, Yamada T, Uchikura A, Osafune K, Yokota H, Nagaoka T, Matsunari H, Umeyama K, Kobayashi E, Nakauchi H, Nagashima H. Genetically engineered pigs manifesting pancreatic agenesis with severe diabetes. BMJ Open Diabetes Res Care 2020; 8:8/2/e001792. [PMID: 33257422 PMCID: PMC7705540 DOI: 10.1136/bmjdrc-2020-001792] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/22/2020] [Revised: 10/08/2020] [Accepted: 10/18/2020] [Indexed: 12/11/2022] Open
Abstract
INTRODUCTION Pancreatic duodenum homeobox 1 (Pdx1) expression is crucial for pancreatic organogenesis and is a key regulator of insulin gene expression. Hairy and enhancer of split 1 (Hes1) controls tissue morphogenesis by maintaining undifferentiated cells. Hes1 encodes a basic helix loop helix (bHLH) transcriptional repressor and functionally antagonizes positive bHLH genes, such as the endocrine determination gene neurogenin-3. Here, we generated a new pig model for diabetes by genetic engineering Pdx1 and Hes1 genes. RESEARCH DESIGN AND METHODS A transgenic (Tg) chimera pig with germ cells carrying a construct expressing Hes1 under the control of the Pdx1 promoter was used to mate with wild-type gilts to obtain Tg piglets. RESULTS The Tg pigs showed perinatal death; however, this phenotype could be rescued by insulin treatment. The duodenal and splenic lobes of the Tg pigs were slender and did not fully develop, whereas the connective lobe was absent. β cells were not detected, even in the adult pancreas, although other endocrine cells were detected, and exocrine cells functioned normally. The pigs showed no irregularities in any organs, except diabetes-associated pathological alterations, such as retinopathy and renal damage. CONCLUSION Pdx1-Hes1 Tg pigs were an attractive model for the analysis of pancreatic development and testing of novel treatment strategies for diabetes.
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Affiliation(s)
- Masaki Nagaya
- Meiji University International Institute for Bio-Resource Research, Meiji University - Ikuta Campus, Kawasaki, Japan
- Department of Immunology, St. Marianna University School of Medicine, Kawasaki, Japan
| | - Koki Hasegawa
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan
| | - Masahito Watanabe
- Meiji University International Institute for Bio-Resource Research, Meiji University - Ikuta Campus, Kawasaki, Japan
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan
| | - Kazuaki Nakano
- Meiji University International Institute for Bio-Resource Research, Meiji University - Ikuta Campus, Kawasaki, Japan
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan
| | - Kazutoshi Okamoto
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan
| | - Takeshi Yamada
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan
| | - Ayuko Uchikura
- Meiji University International Institute for Bio-Resource Research, Meiji University - Ikuta Campus, Kawasaki, Japan
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan
| | - Kenji Osafune
- Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Harumasa Yokota
- Division of Ophthalmology, Department of Visual Sciences, Nihon University School of Medicine, Tokyo, Japan
| | - Taiji Nagaoka
- Division of Ophthalmology, Department of Visual Sciences, Nihon University School of Medicine, Tokyo, Japan
| | - Hitomi Matsunari
- Meiji University International Institute for Bio-Resource Research, Meiji University - Ikuta Campus, Kawasaki, Japan
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan
| | - Kazuhiro Umeyama
- Meiji University International Institute for Bio-Resource Research, Meiji University - Ikuta Campus, Kawasaki, Japan
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan
| | - Eiji Kobayashi
- Department of Organ Fabrication, Keio University, School of Medicine, Tokyo, Japan
| | - Hiromitsu Nakauchi
- Division of Stem Cell Therapy, Institute of Medical Science, Institute of Medical Science, The University of Tokyo, Tokyo, Japan
- Institute for Stem Cell Biology and Regenerative Medicine, Department of Genetics, Stanford University School of Medicine, Stanford, California, USA
| | - Hiroshi Nagashima
- Meiji University International Institute for Bio-Resource Research, Meiji University - Ikuta Campus, Kawasaki, Japan
- Laboratory of Medical Bioengineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan
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17
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Tran R, Moraes C, Hoesli CA. Controlled clustering enhances PDX1 and NKX6.1 expression in pancreatic endoderm cells derived from pluripotent stem cells. Sci Rep 2020; 10:1190. [PMID: 31988329 PMCID: PMC6985188 DOI: 10.1038/s41598-020-57787-0] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2019] [Accepted: 01/07/2020] [Indexed: 01/26/2023] Open
Abstract
Pluripotent stem cell (PSC)-derived insulin-producing cells are a promising cell source for diabetes cellular therapy. However, the efficiency of the multi-step process required to differentiate PSCs towards pancreatic beta cells is variable between cell lines, batches and even within cultures. In adherent pancreatic differentiation protocols, we observed spontaneous local clustering of cells expressing elevated nuclear expression of pancreatic endocrine transcription factors, PDX1 and NKX6.1. Since aggregation has previously been shown to promote downstream differentiation, this local clustering may contribute to the variability in differentiation efficiencies observed within and between cultures. We therefore hypothesized that controlling and directing the spontaneous clustering process would lead to more efficient and consistent induction of pancreatic endocrine fate. Micropatterning cells in adherent microwells prompted clustering, local cell density increases, and increased nuclear accumulation of PDX1 and NKX6.1. Improved differentiation profiles were associated with distinct filamentous actin architectures, suggesting a previously overlooked role for cell-driven morphogenetic changes in supporting pancreatic differentiation. This work demonstrates that confined differentiation in cell-adhesive micropatterns may provide a facile, scalable, and more reproducible manufacturing route to drive morphogenesis and produce well-differentiated pancreatic cell clusters.
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Affiliation(s)
- Raymond Tran
- Department of Chemical Engineering, McGill University, 3610 rue University, Montreal, QC, Canada
| | - Christopher Moraes
- Department of Chemical Engineering, McGill University, 3610 rue University, Montreal, QC, Canada. .,Department of Biomedical Engineering, McGill University, 3775 rue University, Montreal, QC, Canada. .,Rosalind and Morris Goodman Cancer Research Center, McGill University, Montreal, QC, Canada.
| | - Corinne A Hoesli
- Department of Chemical Engineering, McGill University, 3610 rue University, Montreal, QC, Canada. .,Department of Biomedical Engineering, McGill University, 3775 rue University, Montreal, QC, Canada.
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18
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Villani V, Thornton ME, Zook HN, Crook CJ, Grubbs BH, Orlando G, De Filippo R, Ku HT, Perin L. SOX9+/PTF1A+ Cells Define the Tip Progenitor Cells of the Human Fetal Pancreas of the Second Trimester. Stem Cells Transl Med 2019; 8:1249-1264. [PMID: 31631582 PMCID: PMC6877773 DOI: 10.1002/sctm.19-0231] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2019] [Accepted: 09/04/2019] [Indexed: 12/12/2022] Open
Abstract
Significant progress has been made in recent years in characterizing human multipotent progenitor cells (hMPCs) of the early pancreas; however, the identity and persistence of these cells during the second trimester, after the initiation of branching morphogenesis, remain elusive. Additionally, studies on hMPCs have been hindered by few isolation methods that allow for the recovery of live cells. Here, we investigated the tip progenitor domain in the branched epithelium of human fetal pancreas between 13.5 and 17.5 gestational weeks by immunohistological staining. We also used a novel RNA-based technology to isolate live cells followed by gene expression analyses. We identified cells co-expressing SOX9 and PTF1A, two transcription factors known to be important for pancreatic MPCs, within the tips of the epithelium and observed a decrease in their proportions over time. Pancreatic SOX9+/PTF1A+ cells were enriched for MPC markers, including MYC and GATA6. These cells were proliferative and appeared active in branching morphogenesis and matrix remodeling, as evidenced by gene set enrichment analysis. We identified a hub of genes pertaining to the expanding tip progenitor niche, such as FOXF1, GLI3, TBX3, FGFR1, TGFBR2, ITGAV, ITGA2, and ITGB3. YAP1 of the Hippo pathway emerged as a highly enriched component within the SOX9+/PTF1A+ cells. Single-cell RNA-sequencing further corroborated the findings by identifying a cluster of SOX9+/PTF1A+ cells with multipotent characteristics. Based on these results, we propose that the SOX9+/PTF1A+ cells in the human pancreas are uncommitted MPC-like cells that reside at the tips of the expanding pancreatic epithelium, directing self-renewal and inducing pancreatic organogenesis. Stem Cells Translational Medicine 2019;8:1249&1264.
