1: Dev Biol. 2005 Oct 1;286(1):217-24. Genetic determinants of pancreatic epsilon-cell development. Heller RS, Jenny M, Collombat P, Mansouri A, Tomasetto C, Madsen OD, Mellitzer G, Gradwohl G, Serup P. Hagedorn Research Institute, Department of Developmental Biology, DK-2820 Gentofte, Denmark. Recently, the expression of the peptide hormone ghrelin was detected in alpha-cells of the islets of Langerhans as well as in epsilon-cells, a newly discovered endocrine cell type, but it remains unclear how the latter is related in lineage to the four classical islet cell types, alpha-, beta-, delta-, and PP-cells. Here, we provide further evidence that ghrelin is predominantly produced in the alpha-cells of mouse islets but also in single hormone ghrelin-secreting epsilon-cells. We additionally demonstrate that pancreatic epsilon-cells derive from Neurogenin3-expressing precursor cells and their genesis depends on Neurogenin3 activity. Furthermore, our data indicate that the number of ghrelin-producing cells is differentially regulated during pancreas morphogenesis by the homeodomain-containing transcription factors Arx, Pax4, and Pax6. Arx mutants lack ghrelin+ glucagon+ alpha-cells whereas Pax4 mutants develop an excess of these cells. Importantly, the ghrelin+ glucagon- epsilon-cell population is not affected following Arx or Pax4 disruption. In contrast, the loss of Pax6 provokes an unexpected increase of the ghrelin+ glucagon- epsilon-cell number which is not due to increased proliferation. Thus, we demonstrate that the development of ghrelin-producing cells is differentially dependent on Neurogenin3 in different domains of the gastrointestinal tract and that, in the endocrine pancreas, epsilon-cell genesis does not require Arx or Pax4 activities but is antagonized by Pax6. PMID: 16122727 [PubMed - in process] --------------------------------------------------------------- 2: Mol Biol Evol. 2005 Jul;22(7):1569-78. Epub 2005 Apr 27. The Trichoplax PaxB gene: a putative Proto-PaxA/B/C gene predating the origin of nerve and sensory cells. Hadrys T, DeSalle R, Sagasser S, Fischer N, Schierwater B. ITZ, Ecology & Evolution, Hannover, Germany. thorsten.hadrys@uni-oldenburg.de Pax genes play key regulatory roles in embryonic and sensory organ development in metazoans but their evolution and ancestral functions remain widely unresolved. We have isolated a Pax gene from Placozoa, beside Porifera the only metazoan phylum that completely lacks nerve and sensory cells or organs. These simplest known metazoans also lack any kind of symmetry, organs, extracellular matrix, basal lamina, muscle cells, and main body axis. The isolated Pax gene from Trichoplax adhaerens harbors a paired domain, an octapeptide, and a full-length homeodomain. It displays structural features not only of PaxB and Pax2/5/8-like genes but also of PaxC and Pax6 genes. Conserved splice sites between Placozoa, Cnidaria, and triploblasts, mark the ancient origin of intron structures. Phylogenetic analyses demonstrate that the Trichoplax PaxB gene, TriPaxB, is basal not only to all other known PaxB genes but also to PaxA and PaxC genes and their relatives in triploblasts (namely Pax2/5/8, Pax4/6, and Poxneuro). TriPaxB is expressed in distinct cell patches near the outer edge of the animal body, where undifferentiated and possibly multipotent cells are found. This expression pattern indicates a developmental role in cell-type specification and/or differentiation, probably in specifying-determining fiber cells, which are regarded as proto-neural/muscle cells in Trichoplax. While PaxB, Pax2/5/8, and Pax6 genes have been linked to nerve cell and sensory system/organ development in virtually all animals investigated so far, our study suggests that Pax genes predate the origin of nerve and sensory cells. PMID: 15858210 [PubMed - in process] --------------------------------------------------------------- 3: Diabet Med. 2005 May;22(5):641-4. A case of novel de novo paired box gene 6 (PAX6) mutation with early-onset diabetes mellitus and aniridia. Nishi M, Sasahara M, Shono T, Saika S, Yamamoto Y, Ohkawa K, Furuta H, Nakao T, Sasaki H, Nanjo K. The First Department of Medicine, Wakayama Medical University, Wakayama, Japan. mnishi@wakayama-med.ac.jp BACKGROUND: Paired box gene 6 (PAX6) is a transcription factor involved in eye development. Mutations of PAX6 cause congenital eye anomalies, such as aniridia. PAX6 is also involved in the development of the endocrine pancreas, and reported to be a genetic factor common to aniridia and glucose intolerance, although the latter is usually mild. Here, we describe a case of PAX6 mutation with early-onset diabetes mellitus. CASE REPORT: A 27-year-old woman was referred to our clinic. She was diagnosed having diabetes at the age of 15 with negative glutamic acid decarboxylase (GAD) antibody. Insulin treatment was started at age 24. Because she had aniridia, PAX6 gene mutation was investigated and a heterozygous 2-bp deletion (c.402del2) was identified. Her parents did not have aniridia and PAX6 mutations. Heterozygous PAX6 mutation may cause glucose intolerance. However, cases of early-onset diabetes mellitus have not been reported. Her parents did not have diabetes, but their insulinogenic indices were low (0.25 and 0.3, respectively). We thought her early-onset diabetes was partly as a result of PAX6 mutation and partly because of an unknown insulin secretory defect inherited from her parents. We could not find any mutations in HNF-1alpha, -1beta, -4alpha, IPF-1, ISL-1, BEAT2/NeuroD1, PAX4, and amylin genes. CONCLUSIONS: We report a case of PAX6 gene mutation with early-onset diabetes mellitus and aniridia. Low insulin secretory capacity in her parents suggested that her insulin secretory defect is as a result of not only PAX6 mutation but other genetic factors inherited from her parents. Publication Types: Case Reports PMID: 15842522 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Mol Cells. 