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Affiliation(s)
- Valentina Villani
- GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics, Division of UrologySaban Research Institute, Children's Hospital Los AngelesLos AngelesCaliforniaUSA
| | - Matthew E. Thornton
- Maternal‐Fetal Medicine Division, Department of Obstetrics and Gynecology, Keck School of MedicineUniversity of Southern CaliforniaLos AngelesCaliforniaUSA
| | - Heather N. Zook
- Department of Translational Research and Cellular TherapeuticsDiabetes and Metabolism Research Institute of City of HopeDuarteCaliforniaUSA
- Irell & Manella Graduate School of Biological SciencesBeckman Research Institute of City of HopeDuarteCaliforniaUSA
| | - Christiana J. Crook
- Department of Translational Research and Cellular TherapeuticsDiabetes and Metabolism Research Institute of City of HopeDuarteCaliforniaUSA
- Irell & Manella Graduate School of Biological SciencesBeckman Research Institute of City of HopeDuarteCaliforniaUSA
| | - Brendan H. Grubbs
- Maternal‐Fetal Medicine Division, Department of Obstetrics and Gynecology, Keck School of MedicineUniversity of Southern CaliforniaLos AngelesCaliforniaUSA
| | - Giuseppe Orlando
- Department of SurgeryWake Forest School of MedicineWinston‐SalemNorth CarolinaUSA
| | - Roger De Filippo
- GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics, Division of UrologySaban Research Institute, Children's Hospital Los AngelesLos AngelesCaliforniaUSA
- Department of Urology, Keck School of MedicineUniversity of Southern CaliforniaLos AngelesCaliforniaUSA
| | - Hsun Teresa Ku
- Department of Translational Research and Cellular TherapeuticsDiabetes and Metabolism Research Institute of City of HopeDuarteCaliforniaUSA
- Irell & Manella Graduate School of Biological SciencesBeckman Research Institute of City of HopeDuarteCaliforniaUSA
| | - Laura Perin
- GOFARR Laboratory for Organ Regenerative Research and Cell Therapeutics, Division of UrologySaban Research Institute, Children's Hospital Los AngelesLos AngelesCaliforniaUSA
- Department of Urology, Keck School of MedicineUniversity of Southern CaliforniaLos AngelesCaliforniaUSA
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19
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Baeyens L, Lemper M, Staels W, De Groef S, De Leu N, Heremans Y, German MS, Heimberg H. (Re)generating Human Beta Cells: Status, Pitfalls, and Perspectives. Physiol Rev 2018; 98:1143-1167. [PMID: 29717931 DOI: 10.1152/physrev.00034.2016] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Diabetes mellitus results from disturbed glucose homeostasis due to an absolute (type 1) or relative (type 2) deficiency of insulin, a peptide hormone almost exclusively produced by the beta cells of the endocrine pancreas in a tightly regulated manner. Current therapy only delays disease progression through insulin injection and/or oral medications that increase insulin secretion or sensitivity, decrease hepatic glucose production, or promote glucosuria. These drugs have turned diabetes into a chronic disease as they do not solve the underlying beta cell defects or entirely prevent the long-term complications of hyperglycemia. Beta cell replacement through islet transplantation is a more physiological therapeutic alternative but is severely hampered by donor shortage and immune rejection. A curative strategy should combine newer approaches to immunomodulation with beta cell replacement. Success of this approach depends on the development of practical methods for generating beta cells, either in vitro or in situ through beta cell replication or beta cell differentiation. This review provides an overview of human beta cell generation.
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Affiliation(s)
- Luc Baeyens
- Beta Cell Neogenesis (BENE), Vrije Universiteit Brussel, Brussels , Belgium ; Diabetes Center, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, and Department of Medicine, University of California San Francisco , San Francisco, California ; Genentech Safety Assessment, South San Francisco, California ; Investigative Toxicology, UCB BioPharma, Braine-l'Alleud, Belgium ; Department of Pediatrics, Division of Pediatric Endocrinology, Ghent University, Hospital and Department of Pediatrics and Genetics , Ghent , Belgium ; Department of Endocrinology, Universitair Ziekenhuis Brussel, Brussels , Belgium ; and Department of Endocrinology, Algemeen Stedelijk Ziekenhuis Aalst, Aalst, Belgium
| | - Marie Lemper
- Beta Cell Neogenesis (BENE), Vrije Universiteit Brussel, Brussels , Belgium ; Diabetes Center, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, and Department of Medicine, University of California San Francisco , San Francisco, California ; Genentech Safety Assessment, South San Francisco, California ; Investigative Toxicology, UCB BioPharma, Braine-l'Alleud, Belgium ; Department of Pediatrics, Division of Pediatric Endocrinology, Ghent University, Hospital and Department of Pediatrics and Genetics , Ghent , Belgium ; Department of Endocrinology, Universitair Ziekenhuis Brussel, Brussels , Belgium ; and Department of Endocrinology, Algemeen Stedelijk Ziekenhuis Aalst, Aalst, Belgium
| | - Willem Staels
- Beta Cell Neogenesis (BENE), Vrije Universiteit Brussel, Brussels , Belgium ; Diabetes Center, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, and Department of Medicine, University of California San Francisco , San Francisco, California ; Genentech Safety Assessment, South San Francisco, California ; Investigative Toxicology, UCB BioPharma, Braine-l'Alleud, Belgium ; Department of Pediatrics, Division of Pediatric Endocrinology, Ghent University, Hospital and Department of Pediatrics and Genetics , Ghent , Belgium ; Department of Endocrinology, Universitair Ziekenhuis Brussel, Brussels , Belgium ; and Department of Endocrinology, Algemeen Stedelijk Ziekenhuis Aalst, Aalst, Belgium
| | - Sofie De Groef
- Beta Cell Neogenesis (BENE), Vrije Universiteit Brussel, Brussels , Belgium ; Diabetes Center, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, and Department of Medicine, University of California San Francisco , San Francisco, California ; Genentech Safety Assessment, South San Francisco, California ; Investigative Toxicology, UCB BioPharma, Braine-l'Alleud, Belgium ; Department of Pediatrics, Division of Pediatric Endocrinology, Ghent University, Hospital and Department of Pediatrics and Genetics , Ghent , Belgium ; Department of Endocrinology, Universitair Ziekenhuis Brussel, Brussels , Belgium ; and Department of Endocrinology, Algemeen Stedelijk Ziekenhuis Aalst, Aalst, Belgium
| | - Nico De Leu
- Beta Cell Neogenesis (BENE), Vrije Universiteit Brussel, Brussels , Belgium ; Diabetes Center, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, and Department of Medicine, University of California San Francisco , San Francisco, California ; Genentech Safety Assessment, South San Francisco, California ; Investigative Toxicology, UCB BioPharma, Braine-l'Alleud, Belgium ; Department of Pediatrics, Division of Pediatric Endocrinology, Ghent University, Hospital and Department of Pediatrics and Genetics , Ghent , Belgium ; Department of Endocrinology, Universitair Ziekenhuis Brussel, Brussels , Belgium ; and Department of Endocrinology, Algemeen Stedelijk Ziekenhuis Aalst, Aalst, Belgium
| | - Yves Heremans
- Beta Cell Neogenesis (BENE), Vrije Universiteit Brussel, Brussels , Belgium ; Diabetes Center, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, and Department of Medicine, University of California San Francisco , San Francisco, California ; Genentech Safety Assessment, South San Francisco, California ; Investigative Toxicology, UCB BioPharma, Braine-l'Alleud, Belgium ; Department of Pediatrics, Division of Pediatric Endocrinology, Ghent University, Hospital and Department of Pediatrics and Genetics , Ghent , Belgium ; Department of Endocrinology, Universitair Ziekenhuis Brussel, Brussels , Belgium ; and Department of Endocrinology, Algemeen Stedelijk Ziekenhuis Aalst, Aalst, Belgium
| | - Michael S German
- Beta Cell Neogenesis (BENE), Vrije Universiteit Brussel, Brussels , Belgium ; Diabetes Center, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, and Department of Medicine, University of California San Francisco , San Francisco, California ; Genentech Safety Assessment, South San Francisco, California ; Investigative Toxicology, UCB BioPharma, Braine-l'Alleud, Belgium ; Department of Pediatrics, Division of Pediatric Endocrinology, Ghent University, Hospital and Department of Pediatrics and Genetics , Ghent , Belgium ; Department of Endocrinology, Universitair Ziekenhuis Brussel, Brussels , Belgium ; and Department of Endocrinology, Algemeen Stedelijk Ziekenhuis Aalst, Aalst, Belgium
| | - Harry Heimberg
- Beta Cell Neogenesis (BENE), Vrije Universiteit Brussel, Brussels , Belgium ; Diabetes Center, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, and Department of Medicine, University of California San Francisco , San Francisco, California ; Genentech Safety Assessment, South San Francisco, California ; Investigative Toxicology, UCB BioPharma, Braine-l'Alleud, Belgium ; Department of Pediatrics, Division of Pediatric Endocrinology, Ghent University, Hospital and Department of Pediatrics and Genetics , Ghent , Belgium ; Department of Endocrinology, Universitair Ziekenhuis Brussel, Brussels , Belgium ; and Department of Endocrinology, Algemeen Stedelijk Ziekenhuis Aalst, Aalst, Belgium
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20
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Timme-Laragy AR, Hahn ME, Hansen JM, Rastogi A, Roy MA. Redox stress and signaling during vertebrate embryonic development: Regulation and responses. Semin Cell Dev Biol 2018; 80:17-28. [PMID: 28927759 PMCID: PMC5650060 DOI: 10.1016/j.semcdb.2017.09.019] [Citation(s) in RCA: 44] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2017] [Revised: 09/08/2017] [Accepted: 09/11/2017] [Indexed: 12/21/2022]
Abstract
Vertebrate embryonic development requires specific signaling events that regulate cell proliferation and differentiation to occur at the correct place and the correct time in order to build a healthy embryo. Signaling pathways are sensitive to perturbations of the endogenous redox state, and are also susceptible to modulation by reactive species and antioxidant defenses, contributing to a spectrum of passive vs. active effects that can affect redox signaling and redox stress. Here we take a multi-level, integrative approach to discuss the importance of redox status for vertebrate developmental signaling pathways and cell fate decisions, with a focus on glutathione/glutathione disulfide, thioredoxin, and cysteine/cystine redox potentials and the implications for protein function in development. We present a tissue-specific example of the important role that reactive species play in pancreatic development and metabolic regulation. We discuss NFE2L2 (also known as NRF2) and related proteins, their roles in redox signaling, and their regulation of glutathione during development. Finally, we provide examples of xenobiotic compounds that disrupt redox signaling in the context of vertebrate embryonic development. Collectively, this review provides a systems-level perspective on the innate and inducible antioxidant defenses, as well as their roles in maintaining redox balance during chemical exposures that occur in critical windows of development.
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Affiliation(s)
- Alicia R Timme-Laragy
- Department of Environmental Health Sciences, School of Public Health and Health Sciences, University of Massachusetts, Amherst, MA 01003, USA.
| | - Mark E Hahn
- Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
| | - Jason M Hansen
- Department of Physiology and Developmental Biology, Brigham Young University, Provo, UT 84602, USA
| | - Archit Rastogi
- Molecular & Cellular Biology Graduate Program, University of Massachusetts, Amherst, MA 01003, USA
| | - Monika A Roy
- Department of Environmental Health Sciences, School of Public Health and Health Sciences, University of Massachusetts, Amherst, MA 01003, USA; Biotechnology Training Program, University of Massachusetts, Amherst, MA 01003, USA
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21
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Li WC, Chen CY, Kao CW, Huang PC, Hsieh YT, Kuo TY, Chen TY, Chia HY, Juang JH. Porcine Neonatal Pancreatic Cell Clusters Maintain Their Multipotency in Culture and After Transplantation. Sci Rep 2018; 8:8212. [PMID: 29844347 PMCID: PMC5974285 DOI: 10.1038/s41598-018-26404-6] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2017] [Accepted: 05/11/2018] [Indexed: 01/22/2023] Open
Abstract
Ductal epithelium is primarily detected in porcine neonatal pancreatic cell clusters (NPCCs) bearing grafts, suggesting that transplants might exhibit progenitor-like phenotypes. Here we found that soon after NPCC isolation, PDX1+/insulin− and SOX9+ pancreatic progenitor-like cells dramatically increased while dual-hormonal progenitor-like cells were routinely observed in NPCC culture. After transplantation (Tx), insulin+ cells increased and PDX1+ and SOX9+ cells gradually decreased in both non-diabetic (NDM) and streptozotocin-induced diabetic (DM) grafts over 2 months. Strikingly, a significantly higher percentage of insulin+ cells were detected in 9-day and 16-day, but not in 23-day, 30-day and 60-day grafts implying that hyperglycemia could only facilitate NPCC-derived β cells early post-Tx. A higher percentage of NPCC-derived β cells in early DM grafts was determined via an enhanced neogenic differentiation based on the detection of insulin+ cells budding out from PDX1+/SOX9+ epithelium. Interestingly, a drop in SOX9+ progenitor-like cells was detected 16 days post-Tx in DM grafts whilst PDX1+ cells do not show a significant difference until 60 days post-Tx between DM and NDM grafts, demonstrating that distinct progenitor-like populations fuel new β cells post-Tx. In conclusion, PDX1+/SOX9+ cells could be quickly activated after NPCC isolation, maintain their multipotency in culture and differentiate into new β cell post-Tx.
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Affiliation(s)
- Wan-Chun Li
- Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan.,Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan.,Genome Research Center, National Yang-Ming University, Taipei, Taiwan
| | - Chen-Yi Chen
- Division of Endocrinology and Metabolism and Center for Tissue Engineering, Chang Gung Memorial Hospital, Taoyuan, Taiwan
| | - Chen-Wei Kao
- Division of Endocrinology and Metabolism and Center for Tissue Engineering, Chang Gung Memorial Hospital, Taoyuan, Taiwan
| | - Pei-Chun Huang
- Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan
| | - Yi-Ta Hsieh
- Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan
| | - Tz-Yu Kuo
- Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan
| | - Tsai-Ying Chen
- Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan
| | - Hao-Yuan Chia
- Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan
| | - Jyuhn-Huarng Juang
- Division of Endocrinology and Metabolism and Center for Tissue Engineering, Chang Gung Memorial Hospital, Taoyuan, Taiwan. .,Department of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
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22
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Knocking down Insulin Receptor in Pancreatic Beta Cell lines with Lentiviral-Small Hairpin RNA Reduces Glucose-Stimulated Insulin Secretion via Decreasing the Gene Expression of Insulin, GLUT2 and Pdx1. Int J Mol Sci 2018; 19:ijms19040985. [PMID: 29587416 PMCID: PMC5979368 DOI: 10.3390/ijms19040985] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2018] [Revised: 03/13/2018] [Accepted: 03/21/2018] [Indexed: 12/18/2022] Open
Abstract
Type 2 diabetes (T2D) is a metabolic disorder characterized by beta cell dysfunction and insulin resistance in fat, muscle and liver cells. Recent studies have shown that the development of insulin resistance in pancreatic beta cell lines may contribute to beta cell dysfunction in T2D. However, there still is a lack of detailed investigations regarding the mechanisms by which insulin deficiency may contribute in diabetes. In this study, we firstly established a stable insulin receptor knockdown cell line in pancreatic beta cells INS-1 (InsRβKD cells) using anti InsRβ small hairpin RNA (InsRβ-shRNA) encoded by lentiviral vectors. The resultant InsRβKD cells demonstrated a significantly reduced expression of InsRβ as determined by real-time PCR and Western blotting analyses. Upon removing glucose from the medium, these cells exhibited a significant decrease in insulin gene expression and protein secretion in response to 20 mM glucose stimulation. In accordance with this insulin reduction, the glucose uptake efficiency as indicated by a 3[H]-2-deoxy-d-glucose assay also decreased. Furthermore, InsRβKD cells showed a dramatic decrease in glucose transporter 2 (GLUT2, encoded by SLC2A2) and pancreatic duodenal homeobox (Pdx1) mRNA expression compared to the controls. These data collectively suggest that pancreatic beta cell insulin resistance contributes to the development of beta cell dysfunction by impairing pancreatic beta cell glucose sensation through the Pdx1- GLUT2 pathway. InsRβKD cells provide a good model to further investigate the mechanism of β-cell dysfunction in T2D.
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23
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Angelo JR, Tremblay KD. Identification and fate mapping of the pancreatic mesenchyme. Dev Biol 2018; 435:15-25. [PMID: 29329912 DOI: 10.1016/j.ydbio.2018.01.003] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2017] [Revised: 01/06/2018] [Accepted: 01/06/2018] [Indexed: 12/25/2022]
Abstract
The murine pancreas buds from the ventral embryonic endoderm at approximately 8.75 dpc and a second pancreas bud emerges from the dorsal endoderm by 9.0 dpc. Although it is clear that secreted signals from adjacent mesoderm-derived sources are required for both the appropriate emergence and further refinement of the pancreatic endoderm, neither the exact signals nor the requisite tissue sources have been defined in mammalian systems. Herein we use DiI fate mapping of cultured murine embryos to identify the embryonic sources of both the early inductive and later condensed pancreatic mesenchyme. Despite being capable of supporting pancreas induction from dorsal endoderm in co-culture experiments, we find that in the context of the developing embryo, the dorsal aortae as well as the paraxial, intermediate, and lateral mesoderm derivatives only transiently associate with the dorsal pancreas bud, producing descendants that are decidedly anterior to the pancreas bud. Unlike these other mesoderm derivatives, the axial (notochord) descendants maintain association with the dorsal pre-pancreatic endoderm and early pancreas bud. This fate mapping data points to the notochord as the likely inductive source in vivo while also revealing dynamic morphogenetic movements displayed by individual mesodermal subtypes. Because none of the mesoderm examined above produced the pancreatic mesenchyme that condenses around the induced bud to support exocrine and endocrine differentiation, we also sought to identify the mesodermal origins of this mesenchyme. We identify a portion of the coelomic mesoderm that contributes to the condensed pancreatic mesenchyme. In conclusion, we identify a portion of the notochord as a likely source of the signals required to induce and maintain the early dorsal pancreas bud, demonstrate that the coelomic mesothelium contributes to the dorsal and ventral pancreatic mesenchyme, and provide insight into the dynamic morphological rearrangements of mesoderm-derived tissues during early organogenesis stages of mammalian development.
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Affiliation(s)
- Jesse R Angelo
- Department of Veterinary&Animal Sciences, University of Massachusetts, Amherst, MA, USA
| | - Kimberly D Tremblay
- Department of Veterinary&Animal Sciences, University of Massachusetts, Amherst, MA, USA.
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24
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Luo Y, Xu Y, Wang ZY, Li X, Xing WB, Zhang TC. The Synergy of Two Factors on Insulin Expression. Cell Reprogram 2018; 20:49-54. [PMID: 29303357 DOI: 10.1089/cell.2017.0026] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
As a potential cure for diabetes, more and more attentions have been paid to organ transplants to replace insulin therapy. As a result, many researchers have explored out many programs to get insulin-producing cells (IPCs) to replace the defective β cells. Currently, more and more new induction methods are being proposed, and at the same time, more and more possible induction molecular mechanisms are being revealed. The purpose of this study was to explore whether and how the two factors pdx-1 and myocardin affected the differentiation of rat mesenchymal stem cells (rMSCs) into IPCs. In this study, we investigated the process of transfecting myocardin and/or pdx-1 in rMSCs in vitro. The results showed that rMSCs were able to secrete insulin after cotransfected with myocardin and pdx-1. At the same time, we explored the possible mechanism that myocardin and pdx-1 coinduced rMSCs into IPCs by forming a complex to promote the transcriptional activity of insulin. Our results may provide a theoretical basis to the study of islet transplantation in the future.
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Affiliation(s)
- Ying Luo
- 1 Institute of Biology and Medicine, Wuhan University of Science and Technology , Wuhan, China
| | - Yao Xu
- 1 Institute of Biology and Medicine, Wuhan University of Science and Technology , Wuhan, China
| | - Zhen-Yu Wang
- 1 Institute of Biology and Medicine, Wuhan University of Science and Technology , Wuhan, China
| | - Xi Li
- 1 Institute of Biology and Medicine, Wuhan University of Science and Technology , Wuhan, China
| | - Wei-Bing Xing
- 1 Institute of Biology and Medicine, Wuhan University of Science and Technology , Wuhan, China
| | - Tong-Cun Zhang
- 1 Institute of Biology and Medicine, Wuhan University of Science and Technology , Wuhan, China .,2 Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, College of Biotechnology, Tianjin University of Science and Technology , Tianjin, China
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25
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Affiliation(s)
- Aaron R Cox
- McNair Medical Institute and Pediatric Diabetes and Endocrinology, Texas Children's Hospital, Baylor College of Medicine, Houston, TX
| | - Jake A Kushner
- McNair Medical Institute and Pediatric Diabetes and Endocrinology, Texas Children's Hospital, Baylor College of Medicine, Houston, TX
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26
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Spaeth JM, Gupte M, Perelis M, Yang YP, Cyphert H, Guo S, Liu JH, Guo M, Bass J, Magnuson MA, Wright C, Stein R. Defining a Novel Role for the Pdx1 Transcription Factor in Islet β-Cell Maturation and Proliferation During Weaning. Diabetes 2017; 66:2830-2839. [PMID: 28705881 PMCID: PMC5652607 DOI: 10.2337/db16-1516] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/08/2016] [Accepted: 07/03/2017] [Indexed: 01/02/2023]
Abstract
The transcription factor encoded by the Pdx1 gene is a critical transcriptional regulator, as it has fundamental actions in the formation of all pancreatic cell types, islet β-cell development, and adult islet β-cell function. Transgenic- and cell line-based experiments have identified 5'-flanking conserved sequences that control pancreatic and β-cell type-specific transcription, which are found within areas I (bp -2694 to -2561), II (bp -2139 to -1958), III (bp -1879 to -1799), and IV (bp -6200 to -5670). Because of the presence in area IV of binding sites for transcription factors associated with pancreas development and islet cell function, we analyzed how an endogenous deletion mutant affected Pdx1 expression embryonically and postnatally. The most striking result was observed in male Pdx1ΔIV mutant mice after 3 weeks of birth (i.e., the onset of weaning), with only a small effect on pancreas organogenesis and no deficiencies in their female counterparts. Compromised Pdx1 mRNA and protein levels in weaned male mutant β-cells were tightly linked with hyperglycemia, decreased β-cell proliferation, reduced β-cell area, and altered expression of Pdx1-bound genes that are important in β-cell replication, endoplasmic reticulum function, and mitochondrial activity. We discuss the impact of these novel findings to Pdx1 gene regulation and islet β-cell maturation postnatally.