2004 Dec 31;18(3):289-94. The gene Pax4 is an essential regulator of pancreatic beta-cell development. Sosa-Pineda B. Department of Genetics, St. Jude Children's Research Hospital, 332 N Lauderdale, Memphis, Tennessee 38105, USA. beatriz.sosa-pineda@stjude.org The Pax-gene family encodes a group of transcription factors characterized by the presence of a highly conserved DNA-binding motif, the paired domain. Pax proteins are key regulators of vertebrate organogenesis since they play major roles in embryonic pattern formation, cell proliferation and cell differentiation (Chi and Epstein, 2002; Dahl et al., 1997; Dohrman et al., 2000; Epstein et al., 1994). Indeed, mutations in Pax genes lead to profound defects in organisms as diverse as flies, mice and humans (Chi and Epstein, 2002; Dahl et al., 1997). To date, nine mammalian Pax genes are known and these are grouped into five different subclasses according to their structural similarities. One of these subclasses comprises two close homologues, Pax4 and Pax6, that contain a second DNA-binding domain: the homeodomain (Dahl et al., 1997; Dohrman et al., 2000). Previous studies showed that Pax4 is a crucial regulator of mammalian pancreas development since lack of its activity prevents the formation of mature pancreatic insulin-producing (beta) cells (Dohrman et al., 2000; Sosa-Pineda et al., 1997; Wang et al., 2004). Presently, it is not yet clear how Pax4 is specifically required for the development of beta cells. Nonetheless, evidence gathered from recent studies has begun to unravel important aspects of the molecular function of Pax4 in pancreatic endocrine cells. Here, I will try to summarize the results of different efforts aimed at understanding how Pax4 is required for both, beta cell development and beta cell function. Publication Types: Review Review, Tutorial PMID: 15650323 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Diabetes. 2004 Dec;53(12):3168-78. High glucose is necessary for complete maturation of Pdx1-VP16-expressing hepatic cells into functional insulin-producing cells. Cao LZ, Tang DQ, Horb ME, Li SW, Yang LJ. Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida, USA. Pdx1 has been shown to convert hepatocytes into both exocrine and endocrine pancreatic cells in mice, but it fails to selectively convert hepatocytes into pure insulin-producing cells (IPCs). The molecular mechanisms underlying the transdifferentiation remain unclear. In this study, we generated a stably transfected rat hepatic cell line named WB-1 that expresses an active form of Pdx1 along with a reporter gene, RIP-eGFP. Our results demonstrate that Pdx1 induces the expression of multiple genes related to endocrine pancreas development and islet function in these liver cells. We do not however find any expression of the late-stage genes (Pax4, Pax6, Isl-1, and MafA) related to beta-cell development, and the cells do not secrete insulin upon the glucose challenge. Yet when WB-1 cells are transplanted into diabetic NOD-scid mice, these genes become activated and hyperglycemia is completely reversed. Detailed comparison of gene expression profiles between pre- and posttransplanted WB-1 cells demonstrates that the WB-1 cells have similar properties as that seen in pancreatic beta-cells. In addition, in vitro culture in high-glucose medium is sufficient to induce complete maturation of WB-1 cells into functional IPCs. In summary, we find that Pdx1-VP16 is able to selectively convert hepatic cells into pancreatic endocrine precursor cells. However, complete transdifferentiation into functional IPCs requires additional external factors, including high glucose or hyperglycemia. Thus, transdifferentiation of hepatocytes into functional IPCs may serve as a viable therapeutic option for patients with type 1 diabetes. PMID: 15561947 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Pancreas. 