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Affiliation(s)
- Jason M Spaeth
- Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN
| | - Manisha Gupte
- Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN
| | - Mark Perelis
- Division of Endocrinology, Metabolism and Molecular Medicine, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL
| | - Yu-Ping Yang
- Program in Developmental Biology, Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN
| | - Holly Cyphert
- Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN
| | - Shuangli Guo
- Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN
| | - Jin-Hua Liu
- Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN
| | - Min Guo
- Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN
| | - Joseph Bass
- Division of Endocrinology, Metabolism and Molecular Medicine, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL
| | - Mark A Magnuson
- Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN
- Center for Stem Cell Biology, Vanderbilt University, Nashville, TN
| | - Christopher Wright
- Program in Developmental Biology, Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN
- Center for Stem Cell Biology, Vanderbilt University, Nashville, TN
| | - Roland Stein
- Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN
- Program in Developmental Biology, Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN
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27
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Abstract
A small number of cells in the adult pancreas are endocrine cells. They are arranged in clusters called islets of Langerhans. The islets make insulin, glucagon, and other endocrine hormones, and release them into the blood circulation. These hormones help control the level of blood glucose. Therefore, a dysfunction of endocrine cells in the pancreas results in impaired glucose homeostasis, or diabetes mellitus. The pancreas is an organ that originates from the evaginations of pancreatic progenitor cells in the epithelium of the foregut endoderm. Pancreas organogenesis and maturation of the islets of Langerhans occurs via a coordinated and complex interplay of transcriptional networks and signaling molecules, which guide a stepwise and repetitive process of the propagation of progenitor cells and their maturation, eventually resulting in a fully functional organ. Increasing our understanding of the extrinsic, as well as intrinsic mechanisms that control these processes should facilitate the efforts to generate surrogate β cells from ES or iPS cells, or to reactivate the function of important cell types within pancreatic islets that are lost in diabetes.
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Affiliation(s)
- Yoshio Fujitani
- Laboratory of Developmental Biology & Metabolism, Institute for Molecular and Cellular Regulation, Gunma University, Gunma 371-8512, Japan
- AMED-CREST Program, Institute for Molecular and Cellular Regulation, Gunma University, Gunma 371-8512, Japan
- Department of Metabolism and Endocrinology, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan
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28
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Bastidas-Ponce A, Roscioni SS, Burtscher I, Bader E, Sterr M, Bakhti M, Lickert H. Foxa2 and Pdx1 cooperatively regulate postnatal maturation of pancreatic β-cells. Mol Metab 2017; 6:524-534. [PMID: 28580283 PMCID: PMC5444078 DOI: 10.1016/j.molmet.2017.03.007] [Citation(s) in RCA: 63] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/20/2017] [Revised: 03/16/2017] [Accepted: 03/21/2017] [Indexed: 01/04/2023] Open
Abstract
OBJECTIVE The transcription factors (TF) Foxa2 and Pdx1 are key regulators of beta-cell (β-cell) development and function. Mutations of these TFs or their respective cis-regulatory consensus binding sites have been linked to maturity diabetes of the young (MODY), pancreas agenesis, or diabetes susceptibility in human. Although Foxa2 has been shown to directly regulate Pdx1 expression during mouse embryonic development, the impact of this gene regulatory interaction on postnatal β-cell maturation remains obscure. METHODS In order to easily monitor the expression domains of Foxa2 and Pdx1 and analyze their functional interconnection, we generated a novel double knock-in homozygous (FVFPBFDHom) fluorescent reporter mouse model by crossing the previously described Foxa2-Venus fusion (FVF) with the newly generated Pdx1-BFP (blue fluorescent protein) fusion (PBF) mice. RESULTS Although adult PBF homozygous animals exhibited a reduction in expression levels of Pdx1, they are normoglycemic. On the contrary, despite normal pancreas and endocrine development, the FVFPBFDHom reporter male animals developed hyperglycemia at weaning age and displayed a reduction in Pdx1 levels in islets, which coincided with alterations in β-cell number and islet architecture. The failure to establish mature β-cells resulted in loss of β-cell identity and trans-differentiation towards other endocrine cell fates. Further analysis suggested that Foxa2 and Pdx1 genetically and functionally cooperate to regulate maturation of adult β-cells. CONCLUSIONS Our data show that the maturation of pancreatic β-cells requires the cooperative function of Foxa2 and Pdx1. Understanding the postnatal gene regulatory network of β-cell maturation will help to decipher pathomechanisms of diabetes and identify triggers to regenerate dedifferentiated β-cell mass.
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Affiliation(s)
- Aimée Bastidas-Ponce
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Germany.,Institute of Stem Cell Research, Helmholtz Zentrum München, Germany.,German Center for Diabetes Research (DZD), Germany
| | - Sara S Roscioni
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Germany.,Institute of Stem Cell Research, Helmholtz Zentrum München, Germany
| | - Ingo Burtscher
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Germany.,Institute of Stem Cell Research, Helmholtz Zentrum München, Germany.,German Center for Diabetes Research (DZD), Germany
| | - Erik Bader
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Germany.,Institute of Stem Cell Research, Helmholtz Zentrum München, Germany
| | - Michael Sterr
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Germany.,Institute of Stem Cell Research, Helmholtz Zentrum München, Germany
| | - Mostafa Bakhti
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Germany.,Institute of Stem Cell Research, Helmholtz Zentrum München, Germany.,German Center for Diabetes Research (DZD), Germany
| | - Heiko Lickert
- Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Germany.,Institute of Stem Cell Research, Helmholtz Zentrum München, Germany.,Technical University of Munich, Germany.,German Center for Diabetes Research (DZD), Germany
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29
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McCracken KW, Wells JM. Mechanisms of embryonic stomach development. Semin Cell Dev Biol 2017; 66:36-42. [PMID: 28238948 DOI: 10.1016/j.semcdb.2017.02.004] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2017] [Accepted: 02/20/2017] [Indexed: 12/18/2022]
Abstract
The stomach is a digestive organ that has important roles in human physiology and pathophysiology. The developmental origin of the stomach is the embryonic foregut, which also gives rise a number of other structures. There are several signaling pathways and transcription factors that are known to regulate stomach development at different stages, including foregut patterning, stomach specification, and gastric regionalization. These developmental events have important implications in later homeostasis and disease in the adult stomach. Here we will review the literature that has shaped our current understanding of the molecular mechanisms that coordinate gastric organogenesis. Further we will discuss how developmental paradigms have guided recent efforts to differentiate stomach tissue from pluripotent stem cells.
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Affiliation(s)
- Kyle W McCracken
- Division of Developmental Biology, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA; Boston Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA.
| | - James M Wells
- Division of Developmental Biology, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA; Division of Endocrinology Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA.
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van Arensbergen J, Dussaud S, Pardanaud-Glavieux C, García-Hurtado J, Sauty C, Guerci A, Ferrer J, Ravassard P. A distal intergenic region controls pancreatic endocrine differentiation by acting as a transcriptional enhancer and as a polycomb response element. PLoS One 2017; 12:e0171508. [PMID: 28225770 PMCID: PMC5321433 DOI: 10.1371/journal.pone.0171508] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2016] [Accepted: 01/02/2017] [Indexed: 12/11/2022] Open
Abstract
Lineage-selective expression of developmental genes is dependent on the interplay between activating and repressive mechanisms. Gene activation is dependent on cell-specific transcription factors that recognize transcriptional enhancer sequences. Gene repression often depends on the recruitment of Polycomb group (PcG) proteins, although the sequences that underlie the recruitment of PcG proteins, also known as Polycomb response elements (PREs), remain poorly understood in vertebrates. While distal PREs have been identified in mammals, a role for positive-acting enhancers in PcG-mediated repression has not been described. Here we have used a highly efficient procedure based on lentiviral-mediated transgenesis to carry out in vivo fine-mapping of, cis-regulatory sequences that control lineage-specific activation of Neurog3, a master regulator of pancreatic endocrine differentiation. Our findings reveal an enhancer region that is sufficient to drive correct spacio-temporal expression of Neurog3 and demonstrate that this same region serves as a PRE in alternative lineages where Neurog3 is inactive.
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Affiliation(s)
- Joris van Arensbergen
- Genomic Programming of Beta-Cells Laboratory, IDIBAPS, Barcelona, Spain
- CIBER de Diabetes y Enfermedades Metabólicas, Barcelona, Spain
| | - Sebastien Dussaud
- Sorbonne Universités, UPMC Univ Paris 06, Inserm, CNRS, Institut du cerveau et de la moelle (ICM)–Hôpital Pitié-Salpêtrière, Boulevard de l’Hôpital, Paris, France
| | - Corinne Pardanaud-Glavieux
- Sorbonne Universités, UPMC Univ Paris 06, Inserm, CNRS, Institut du cerveau et de la moelle (ICM)–Hôpital Pitié-Salpêtrière, Boulevard de l’Hôpital, Paris, France
| | - Javier García-Hurtado
- Genomic Programming of Beta-Cells Laboratory, IDIBAPS, Barcelona, Spain
- CIBER de Diabetes y Enfermedades Metabólicas, Barcelona, Spain
| | - Claire Sauty
- Sorbonne Universités, UPMC Univ Paris 06, Inserm, CNRS, Institut du cerveau et de la moelle (ICM)–Hôpital Pitié-Salpêtrière, Boulevard de l’Hôpital, Paris, France
| | - Aline Guerci
- Sorbonne Universités, UPMC Univ Paris 06, Inserm, CNRS, Institut du cerveau et de la moelle (ICM)–Hôpital Pitié-Salpêtrière, Boulevard de l’Hôpital, Paris, France
| | - Jorge Ferrer
- Genomic Programming of Beta-Cells Laboratory, IDIBAPS, Barcelona, Spain
- CIBER de Diabetes y Enfermedades Metabólicas, Barcelona, Spain
- Department of Medicine, Imperial Centre for Translational and Experimental Medicine, Imperial College, London, United Kingdom
- * E-mail: (PR); (JF)
| | - Philippe Ravassard
- Sorbonne Universités, UPMC Univ Paris 06, Inserm, CNRS, Institut du cerveau et de la moelle (ICM)–Hôpital Pitié-Salpêtrière, Boulevard de l’Hôpital, Paris, France
- * E-mail: (PR); (JF)
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Zinovyeva MV, Kuzmich AI, Monastyrskaya GS, Sverdlov ED. The role of FOXA subfamily factors in embryonic development and carcinogenesis of the pancreas. MOLECULAR GENETICS MICROBIOLOGY AND VIROLOGY 2017. [DOI: 10.3103/s0891416816030113] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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Larsen HL, Grapin-Botton A. The molecular and morphogenetic basis of pancreas organogenesis. Semin Cell Dev Biol 2017; 66:51-68. [PMID: 28089869 DOI: 10.1016/j.semcdb.2017.01.005] [Citation(s) in RCA: 93] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2016] [Revised: 01/06/2017] [Accepted: 01/09/2017] [Indexed: 01/08/2023]
Abstract
The pancreas is an essential endoderm-derived organ that ensures nutrient metabolism via its endocrine and exocrine functions. Here we review the essential processes governing the embryonic and early postnatal development of the pancreas discussing both the mechanisms and molecules controlling progenitor specification, expansion and differentiation. We elaborate on how these processes are orchestrated in space and coordinated with morphogenesis. We draw mainly from experiments conducted in the mouse model but also from investigations in other model organisms, complementing a recent comprehensive review of human pancreas development (Jennings et al., 2015) [1]. The understanding of pancreas development in model organisms provides a framework to interpret how human mutations lead to neonatal diabetes and may contribute to other forms of diabetes and to guide the production of desired pancreatic cell types from pluripotent stem cells for therapeutic purposes.
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Affiliation(s)
- Hjalte List Larsen
- DanStem, University of Copenhagen, 3 B Blegdamsvej, DK-2200 Copenhagen N, Denmark
| | - Anne Grapin-Botton
- DanStem, University of Copenhagen, 3 B Blegdamsvej, DK-2200 Copenhagen N, Denmark.
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Yang YP, Magnuson MA, Stein R, Wright CVE. The mammal-specific Pdx1 Area II enhancer has multiple essential functions in early endocrine cell specification and postnatal β-cell maturation. Development 2016; 144:248-257. [PMID: 27993987 DOI: 10.1242/dev.143123] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2016] [Accepted: 12/07/2016] [Indexed: 01/19/2023]
Abstract
The transcription factor Pdx1 is required for multiple aspects of pancreatic organogenesis. It remains unclear to what extent Pdx1 expression and function depend upon trans-activation through 5' conserved cis-regulatory regions and, in particular, whether the mammal-specific Area II (-2139 to -1958 bp) affects minor or major aspects of organogenesis. We show that Area II is a primary effector of endocrine-selective transcription in epithelial multipotent cells, nascent endocrine progenitors, and differentiating and mature β cells in vivo Pdx1ΔAREAII/- mice exhibit a massive reduction in endocrine progenitor cells and progeny hormone-producing cells, indicating that Area II activity is fundamental to mounting an effective endocrine lineage-specification program within the multipotent cell population. Creating an Area II-deleted state within already specified Neurog3-expressing endocrine progenitor cells increased the proportion of glucagon+ α relative to insulin+ β cells, associated with the transcriptional and epigenetic derepression of the α-cell-determining Arx gene in endocrine progenitors. There were also glucagon and insulin co-expressing cells, and β cells that were incapable of maturation. Creating the Pdx1ΔAREAII state after cells entered an insulin-expressing stage led to immature and dysfunctional islet β cells carrying abnormal chromatin marking in vital β-cell-associated genes. Therefore, trans-regulatory integration through Area II mediates a surprisingly extensive range of progenitor and β-cell-specific Pdx1 functions.
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Affiliation(s)
- Yu-Ping Yang
- Vanderbilt University Program in Developmental Biology and Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232 USA.,Vanderbilt Center for Stem Cell Biology, Vanderbilt University, Nashville, TN 37232, USA
| | - Mark A Magnuson
- Vanderbilt University Program in Developmental Biology and Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232 USA.,Vanderbilt Center for Stem Cell Biology, Vanderbilt University, Nashville, TN 37232, USA.,Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, USA
| | - Roland Stein
- Vanderbilt University Program in Developmental Biology and Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232 USA.,Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, USA
| | - Christopher V E Wright
- Vanderbilt University Program in Developmental Biology and Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232 USA .,Vanderbilt Center for Stem Cell Biology, Vanderbilt University, Nashville, TN 37232, USA
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The Chromatin Modifier MSK1/2 Suppresses Endocrine Cell Fates during Mouse Pancreatic Development. PLoS One 2016; 11:e0166703. [PMID: 27973548 PMCID: PMC5156359 DOI: 10.1371/journal.pone.0166703] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2016] [Accepted: 11/02/2016] [Indexed: 11/24/2022] Open
Abstract
Type I diabetes is caused by loss of insulin-secreting beta cells. To identify novel, pharmacologically-targetable histone-modifying proteins that enhance beta cell production from pancreatic progenitors, we performed a screen for histone modifications induced by signal transduction pathways at key pancreatic genes. The screen led us to investigate the temporal dynamics of ser-28 phosphorylated histone H3 (H3S28ph) and its upstream kinases, MSK1 and MSK2 (MSK1/2). H3S28ph and MSK1/2 were enriched at the key endocrine and acinar promoters in E12.5 multipotent pancreatic progenitors. Pharmacological inhibition of MSK1/2 in embryonic pancreatic explants promoted the specification of endocrine fates, including the beta-cell lineage, while depleting acinar fates. Germline knockout of both Msk isoforms caused enhancement of alpha cells and a reduction in acinar differentiation, while monoallelic loss of Msk1 promoted beta cell mass. Our screen of chromatin state dynamics can be applied to other developmental contexts to reveal new pathways and approaches to modulate cell fates.
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Abstract
The stomach, an organ derived from foregut endoderm, secretes acid and enzymes and plays a key role in digestion. During development, mesenchymal-epithelial interactions drive stomach specification, patterning, differentiation and growth through selected signaling pathways and transcription factors. After birth, the gastric epithelium is maintained by the activity of stem cells. Developmental signals are aberrantly activated and stem cell functions are disrupted in gastric cancer and other disorders. Therefore, a better understanding of stomach development and stem cells can inform approaches to treating these conditions. This Review highlights the molecular mechanisms of stomach development and discusses recent findings regarding stomach stem cells and organoid cultures, and their roles in investigating disease mechanisms.
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Affiliation(s)
- Tae-Hee Kim
- Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 0A4 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8
| | - Ramesh A Shivdasani
- Department of Medical Oncology and Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, MA 02215, USA Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA 02215, USA
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36
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Pan FC, Brissova M, Powers AC, Pfaff S, Wright CVE. Inactivating the permanent neonatal diabetes gene Mnx1 switches insulin-producing β-cells to a δ-like fate and reveals a facultative proliferative capacity in aged β-cells. Development 2016; 142:3637-48. [PMID: 26534984 DOI: 10.1242/dev.126011] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Homozygous Mnx1 mutation causes permanent neonatal diabetes in humans, but via unknown mechanisms. Our systematic and longitudinal analysis of Mnx1 function during murine pancreas organogenesis and into the adult uncovered novel stage-specific roles for Mnx1 in endocrine lineage allocation and β-cell fate maintenance. Inactivation in the endocrine-progenitor stage shows that Mnx1 promotes β-cell while suppressing δ-cell differentiation programs, and is crucial for postnatal β-cell fate maintenance. Inactivating Mnx1 in embryonic β-cells (Mnx1(Δbeta)) caused β-to-δ-like cell transdifferentiation, which was delayed until postnatal stages. In the latter context, β-cells escaping Mnx1 inactivation unexpectedly upregulated Mnx1 expression and underwent an age-independent persistent proliferation. Escaper β-cells restored, but then eventually surpassed, the normal pancreatic β-cell mass, leading to islet hyperplasia in aged mice. In vitro analysis of islets isolated from Mnx1(Δbeta) mice showed higher insulin secretory activity and greater insulin mRNA content than in wild-type islets. Mnx1(Δbeta) mice also showed a much faster return to euglycemia after β-cell ablation, suggesting that the new β-cells derived from the escaper population are functional. Our findings identify Mnx1 as an important factor in β-cell differentiation and proliferation, with the potential for targeting to increase the number of endogenous β-cells for diabetes therapy.