2004 Oct;29(3):e64-76. Redox-mediated enrichment of self-renewing adult human pancreatic cells that possess endocrine differentiation potential. Linning KD, Tai MH, Madhukar BV, Chang CC, Reed DN Jr, Ferber S, Trosko JE, Olson LK. Department of Physiology, Michigan State University, East Lansing, Michigan 48824, USA. OBJECTIVES: The limited availability of transplantable human islets has stimulated the development of methods needed to isolate adult pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation. The objective of this study was to determine whether modulation of intracellular redox state with N-acetyl-L-cysteine (NAC) would allow for the propagation of pancreatic stem/progenitor cells from adult human pancreatic tissue. METHODS: Cells were propagated from human pancreatic tissue using a serum-free, low-calcium medium supplemented with NAC and tested for their ability to differentiate when cultured under different growth conditions. RESULTS: Human pancreatic cell (HPC) cultures coexpressed alpha-amylase, albumin, vimentin, and nestin. The HPC cultures, however, did not express other genes associated with differentiated pancreatic exocrine, duct, or endocrine cells. A number of transcription factors involved in endocrine cell development including Beta 2, Islet-1, Nkx6.1, Pax4, and Pax6 were expressed at variable levels in HPC cultures. In contrast, pancreatic duodenal homeobox factor 1 (Pdx-1) expression was extremely low and at times undetectable. Overexpression of Pdx-1 in HPC cultures stimulated somatostatin, glucagon, and carbonic anhydrase expression but had no effect on insulin gene expression. HPC cultures could form 3-dimensional islet-like cell aggregates, and this was associated with expression of somatostatin and glucagon but not insulin. Cultivation of HPCs in a differentiation medium supplemented with nicotinamide, exendin-4, and/or LY294002, an inhibitor of phosphatidylinositol-3 kinase, stimulated expression of insulin mRNA and protein. CONCLUSION: These data support the use of intracellular redox modulation for the enrichment of pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation. PMID: 15367896 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Transplant Proc. 2004 May;36(4):1151-8. Expression in murine teratocarcinoma F9 cells of transcription factors involved in pancreas development. O'Driscoll L, Gammell P, Clynes M. National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin, Ireland. Lorraine.ODriscoll@dcu.ie BACKGROUND: Although it has been established that formation and functional differentiation of the pancreas from embryonic endoderm is associated with activation/inactivation of many genes controlled by specific sets of transcription factors, the role and activation sequence of individual transcription factors has not yet been fully elucidated. This study sought to differentiate a murine teratocarcinoma cell line, F9, to endodermal-like cells and, subsequently; to investigate the effects of regulated expression of transcription factors in pancreas development. METHODS: Following differentiation using retinoic acid and db cAMP (RAC), resulting F9 cells (F9-RAC) were transfected with cDNAs for PDX-1, ngn3, beta 2/NeuroD (beta 2), and Nkx2.2, singly or in combination. Expression of these transcription factors was investigated using RT-PCR and immunofluorescence techniques. RT-PCR analysis was used to assess the subsequent effects of expression of these factors on endogenous genes related to pancreas development. RESULTS: Regulated differentiation of F9 cells generated endodermal-like cell types. Following transfection, PDX-1, ngn3, beta 2, and Nkx2.2 were expressed in F9-RAC cells, with their proteins localized mainly in cellular nuclei. Expression of these factors apparently did not affect the endogenous expression of preproinsulin, PDX-1, beta 2, Isl1, Pax4, Pax6, Sonic hedgehog, and Indian hedgehog. CONCLUSION: This study describes the successful transient expression of transcription factors related to pancreas development, following directed differentiation of F9 cells to endoderm-like cells, and shows that treatment of F9 cells with a combination of RAC causes up-regulation of genes relevant to pancreatic development. The lack of further effect of regulated transcription factor expression on these genes may suggest that parietal endoderm- like cells derived from F9 cells is not the optimal lineage from which to develop beta cells. It may be useful to include F9-derived visceral endoderm in future studies. PMID: 15194401 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Endocrinology. 2004 Jun;145(6):2639-44. Epub 2004 Mar 24. Minireview: Development and differentiation of gut endocrine cells. Schonhoff SE, Giel-Moloney M, Leiter AB. Division of Gastroenterology No. 218, New England Medical Center, 750 Washington Street, Boston, Massachusetts 02111, USA. For over 30 yr, it has been known that enteroendocrine cells derive from common precursor cells in the intestinal crypts. Until recently, relatively little was understood about the events that result in commitment to endocrine differentiation or the segregation of over 10 different hormone-expressing cell types in the gastrointestinal tract. The earliest cell fate decisions appear to be regulated by the Notch signaling pathway. Notch is inactive in endocrine precursor cells, allowing for expression of the proendocrine basic helix-loop-helix proteins Math1 and neurogenin3. Differentiating precursor cells activate Notch in neighboring cells to switch off expression of proendocrine factors and inhibit endocrine differentiation. Math1 is the first factor involved in endocrine specification, committing cells to become one of three secretory lineages-goblet, Paneth, and enteroendocrine. Neurogenin3 appears to be a downstream target that is essential for endocrine cell differentiation. Events that control the segregation of