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Affiliation(s)
- Fong Cheng Pan
- Vanderbilt University Program in Developmental Biology, Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
| | - Marcela Brissova
- Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA
| | - Alvin C Powers
- Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA VA Tennessee Valley Healthcare System, Nashville, TN 37212, USA
| | - Samuel Pfaff
- Gene Expression Laboratory, The Salk Institute, La Jolla, CA 92037, USA
| | - Christopher V E Wright
- Vanderbilt University Program in Developmental Biology, Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
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Xu J, Cui J, Del Campo A, Shin CH. Four and a Half LIM Domains 1b (Fhl1b) Is Essential for Regulating the Liver versus Pancreas Fate Decision and for β-Cell Regeneration. PLoS Genet 2016; 12:e1005831. [PMID: 26845333 PMCID: PMC4741517 DOI: 10.1371/journal.pgen.1005831] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2015] [Accepted: 01/06/2016] [Indexed: 12/12/2022] Open
Abstract
The liver and pancreas originate from overlapping embryonic regions, and single-cell lineage tracing in zebrafish has shown that Bone morphogenetic protein 2b (Bmp2b) signaling is essential for determining the fate of bipotential hepatopancreatic progenitors towards the liver or pancreas. Despite its pivotal role, the gene regulatory networks functioning downstream of Bmp2b signaling in this process are poorly understood. We have identified four and a half LIM domains 1b (fhl1b), which is primarily expressed in the prospective liver anlage, as a novel target of Bmp2b signaling. fhl1b depletion compromised liver specification and enhanced induction of pancreatic cells from endodermal progenitors. Conversely, overexpression of fhl1b favored liver specification and inhibited induction of pancreatic cells. By single-cell lineage tracing, we showed that fhl1b depletion led lateral endodermal cells, destined to become liver cells, to become pancreatic cells. Reversely, when fhl1b was overexpressed, medially located endodermal cells, fated to differentiate into pancreatic and intestinal cells, contributed to the liver by directly or indirectly modulating the discrete levels of pdx1 expression in endodermal progenitors. Moreover, loss of fhl1b increased the regenerative capacity of β-cells by increasing pdx1 and neurod expression in the hepatopancreatic ductal system. Altogether, these data reveal novel and critical functions of Fhl1b in the hepatic versus pancreatic fate decision and in β-cell regeneration. Lineage-specific multipotent progenitors play crucial roles in embryonic development, regeneration in adult tissues, and diseases such as cancer. Bone morphogenetic protein (Bmp) signaling is critical for regulating the cell fate choice of liver versus pancreas, two essential organs of body metabolism. Through transcriptome profiling of endodermal tissues exposed to increased or decreased Bmp2b signaling, we have discovered the zebrafish gene four and a half LIM domains 1b (fhl1b) as a novel target of Bmp2b signaling. fhl1b is primarily expressed in the prospective liver anlage. Loss- and gain-of-function analyses indicate that Fhl1b suppresses specification of the pancreas and induces the liver. By single-cell lineage tracing, we showed that depletion of fhl1b caused a liver-to-pancreas fate switch, while fhl1b overexpression redirected pancreatic progenitors to become liver cells. At later stages, Fhl1b regulates regeneration of insulin-secreting β-cells by directly or indirectly modulating pdx1 and neurod expression in the hepatopancreatic ductal system. Therefore, our work provides a novel paradigm of how Bmp signaling regulates the hepatic versus pancreatic fate decision and β-cell regeneration through its novel target Fhl1b.
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Affiliation(s)
- Jin Xu
- School of Biology and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, United States of America
| | - Jiaxi Cui
- Max Planck Institute for Polymer Research, Mainz, Germany
| | | | - Chong Hyun Shin
- School of Biology and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, United States of America
- * E-mail:
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Kropp PA, Gannon M. Onecut transcription factors in development and disease. TRENDS IN DEVELOPMENTAL BIOLOGY 2016; 9:43-57. [PMID: 28018056 PMCID: PMC5176019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Subscribe] [Scholar Register] [Indexed: 06/06/2023]
Abstract
Developmental processes are remarkably well conserved among species, and among the most highly conserved developmental regulators are transcription factor families. The Onecut transcription factor family consists of three members known for their single "cut" DNA-binding domain and an aberrant homeodomain. The three members of the Onecut family are highly conserved from Drosophila to humans and have significant roles in regulating the development of diverse tissues derived from the ectoderm or endoderm, where they activate a number of gene families. Of note, the genetic interaction between Onecut family members and Neurogenin genes appears to be essential in multiple tissues for proper specification and development of unique cell types. This review highlights the importance of the Onecut factors in cell fate specification and organogenesis, highlighting their role in vertebrates, and discusses their role in the maintenance of cell fate and prevention of disease. We cover the essential spatial and temporal control of Onecut factor expression and how this tight regulation is required for proper specification and subsequent terminal differentiation of multiple tissue types including those within the retina, central nervous system, liver and pancreas. Beyond development, Onecut factors perform necessary functions in mature cell types; their misregulation can contribute to diseases such as pancreatic cancer. Given the importance of this family of transcription factors in development and disease, their consideration in essential transcription factor networks is underappreciated.
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Affiliation(s)
- Peter A. Kropp
- Department of Molecular Physiology and Biophysicsm Vanderbilt University, Nashville, TN
- Program in Developmental Biology, Vanderbilt University, Nashville, TN
| | - Maureen Gannon
- Department of Molecular Physiology and Biophysicsm Vanderbilt University, Nashville, TN
- Department of Medicine, Vanderbilt University, Nashville, TN
- Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN
- Program in Developmental Biology, Vanderbilt University, Nashville, TN
- Department of Veterans Affairs, Tennessee Valley Health Authority, Nashville, TN, USA
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Kofent J, Zhang J, Spagnoli FM. The histone methyltransferase Setd7 promotes pancreatic progenitor identity. Development 2016; 143:3573-3581. [DOI: 10.1242/dev.136226] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2016] [Accepted: 08/08/2016] [Indexed: 11/20/2022]
Abstract
Cell fate specification depends on transcriptional activation driven by lineage-specific transcription factors as well as changes in chromatin organization. To date, the interplay between transcription factors and chromatin modifiers during development is not well understood. We focus here on the initiation of the pancreatic program from multipotent endodermal progenitors. Transcription factors that play key roles in regulating pancreatic progenitor state have been identified, but the chromatin regulators that help establishing and maintaining pancreatic fate are less well known. Using a comparative approach, we identify a critical role for the histone methyltransferase Setd7 in establishing pancreatic cell identity. We show that Setd7 is expressed in the prospective pancreatic endoderm of Xenopus and mouse embryos prior to Pdx1 induction. Importantly, we demonstrate that setd7 is sufficient and required for pancreatic cell fate specification in Xenopus. Functional and biochemical approaches in Xenopus and mouse endoderm support that Setd7 modulates methylation marks at pancreatic regulatory regions, possibly through interaction with the transcription factor Foxa2. Together, these results demonstrate that Setd7 acts as a central component of the transcription complex initiating the pancreatic program.
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Affiliation(s)
- Julia Kofent
- Lab. of Molecular and Cellular Basis of Embryonic Development, Max-Delbrück Center for Molecular Medicine, Robert-Roessle strasse 10, Berlin 13125, Germany
| | - Juan Zhang
- Lab. of Molecular and Cellular Basis of Embryonic Development, Max-Delbrück Center for Molecular Medicine, Robert-Roessle strasse 10, Berlin 13125, Germany
| | - Francesca M. Spagnoli
- Lab. of Molecular and Cellular Basis of Embryonic Development, Max-Delbrück Center for Molecular Medicine, Robert-Roessle strasse 10, Berlin 13125, Germany
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40
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Abstract
Lineage tracing studies have revealed that transcription factors play a cardinal role in pancreatic development, differentiation and function. Three transitions define pancreatic organogenesis, differentiation and maturation. In the primary transition, when pancreatic organogenesis is initiated, there is active proliferation of pancreatic progenitor cells. During the secondary transition, defined by differentiation, there is growth, branching, differentiation and pancreatic cell lineage allocation. The tertiary transition is characterized by differentiated pancreatic cells that undergo further remodeling, including apoptosis, replication and neogenesis thereby establishing a mature organ. Transcription factors function at multiple levels and may regulate one another and auto-regulate. The interaction between extrinsic signals from non-pancreatic tissues and intrinsic transcription factors form a complex gene regulatory network ultimately culminating in the different cell lineages and tissue types in the developing pancreas. Mutations in these transcription factors clinically manifest as subtypes of diabetes mellitus. Current treatment for diabetes is not curative and thus, developmental biologists and stem cell researchers are utilizing knowledge of normal pancreatic development to explore novel therapeutic alternatives. This review summarizes current knowledge of transcription factors involved in pancreatic development and β-cell differentiation in rodents.
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Affiliation(s)
- Reshmi Dassaye
- a Discipline of Pharmaceutical Sciences; Nelson R. Mandela School of Medicine, University of KwaZulu-Natal , Durban , South Africa
| | - Strini Naidoo
- a Discipline of Pharmaceutical Sciences; Nelson R. Mandela School of Medicine, University of KwaZulu-Natal , Durban , South Africa
| | - Marlon E Cerf
- b Diabetes Discovery Platform, South African Medical Research Council , Cape Town , South Africa
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Abstract
PURPOSE OF REVIEW This review will discuss recent advances in understanding mouse and human pancreatic islet cell development, novel concepts related to β cell dysfunction and improved approaches for replenishing β cells to treat diabetes. RECENT FINDINGS Considerable knowledge about pancreatic islet development and function has been gained using model systems with subsequent validation in human tissues. Recently, several rodent studies have revealed that differentiated adult islet cells retain remarkable plasticity and can be converted to other islet cell types by perturbing their transcription factor profiles. Furthermore, significant advances have been made in the generation of β-like cells from stem cell populations. Therefore, the generation of functionally mature β cells by the in-situ conversion of non-β cell populations or by the directed differentiation of human pluripotent stem cells could represent novel mechanisms for replenishing β cells in diabetic patients. SUMMARY The overall conservation between mouse and human pancreatic development, islet physiology and etiology of diabetes encourages the translation of novel β cell replacement therapies to humans. Further deciphering the molecular mechanisms that direct islet cell regeneration, plasticity and function could improve and expand the β cell replacement strategies for treating diabetes.
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Affiliation(s)
- Anthony I Romer
- Department of Genetics and Development, Columbia University, New York, New York, USA
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Raum JC, Soleimanpour SA, Groff DN, Coré N, Fasano L, Garratt AN, Dai C, Powers AC, Stoffers DA. Tshz1 Regulates Pancreatic β-Cell Maturation. Diabetes 2015; 64:2905-14. [PMID: 25918232 PMCID: PMC4512227 DOI: 10.2337/db14-1443] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/18/2014] [Accepted: 04/08/2015] [Indexed: 01/09/2023]
Abstract
The homeodomain transcription factor Pdx1 controls pancreas organogenesis, specification of endocrine pancreas progenitors, and the postnatal growth and function of pancreatic β-cells. Pdx1 expression in human-derived stem cells is used as a marker for induced pancreatic precursor cells. Unfortunately, the differentiation efficiency of human pancreatic progenitors into functional β-cells is poor. In order to gain insight into the genes that Pdx1 regulates during differentiation, we performed Pdx1 chromatin immunoprecipitation followed by high-throughput sequencing of embryonic day (e) 13.5 and 15.5 mouse pancreata. From this, we identified the transcription factor Teashirt zinc finger 1 (Tshz1) as a direct Pdx1 target. Tshz1 is expressed in developing and adult insulin- and glucagon-positive cells. Endocrine cells are properly specified in Tshz1-null embryos, but critical regulators of β-cell (Pdx1 and Nkx6.1) and α-cell (MafB and Arx) formation and function are downregulated. Adult Tshz1(+/-) mice display glucose intolerance due to defects in glucose-stimulated insulin secretion associated with reduced Pdx1 and Clec16a expression in Tshz1(+/-) islets. Lastly, we demonstrate that TSHZ1 levels are reduced in human islets of donors with type 2 diabetes. Thus, we position Tshz1 in the transcriptional network of maturing β-cells and suggest that its dysregulation could contribute to the islet phenotype of human type 2 diabetes.
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Affiliation(s)
- Jeffrey C Raum
- Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
| | - Scott A Soleimanpour
- Division of Metabolism, Endocrinology & Diabetes, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI
| | - David N Groff
- Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
| | - Nathalie Coré
- Institut de Biologie du Développement de Marseille, UMR 7288, Aix-Marseille Université, CNRS, Marseille, France
| | - Laurent Fasano
- Institut de Biologie du Développement de Marseille, UMR 7288, Aix-Marseille Université, CNRS, Marseille, France
| | - Alistair N Garratt
- Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité University Hospital Berlin, Berlin, Germany
| | - Chunhua Dai
- Division of Diabetes, Endocrinology & Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN
| | - Alvin C Powers
- Division of Diabetes, Endocrinology & Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN VA Tennessee Valley Healthcare System, Nashville, TN
| | - Doris A Stoffers
- Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
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Martin D, Kim YH, Sever D, Mao CA, Haefliger JA, Grapin-Botton A. REST represses a subset of the pancreatic endocrine differentiation program. Dev Biol 2015; 405:316-27. [PMID: 26156633 DOI: 10.1016/j.ydbio.2015.07.002] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2014] [Revised: 07/01/2015] [Accepted: 07/02/2015] [Indexed: 12/20/2022]
Abstract
To contribute to devise successful beta-cell differentiation strategies for the cure of Type 1 diabetes we sought to uncover barriers that restrict endocrine fate acquisition by studying the role of the transcriptional repressor REST in the developing pancreas. Rest expression is prevented in neurons and in endocrine cells, which is necessary for their normal function. During development, REST represses a subset of genes in the neuronal differentiation program and Rest is down-regulated as neurons differentiate. Here, we investigate the role of REST in the differentiation of pancreatic endocrine cells, which are molecularly close to neurons. We show that Rest is widely expressed in pancreas progenitors and that it is down-regulated in differentiated endocrine cells. Sustained expression of REST in Pdx1(+) progenitors impairs the differentiation of endocrine-committed Neurog3(+) progenitors, decreases beta and alpha cell mass by E18.5, and triggers diabetes in adulthood. Conditional inactivation of Rest in Pdx1(+) progenitors is not sufficient to trigger endocrine differentiation but up-regulates a subset of differentiation genes. Our results show that the transcriptional repressor REST is active in pancreas progenitors where it gates the activation of part of the beta cell differentiation program.
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Affiliation(s)
- David Martin
- Swiss Institute for Experimental Cancer Research, Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Station 19, 1015 Lausanne, Switzerland
| | - Yung-Hae Kim
- DanStem, University of Copenhagen, 3B Blegdamsvej, DK-2200 Copenhagen N, Denmark
| | - Dror Sever
- DanStem, University of Copenhagen, 3B Blegdamsvej, DK-2200 Copenhagen N, Denmark
| | - Chai-An Mao
- Department of Systems Biology, The University of MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Jacques-Antoine Haefliger
- Department of Medicine, Laboratory of Experimental Medicine, C/O Department of Physiology, Bugnon 7a, 1005 Lausanne, Switzerland
| | - Anne Grapin-Botton
- Swiss Institute for Experimental Cancer Research, Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Station 19, 1015 Lausanne, Switzerland; DanStem, University of Copenhagen, 3B Blegdamsvej, DK-2200 Copenhagen N, Denmark.
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Cebola I, Rodríguez-Seguí SA, Cho CHH, Bessa J, Rovira M, Luengo M, Chhatriwala M, Berry A, Ponsa-Cobas J, Maestro MA, Jennings RE, Pasquali L, Morán I, Castro N, Hanley NA, Gomez-Skarmeta JL, Vallier L, Ferrer J. TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors. Nat Cell Biol 2015; 17:615-626. [PMID: 25915126 PMCID: PMC4434585 DOI: 10.1038/ncb3160] [Citation(s) in RCA: 163] [Impact Index Per Article: 16.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2014] [Accepted: 03/13/2015] [Indexed: 02/02/2023]
Abstract
The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.
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Affiliation(s)
- Inês Cebola
- Department of Medicine, Imperial College London, London W12 0NN, United Kingdom
| | - Santiago A. Rodríguez-Seguí
- Genomic Programming of Beta-cells Laboratory, Institut d’Investigacions August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain
- CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 08036 Barcelona, Spain
- Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología, Biología Molecular y Celular, IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EGA Buenos Aires, Argentina
| | - Candy H.-H. Cho
- Wellcome Trust and MRC Stem Cells Centre, Anne McLaren Laboratory for Regenerative Medicine, Department of Surgery and Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0SZ, United Kingdom
| | - José Bessa
- Instituto de Biologia Molecular e Celular (IBMC), 4150-180 Porto, Portugal
- Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal
| | - Meritxell Rovira
- Genomic Programming of Beta-cells Laboratory, Institut d’Investigacions August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain
- CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 08036 Barcelona, Spain
| | - Mario Luengo
- Centro Andaluz de Biología del Desarrollo, Consejo Superior de Investigaciones Científicas/Universidad Pablo de Olavide, 41013 Sevilla, Spain
| | - Mariya Chhatriwala
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom
| | - Andrew Berry
- Centre for Endocrinology and Diabetes, Institute of Human Development, Faculty of Medical & Human Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester M13 9PT, United Kingdom
| | - Joan Ponsa-Cobas
- Department of Medicine, Imperial College London, London W12 0NN, United Kingdom
| | - Miguel Angel Maestro
- Genomic Programming of Beta-cells Laboratory, Institut d’Investigacions August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain
- CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 08036 Barcelona, Spain
| | - Rachel E. Jennings
- Centre for Endocrinology and Diabetes, Institute of Human Development, Faculty of Medical & Human Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester M13 9PT, United Kingdom
| | - Lorenzo Pasquali
- Genomic Programming of Beta-cells Laboratory, Institut d’Investigacions August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain
- CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 08036 Barcelona, Spain
| | - Ignasi Morán
- Department of Medicine, Imperial College London, London W12 0NN, United Kingdom
| | - Natalia Castro
- Genomic Programming of Beta-cells Laboratory, Institut d’Investigacions August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain
- CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 08036 Barcelona, Spain
| | - Neil A. Hanley
- Centre for Endocrinology and Diabetes, Institute of Human Development, Faculty of Medical & Human Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester M13 9PT, United Kingdom
- Endocrinology Department, Central Manchester University Hospitals NHS Foundation Trust, Manchester M13 9WU, United Kingdom
| | - Jose Luis Gomez-Skarmeta
- Centro Andaluz de Biología del Desarrollo, Consejo Superior de Investigaciones Científicas/Universidad Pablo de Olavide, 41013 Sevilla, Spain
| | - Ludovic Vallier
- Wellcome Trust and MRC Stem Cells Centre, Anne McLaren Laboratory for Regenerative Medicine, Department of Surgery and Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0SZ, United Kingdom
- Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom
| | - Jorge Ferrer
- Department of Medicine, Imperial College London, London W12 0NN, United Kingdom
- Genomic Programming of Beta-cells Laboratory, Institut d’Investigacions August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain
- CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), 08036 Barcelona, Spain
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van der Meulen T, Huising MO. Role of transcription factors in the transdifferentiation of pancreatic islet cells. J Mol Endocrinol 2015; 54:R103-17. [PMID: 25791577 PMCID: PMC4373662 DOI: 10.1530/jme-14-0290] [Citation(s) in RCA: 90] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The α and β cells act in concert to maintain blood glucose. The α cells release glucagon in response to low levels of glucose to stimulate glycogenolysis in the liver. In contrast, β cells release insulin in response to elevated levels of glucose to stimulate peripheral glucose disposal. Despite these opposing roles in glucose homeostasis, α and β cells are derived from a common progenitor and share many proteins important for glucose sensing and hormone secretion. Results from recent work have underlined these similarities between the two cell types by revealing that β-to-α as well as α-to-β transdifferentiation can take place under certain experimental circumstances. These exciting findings highlight unexpected plasticity of adult islets and offer hope of novel therapeutic paths to replenish β cells in diabetes. In this review, we focus on the transcription factor networks that establish and maintain pancreatic endocrine cell identity and how they may be perturbed to facilitate transdifferentiation.