each mature lineage from progenitor cells have not been characterized in detail. The transcription factors Pax4, Pax6, BETA2/NeuroD, and pancreatic-duodenal homeobox 1 have all been implicated in enteroendocrine differentiation. BETA2/NeuroD appears to coordinate secretin gene expression in S-type enteroendocrine cells with cell cycle arrest as cells terminally differentiate. Powerful genetic approaches have established the murine intestine as the most important model for studying enteroendocrine differentiation. Enteroendocrine cells in the mouse are remarkably similar to those in humans, making it likely that insights learned from the mouse may contribute to both our understanding and treatment of a variety of human disorders. Publication Types: Review Review, Tutorial PMID: 15044355 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Cell Mol Life Sci. 2003 Jun;60(6):1033-48. Promoter elements in endocrine pancreas development and hormone regulation. Brink C. Biozentrum, Klingelbergstrasse 70, 4056 Basel, Switzerland. christopher.brink@unibas.ch The research upon the genetics of mammalian endocrine pancreas development gave rise to the detection of several genes that mediate decisions between different cell lineages that finally lead to four different hormone-producing cell types. Transcription factors such as Pdx1, Hnf6, ngn3, NeuroD/BETA2, Pax6 or Pax4 act within regulatory cascades and networks of transcriptional regulations that provide the genetic background for endocrine pancreas development. In adult animals the anatomical unit of the endocrine organ, the Islets of Langerhans, is built out of alpha, beta, delta and PP cells producing the peptide hormones glucagon, insulin, somatostatin and pancreatic polypeptide, respectively. Numerous promoter analyses of genes expressed in endocrine cells during development and adulthood have been performed. It turns out that the sequences of cis-regulatory elements within promoters of both, developmental control genes and peptide hormones, can show significant similarities. The relevance of such elements has been demonstrated by several deletion experiments and protein-DNA interaction assays. This review summarizes the currently known cis-regulatory elements that are important for islet development and provides the opportunity of detecting further pancreatic genes by discussing common promoter structures. Publication Types: Review PMID: 12861373 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Nippon Naika Gakkai Zasshi. 2003 May 10;92(5):890-6. [Transcription factors and diabetes mellitus] [Article in Japanese] Horikawa Y, Shihara N, Takeda J. Publication Types: Review Review, Tutorial PMID: 12808917 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: J Cell Biol. 2002 Oct 28;159(2):303-12. Epub 2002 Oct 28. Recapitulation of embryonic neuroendocrine differentiation in adult human pancreatic duct cells expressing neurogenin 3. Heremans Y, Van De Casteele M, in't Veld P, Gradwohl G, Serup P, Madsen O, Pipeleers D, Heimberg H. Diabetes Research Center, Brussels Free University (VUB), Laarbeeklaan 103, B-1090 Brussels, Belgium. Regulatory proteins have been identified in embryonic development of the endocrine pancreas. It is unknown whether these factors can also play a role in the formation of pancreatic endocrine cells from postnatal nonendocrine cells. The present study demonstrates that adult human pancreatic duct cells can be converted into insulin-expressing cells after ectopic, adenovirus-mediated expression of the class B basic helix-loop-helix factor neurogenin 3 (ngn3), which is a critical factor in embryogenesis of the mouse endocrine pancreas. Infection with adenovirus ngn3 (Adngn3) induced gene and/or protein expression of NeuroD/beta2, Pax4, Nkx2.2, Pax6, and Nkx6.1, all known to be essential for beta-cell differentiation in mouse embryos. Expression of ngn3 in adult human duct cells induced Notch ligands Dll1 and Dll4 and neuroendocrine- and beta-cell-specific markers: it increased the percentage of synaptophysin- and insulin-positive cells 15-fold in ngn3-infected versus control cells. Infection with NeuroD/beta2 (a downstream target of ngn3) induced similar effects. These data indicate that the Delta-Notch pathway, which controls embryonic development of the mouse endocrine pancreas, can also operate in adult human duct cells driving them to a neuroendocrine phenotype with the formation of insulin-expressing cells. PMID: 12403815 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Pancreas. 