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Affiliation(s)
- Talitha van der Meulen
- Department of NeurobiologyPhysiology and Behavior, College of Biological SciencesDepartment of Physiology and Membrane BiologySchool of Medicine, University of California, 193 Briggs Hall, One Shields Avenue, Davis, California 95616, USA
| | - Mark O Huising
- Department of NeurobiologyPhysiology and Behavior, College of Biological SciencesDepartment of Physiology and Membrane BiologySchool of Medicine, University of California, 193 Briggs Hall, One Shields Avenue, Davis, California 95616, USA Department of NeurobiologyPhysiology and Behavior, College of Biological SciencesDepartment of Physiology and Membrane BiologySchool of Medicine, University of California, 193 Briggs Hall, One Shields Avenue, Davis, California 95616, USA
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Soggia A, Ramond C, Akiyama H, Scharfmann R, Duvillie B. von Hippel-Lindau gene disruption in mouse pancreatic progenitors and its consequences on endocrine differentiation in vivo: importance of HIF1-α and VEGF-A upregulation. Diabetologia 2014; 57:2348-56. [PMID: 25186293 DOI: 10.1007/s00125-014-3365-y] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/20/2014] [Accepted: 08/11/2014] [Indexed: 12/15/2022]
Abstract
AIM/HYPOTHESIS Different studies have linked hypoxia to embryonic development. Specifically, when embryonic pancreases are cultured ex vivo under hypoxic conditions (3% O2), beta cell development is impaired. Different cellular signalling pathways are involved in adaptation to hypoxia, including the ubiquitous hypoxia-inducible-factor 1-α (HIF1-α) pathway. We aimed to analyse the effects of HIF1-α stabilisation on fetal pancreas development in vivo. METHODS We deleted the Vhl gene, which encodes von Hippel-Lindau protein (pVHL), a factor necessary for HIF1-α degradation, by crossing Vhl-floxed mice with Sox9-Cre mice. RESULTS HIF1-α was stabilised in pancreatic progenitor cells in which the HIF pathway was induced. The number of neurogenin-3 (NGN3)-expressing cells was reduced and consequently endocrine development was altered in Vhl knockout pancreases. HIF1-α stabilisation induced Vegfa upregulation, leading to increased vascularisation. To investigate the impact of increased vascularisation on NGN3 expression, we used a bioassay in which Vhl mutant pancreases were cultured with or without vascular endothelial growth factor (VEGF) receptor 2 (VEGF-R2) inhibitors (e.g. Ki8751). Ex vivo analysis showed that Vhl knockout pancreases developed fewer NGN3-positive cells compared with controls. Interestingly, this effect was blocked when vascularisation was inhibited in the presence of VEGF-R2 inhibitors. CONCLUSIONS/INTERPRETATION Our data demonstrate that HIF1-α negatively controls beta cell differentiation in vivo by regulating NGN3 expression, and that this effect is mediated by signals from blood vessels.
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Affiliation(s)
- Andrea Soggia
- U1016 Inserm/Institut Cochin, Groupe Hospitalier Cochin Port-Royal, Bâtiment Cassini, 123 Boulevard du Port-Royal, 75014, Paris, France
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Li JT, Sun FX. Myocardin and pdx-1 synergistically induce hMSCs to differentiate into insulin secreting cells. Biochem Biophys Res Commun 2014:S0006-291X(14)01747-1. [PMID: 25301554 DOI: 10.1016/j.bbrc.2014.09.110] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2014] [Accepted: 09/26/2014] [Indexed: 01/09/2023]
Abstract
Mesenchymal stem cells (MSCs) have been reported as an attractive source for the generation of transplantable surrogate β cells. The objective of this study was to investigate a new method to induce the differentiation of hMSCs into insulin secretion cells and to explore its molecular mechanisms. In this study, we investigated in vitro differentiation of hMSCs by overexpression of myocardin and pdx-1. Differentiated cells were evaluated by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), quantificational real-time RT-PCR (qRT-PCR) and Western blotting. Furthermore, the molecular mechanisms were evaluated by chip assay, CO-IP and Luciferase assay. This study reported a new method to induce the differentiation of hMSCs into insulin secretion cells. The method is cotransduction of myocardin and pdx-1 for 7days. At the same time, we find myocardin and pdx-1 can form a complex to promote the transactivities of insulin by affecting the formation of the pdx-1/myocardin/SRF/CArG complex both in vitro and in vitro. The present study provided a simple and faithful in vitro model for further investigating the cell replacement therapy for diabetes.
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Affiliation(s)
- Jing-Ting Li
- College of Resource and Environment Science, Pingdingshan University, Pingdingshan 467000, China.
| | - Fang-Xing Sun
- College of Architecture & Urban Planning, Henan University of Urban Construction, Pingdingshan 467036, China
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Osipovich AB, Long Q, Manduchi E, Gangula R, Hipkens SB, Schneider J, Okubo T, Stoeckert CJ, Takada S, Magnuson MA. Insm1 promotes endocrine cell differentiation by modulating the expression of a network of genes that includes Neurog3 and Ripply3. Development 2014; 141:2939-49. [PMID: 25053427 PMCID: PMC4197673 DOI: 10.1242/dev.104810] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
Insulinoma associated 1 (Insm1) plays an important role in regulating the development of cells in the central and peripheral nervous systems, olfactory epithelium and endocrine pancreas. To better define the role of Insm1 in pancreatic endocrine cell development we generated mice with an Insm1GFPCre reporter allele and used them to study Insm1-expressing and null populations. Endocrine progenitor cells lacking Insm1 were less differentiated and exhibited broad defects in hormone production, cell proliferation and cell migration. Embryos lacking Insm1 contained greater amounts of a non-coding Neurog3 mRNA splice variant and had fewer Neurog3/Insm1 co-expressing progenitor cells, suggesting that Insm1 positively regulates Neurog3. Moreover, endocrine progenitor cells that express either high or low levels of Pdx1, and thus may be biased towards the formation of specific cell lineages, exhibited cell type-specific differences in the genes regulated by Insm1. Analysis of the function of Ripply3, an Insm1-regulated gene enriched in the Pdx1-high cell population, revealed that it negatively regulates the proliferation of early endocrine cells. Taken together, these findings indicate that in developing pancreatic endocrine cells Insm1 promotes the transition from a ductal progenitor to a committed endocrine cell by repressing a progenitor cell program and activating genes essential for RNA splicing, cell migration, controlled cellular proliferation, vasculogenesis, extracellular matrix and hormone secretion.
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Affiliation(s)
- Anna B Osipovich
- Center for Stem Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
| | - Qiaoming Long
- Department of Animal Science, Cornell University, Ithaca, NY 14850, USA
| | - Elisabetta Manduchi
- Penn Center for Bioinformatics, Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
| | - Rama Gangula
- Center for Stem Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
| | - Susan B Hipkens
- Center for Stem Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
| | - Judsen Schneider
- Center for Stem Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
| | - Tadashi Okubo
- Department of Laboratory Animal Science, Kitasato University School of Medicine, Sagamihara, 252-0374, Japan
| | - Christian J Stoeckert
- Penn Center for Bioinformatics, Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
| | - Shinji Takada
- Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, 444-8787, Japan
| | - Mark A Magnuson
- Center for Stem Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
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Mulley JF, Holland PW. Genomic organisation of the seven ParaHox genes of coelacanths. JOURNAL OF EXPERIMENTAL ZOOLOGY PART B: MOLECULAR AND DEVELOPMENTAL EVOLUTION 2014; 322:352-8. [PMID: 23775937 PMCID: PMC4471637 DOI: 10.1002/jez.b.22513] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/27/2013] [Revised: 04/25/2013] [Accepted: 04/25/2013] [Indexed: 11/30/2022]
Abstract
Human and mouse genomes contain six ParaHox genes implicated in gut and neural patterning. In coelacanths and cartilaginous fish, an additional ParaHox gene exists—Pdx2—that dates back to the genome duplications in early vertebrate evolution. Here we examine the genomic arrangement and flanking genes of all ParaHox genes in coelacanths, to determine the full complement of these genes. We find that coelacanths have seven ParaHox genes in total, in four chromosomal locations, revealing that five gene losses occurred soon after vertebrate genome duplication. Comparison of intergenic sequences reveals that some Pdx1 regulatory regions associated with development of pancreatic islets are older than tetrapods, that Pdx1 and Pdx2 share few if any conserved non-coding elements, and that there is very high sequence conservation between coelacanth species.
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Affiliation(s)
- John F. Mulley
- School of Biological SciencesBangor UniversityBangorGwynedd, United Kingdom
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50
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Ishkitiev N, Yaegaki K, Kozhuharova A, Tanaka T, Okada M, Mitev V, Fukuda M, Imai T. Pancreatic differentiation of human dental pulp CD117⁺ stem cells. Regen Med 2014; 8:597-612. [PMID: 23998753 DOI: 10.2217/rme.13.42] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
AIM Adult stem cells cannot proliferate to produce enough cells for human transplantation with keeping stem cell characteristics shown in the primary culture. We established a novel culture protocol using human dental pulp stem cells (DPSCs) that can produce quantities sufficient for human transplantation. The present study assessed differentiation of DPSCs toward a pancreatic lineage in serum-free conditions, which is essential for safe transplantation. MATERIALS & METHODS CD117⁺ stem cells were separated from human exfoliated deciduous teeth (stem cells from human exfoliated deciduous teeth; SHED) and adult DPSCs. The cells were characterized with real-time reverse-transcription PCR for a panel of embryonal lineage markers. RESULTS 82 out of 84 markers were expressed in different levels in SHED or DPSCs. After pancreatic differentiation in vitro, we found expression of pancreatic-specific endocrine markers insulin, glucagon, somatostatin and pancreatic polypeptide, and exocrine marker amylase-2a in both cultures. We also found reprogramming in both cell cultures mimicking the embryonal stages of development of the pancreas. Transcription factors PDX1, HHEX, MNX1, NEUROG3, PAX4, PAX6 and NKX6-1, crucial markers for the pancreatic development, were all activated. Expression of these factors strongly implies that the cells differentiated toward a distinguished pancreatic lineage. CONCLUSION Our results show that CD117⁺ SHED and DPSCs are capable of differentiation toward all functional endocrine and exocrine subsets of pancreatic cells in serum-free conditions. SHED and DPSCs may therefore have great potential for future cell therapy of pancreatic disorders.
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Affiliation(s)
- Nikolay Ishkitiev
- Nippon Dental University, School of Life Dentistry at Tokyo, Department of Oral Health, 1-9-20 Chiyoda-ku, 102-8159 Tokyo, Japan
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