2000 Nov;21(4):399-406. IFN-gamma overexpression within the pancreas is not sufficient to rescue Pax4, Pax6, and Pdx-1 mutant mice from death. Krakowski M, Yeung B, Abdelmalik R, Good A, Mocnik L, Sosa-Pineda B, St-Onge L, Gruss P, Sarvetnick N. The Scripps Research Institute, Department of Immunology, La Jolla, California 92037, USA. In the presence of interferon-gamma (IFN-gamma), pancreatic ductal epithelial cells grow continuously, and islets undergo neogenesis. To determine whether these new islets are derived from conventional precursors, we tested whether IFN-gamma can complement the loss of transcription factors known to regulate pancreatic development. We analyzed the effect of a transgene on lethality in mice lacking the transcription factors Pax4, Pax6, or Pdx-1, by intercrossing such mice with transgenic mice whose pancreatic cells make IFN-gamma (ins-IFN-gamma mice). However, IFN-gamma expression did not rescue these mice from the lethal mutations, because no homozygous knockout mice carrying the IFN-gamma transgene survived, despite the survival of all other hemizygous gene combinations. This outcome demonstrates that the pathway for IFN-gamma regeneration requires the participation of Pax4, Pax6, and Pdx-1. We conclude that the striking islet regeneration observed in the ins-IFN-gamma NOD strain is regulated by the same transcription factors that control initial pancreatic development. PMID: 11075995 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Mol Cell Endocrinol. 2000 Jul 25;165(1-2):41-9. Transcription factor expression and hormone production in pancreatic AR42J cells. Palgi J, Stumpf E, Otonkoski T. Transplantation Laboratory, Haartman Institute and the Hospital for Children and Adolescents, University of Helsinki, PO Box 21 (Haartmaninkatu 3), FIN-00014, Helsinki, Finland. jaan.palgi@helsinki.fi AR42J is an exocrine pancreatic cell line that has been reported to differentiate towards an endocrine phenotype when stimulated with various growth factors, such as activin A, hepatocyte growth factor (HGF), betacellulin or glucagon-like peptide 1. In our experiments, AR42J-B13 cells differentiated morphologically in response to the growth factor treatment as reported previously. However, they failed to express the insulin gene. We found that the cells did not express several transcription factors known to be found in the beta-cell, including Nkx6.1, isl-1, Pax4 and Pax6. In addition, the mRNA level for pdx-1 and Nkx2.2 were very low in comparison to the insulinoma cell lines INS-1 and RINm5F. However, some transcription factors typically found in beta-cells and neuroendocrine cells were expressed also in the AR42J-B13 cells. These included BETA2/NeuroD, HNF1alpha, C/EBPbeta and IA-1. Unlike the insulinoma cells, AR42J cells expressed the exocrine transcription factor p48. In order to induce endocrine differentiation, we transfected the AR42J-B13 cells with the full length cDNAs of isl-1, Nkx6.1, Nkx2.2 and pdx-1 under the control of the CMV promoter, both separately and in combinations. The expression of Nkx2.2 led consistently to the appearance of pancreatic polypeptide but not insulin, glucagon or somatostatin mRNA. The PP mRNA expression in Nkx2.2 cDNA transfected cells was independent of the growth factor treatment used for differentiating AR42J cells. In conclusion, the AR42J-B13 line possesses some features of a pancreatic neuroendocrine cell. However, we were unable to confirm the capacity of these cells to differentiate into insulin-producing cells. Our results indicate that Nkx2.2 plays a role in the transcriptional regulation of PP expression. PMID: 10940482 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Mol Cell Biol Res Commun. 2000 Apr;3(4):249-54. Pax4 represses pancreatic glucagon gene expression. Petersen HV, Jorgensen MC, Andersen FG, Jensen J, F-Nielsen T, Jorgensen R, Madsen OD, Serup P. Department of Developmental Biology, Hagedorn Research Institute, Niels Steensensvej 6, Gentofte, DK-2820, Denmark. The paired box and homeodomain containing transcription factors Pax4 and Pax6 are known to be essential for development of the pancreatic endocrine cells. In this report we demonstrate that stable expression of Pax4 in a rat glucagon-producing cell line inhibits the endogenously expressed glucagon gene completely. Furthermore, Pax4 represses Pax6 independent transcription of the insulin promoter, suggesting that Pax4 can actively repress transcription in addition to acting by competition with the transcriptional activator Pax6. Copyright 2000 Academic Press. PMID: 10891400 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Mech Dev. 2000 Mar 15;92(1):47-54. Pax genes and the differentiation of hormone-producing endocrine cells in the pancreas. Dohrmann C, Gruss P, Lemaire L. DeveloGen AG, Rudolf-Wissell-Strasse 28, D-37079, Gottingen, Germany. Despite the pivotal role of the pancreas in hormonally-regulated pathways in the body, e.g. glucose homeostasis, the genetic mechanisms defining it have for many years remained largely enigmatic. After years out of the spotlight, pancreas development has once again come to centre stage. To a large extent, this is due to recent advances made through the detailed analysis of transgenic mice which have been engineered to carry mutations in specific developmental control genes. This review specifically focuses on the specification of the endocrine pancreas lineage and in particular on the role of the developmental control genes Pax4 and Pax6 in the generation of specific endocrine cell types. The comparison of various phenotypes of different mouse mutants affecting endocrine development supports a model in which Pax4 and Pax6 are required for the differentiation of certain endocrine cell lineages and implies a potential for acting at different levels of endocrine development. Publication Types: Review PMID: 10704887 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1607-11. neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Gradwohl G, Dierich A, LeMeur M, Guillemot F. Institut de Genetique et de Biologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Sante et de la Recherche Medicale/Universite Louis Pasteur, F-67404 Illkirch, France. In the mammalian pancreas, the endocrine cell types of the islets of Langerhans, including the alpha-, beta-, delta-, and pancreatic polypeptide cells as well as the exocrine cells, derive from foregut endodermal progenitors. Recent genetic studies have identified a network of transcription factors, including Pdx1, Isl1, Pax4, Pax6, NeuroD, Nkx2.2, and Hlxb9, regulating the development of islet cells at different stages, but the molecular mechanisms controlling the specification of pancreatic endocrine precursors remain unknown. neurogenin3 (ngn3) is a member of a family of basic helix-loop-helix transcription factors that is involved in the determination of neural precursor cells in the neuroectoderm. ngn3 is expressed in discrete regions of the nervous system and in scattered cells in the embryonic pancreas. We show herein that ngn3-positive cells coexpress neither insulin nor glucagon, suggesting that ngn3 marks early precursors of pancreatic endocrine cells. Mice lacking ngn3 function fail to generate any pancreatic endocrine cells and die postnatally from diabetes. Expression of Isl1, Pax4, Pax6, and NeuroD is lost, and endocrine precursors are lacking in the mutant pancreatic epithelium. Thus, ngn3 is required for the specification of a common precursor for the four pancreatic endocrine cell types. PMID: 10677506 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: FEBS Lett. 1999 Nov 19;461(3):287-94. Cloning and DNA-binding properties of the rat pancreatic beta-cell-specific factor Nkx6.1. Jorgensen MC, Vestergard Petersen H, Ericson J, Madsen OD, Serup P. Department of Developmental Biology, Hagedorn Research Institute, Niels Steensensvej 6, DK-2820, Gentofte, Denmark. The homeodomain (HD) protein Nkx6.1 is the most beta-cell-specific transcription factor known in the pancreas and its function is critical for the formation of the insulin-producing beta-cells. However, the target genes, DNA-binding site, and transcriptional properties of Nkx6.1 are unknown. Using in vitro binding site selection we have identified the DNA sequence of the Nkx6.1 binding site to be TTAATTG/A. A reporter plasmid containing four copies of this sequence is activated by an Nkx6.1HD/VP16 fusion construct. Full-length Nkx6.1 fails to activate this reporter plasmid in spite of robust interaction with the binding site in vitro. Stable expression of Nkx6.1 in the glucagon-producing alpha-cell-like MSL-G-AN cells induces expression of the endogenous insulin gene in a subset of the cell population. The expression of other known beta-cell-specific factors such as Pax4, Pax6, Pdx1, GLUT2 and GLP1-R is unchanged by the introduction of Nkx6.1. PMID: 10567713 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Mol Cell Biol. 1999 Dec;19(12):8281-91. Identification of a portable repression domain and an E1A-responsive activation domain in Pax4: a possible role of Pax4 as a transcriptional repressor in the pancreas. Fujitani Y, Kajimoto Y, Yasuda T, Matsuoka TA, Kaneto H, Umayahara Y, Fujita N, Watada H, Miyazaki JI, Yamasaki Y, Hori M. Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Suita 565-0871, Japan. Pax4 is a paired-domain (PD)-containing transcription factor which plays a crucial role in pancreatic beta/delta-cell development. In this study, we characterized the DNA-binding and transactivation properties of mouse Pax4. Repetitive rounds of PCR-based selection led to identification of the optimal DNA-binding sequences for the PD of Pax4. In agreement with the conservation of the optimal binding sequences among the Pax family transcription factors, Pax4 could bind to the potential binding sites for Pax6, another member of the Pax family also involved in endocrine pancreas development. The overexpression of Pax4 in HIT-T15 cells dose dependently inhibited the basal transcriptional activity as well as Pax6-induced activity. Detailed domain mapping analyses using GAL4-Pax4 chimeras revealed that the C-terminal region of Pax4 contains both activation and repression domains. The activation domain was active in the embryonic kidney-derived 293/293T cells and embryonal carcinoma-derived F9 cells, containing adenoviral E1A protein or E1A-like activity, respectively but was inactive or very weakly active in other cells including those of pancreatic beta- and alpha-cell origin. Indeed, the exogenous overexpression of type 13S E1A in heterologous cell types could convert the activation domain to an active one. On the other hand, the repression domain was active regardless of the cell type. When the repression domain was linked to the transactivation domain of a heterologous transcription factor, PDX-1, it could completely abolish the transactivation potential of PDX-1. These observations suggest a primary role of Pax4 as a transcriptional repressor whose function may involve the competitive inhibition of Pax6 function. The identification of the E1A-responsive transactivation domain, however, indicates that the function of Pax4 is subject to posttranslational regulation, providing further support for the complexity of mechanisms that regulate pancreas development. PMID: 10567553 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Mol Cell Biol. 1999 Dec;19(12):8272-80. Paired-homeodomain transcription factor PAX4 acts as a transcriptional repressor in early pancreatic development. Smith SB, Ee HC, Conners JR, German MS. Hormone Research Institute, University of California, San Francisco, San Francisco, California 94143-0534, USA. The paired-homeodomain transcription factor PAX4 is expressed in the developing pancreas and along with PAX6 is required for normal development of the endocrine cells. In the absence of PAX4, the numbers of insulin-producing beta cells and somatostatin-producing delta cells are drastically reduced, while the numbers of glucagon-producing alpha cells are increased. To gain insight into PAX4 function, we cloned a full-length Pax4 cDNA from a beta-cell cDNA library and identified a bipartite consensus DNA binding sequence consisting of a homeodomain binding site separated from a paired domain binding site by 15 nucleotides. The paired half of this consensus sequence has similarities to the PAX6 paired domain consensus binding site, and the two proteins bind to common sequences in several islet genes, although with different relative affinities. When expressed in an alpha-cell line, PAX4 represses transcription through the glucagon or insulin promoters or through an isolated PAX4 binding site. This repression is not simply due to competition with the PAX6 transcriptional activator for the same binding site, since PAX4 fused to the unrelated yeast GAL4 DNA binding domain also represses transcription through the GAL4 binding site in the alpha-cell line and to a lesser degree in beta-cell lines and NIH 3T3 cells. Repressor activity maps to more than one domain within the molecule, although the homeodomain and carboxyl terminus give the strongest repression. PAX4 transcriptional regulation apparently plays a role only early in islet development, since Pax4 mRNA as determined by reverse transcriptase PCR peaks at embryonic day 13.5 in the fetal mouse pancreas and is undetectable in adult islets. In summary, PAX4 can function as a transcriptional repressor and is expressed early in pancreatic development, which may allow it to suppress alpha-cell differentiation and permit beta-cell differentiation. PMID: 10567552 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Nat Genet. 1999 Sep;23(1):71-5. Pancreas dorsal lobe agenesis and abnormal islets of Langerhans in Hlxb9-deficient mice. Harrison KA, Thaler J, Pfaff SL, Gu H, Kehrl JH. Laboratory of Immunoregulation National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. In most mammals the pancreas develops from the foregut endoderm as ventral and dorsal buds. These buds fuse and develop into a complex organ composed of endocrine, exocrine and ductal components. This developmental process depends upon an integrated network of transcription factors. Gene targeting experiments have revealed critical roles for Pdx1, Isl1, Pax4, Pax6 and Nkx2-2 (refs 3,4,5,6,7, 8,9,10). The homeobox gene HLXB9 (encoding HB9) is prominently expressed in adult human pancreas, although its role in pancreas development and function is unknown. To facilitate its study, we isolated the mouse HLXB9 orthologue, Hlxb9. During mouse development, the dorsal and ventral pancreatic buds and mature beta-cells in the islets of Langerhans express Hlxb9. In mice homologous for a null mutation of Hlxb9, the dorsal lobe of the pancreas fails to develop. The remnant Hlxb9-/- pancreas has small islets of Langerhans with reduced numbers of insulin-producing beta-cells. Hlxb9-/- beta-cells express low levels of the glucose transporter Glut2 and homeodomain factor Nkx 6-1. Thus, Hlxb9 is key to normal pancreas development and function. PMID: 10471502 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Biochem Biophys Res Commun. 1999 Jun 16;259(3):510-8. DNA binding and transactivating properties of the paired and homeobox protein Pax4. Kalousova A, Benes V, Paces J, Paces V, Kozmik Z. Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic. Anna@biomed.cas.cz Transcription factors Pax-4 and Pax-6 are known to be key regulators of pancreatic cell differentiation and development. We report on the cloning of a mouse Pax-4 gene, which contains 10 exons, spanning a 4. 7-kbp region. The gene-targeting experiments revealed that Pax-4 and Pax-6 cannot substitute for each other in tissue with overlapping expression of both genes. We identified DNA-binding specificities of Pax-4 paired domain and paired-type homeodomain. Despite the different Pax-4 amino acid residues in positions responsible for Pax-6 paired-domain specificity, the DNA-binding specificities of Pax-4 and Pax-6 are similar. The Pax-4 homeodomain was shown to preferentially dimerize on DNA sequences consisting of an inverted TAAT motif, separated by 4-nucleotide spacing. The Pax-4 transactivation domain was localized within its C-terminal region, which transactivated GAL-based reporter 2.5-fold less than the C-terminal region of Pax-6. We believe that Pax-4 can act as a Pax-6 "repressor," due to the competition for binding sites and lower transactivation potential of Pax-4. Copyright 1999 Academic Press. PMID: 10364449 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Mech Dev. 1998 Dec;79(1-2):153-9. Pax 4 and 6 regulate gastrointestinal endocrine cell development. Larsson LI, St-Onge L, Hougaard DM, Sosa-Pineda B, Gruss P. Department of Molecular Cell Biology, Statens Serum Institut, Copenhagen, Denmark. Larsson@biobase.dk The mechanisms behind the cell-specific and compartmentalized expression of gut and pancreatic hormones is largely unknown. We hereby report that deletion of the Pax 4 gene virtually eliminates duodenal and jejunal hormone-secreting cells, as well as serotonin and somatostatin cells of the distal stomach, while deletion of the Pax 6 gene eliminates duodenal GIP cells as well as gastrin and somatostatin cells of the distal stomach. Thus, together, these two genes regulate the differentiation of all proximal gastrointestinal endocrine cells and reflect common pathways for pancreatic and gastrointestinal endocrine cell differentiation. PMID: 10349628 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Trends Endocrinol Metab. 1999 May;10(4):164-167. Role of Genes in Endoderm-derived Organs. Mansouri A, St-Onge L, Gruss P. Max-Planck-Institute for Biophysical Chemistry, Department of Molecular Cell Biology, Am Fassberg, D-37077 Gottingen, Germany. Pax genes, which encode a family of transcription factors, are essentially required for the formation of several tissues from all germ layers in the mammalian embryo. Specifically, in organogenesis, they are involved in triggering early events of cell differentiation. The differentiation of endoderm-derived endocrine pancreas is mediated through Pax4 and Pax6. In the thyroid gland, Pax8 is essential for the formation of thyroxine-producing follicular cells, also of endodermal origin. The analysis of loss-of-function mutants revealed a common function of Pax genes in organogenesis. PMID: 10322412 [PubMed - as supplied by publisher] --------------------------------------------------------------- 24: Int J Mol Med. 1999 Mar;3(3):247-61. Development of pancreatic islets (review). Yamaoka T, Itakura M. Otsuka Department of Clinical and Molecular Nutrition, School of Medicine, The University of Tokushima, Tokushima 770-8503, Japan. Recent studies have revealed that islet cells differentiate from the epithelial cells of primitive pancreatic ducts during embryogenesis, and can regenerate in response to the loss of islet cells even in adult pancreas. The ability of islet cells to regenerate raises the possibility that impaired and decreased islets of diabetic patients can be restored. In this review, factors regulating islet development including differentiation factors (Shh, activin, follistatin, and TGF alpha), transcriptional factors (PDX1, Isl1, Pax4, Pax6, Nkx2.2, Nkx6.1, BETA2, and HNF), growth factors (the EGF family, HGF, IGF-I, IGF-II, Reg, INGAP, PDGF, FGF, VEGF, and NGF), hormones (insulin, the GH family, PTHrP, TRH, and gastrin), and cell adhesion molecules (N-CAM and cadherins) are described after a short introduction and an outline of pancreatic development. Publication Types: Review Review, Tutorial PMID: 10028048 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8654-9. Targeted oncogenesis of hormone-negative pancreatic islet progenitor cells. Jetton TL, Moates JM, Lindner J, Wright CV, Magnuson MA. Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615, USA. Transgenic mice containing an upstream glucokinase (betaGK) promoter- simian virus 40 T antigen (Tag) fusion gene develop neuroendocrine tumors primarily in the pancreas, gut, and pituitary. Pancreatic tumors from a line with delayed tumorigenesis were of two different types: insulinomas and noninsulinomas. The noninsulinomas are often periductal in location, express none of the four major islet peptide hormones, Glut-2, Pdx1, tyrosine hydroxylase, Pax4, Pax6, or Nkx6.1, but do express glucokinase, Sur1, Isl1, Hnf3beta, Hnf6, Beta2/NeuroD, and Nkx2.2. Cells from two different noninsulinoma tumors, when adapted to culture, began to express either insulin, glucagon, or somatostatin. Given the partial gene expression repertoire of the noninsulinoma tumors, their apparent periductal origin, and the ability of these cells to partially cytodifferentiate in culture, we suggest that these tumors are derived from islet progenitor cells. Thus, betaGK-Tag transgenic mice provide a new model system for studying the events that occur during both islet cell neogenesis and normal embryonic development. PMID: 9671733 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: Biochem Biophys Res Commun. 1998 Feb 13;243(2):628-33. Isolation of full-length cDNA of mouse PAX4 gene and identification of its human homologue. Inoue H, Nomiyama J, Nakai K, Matsutani A, Tanizawa Y, Oka Y. Third Department Internal Medicine, Yamaguchi University School of Medicine, Japan. Recent genetic studies have suggested that PAX4, a member of the paired box (PAX) gene family, is involved in the mechanism regulating the fate of pancreatic islet endocrine progenitor cells. Murine PAX4 was originally identified by genomic screening and, to date, only a partial sequence of PAX4 has been reported. In this study, we cloned the full-length cDNA of mouse PAX4 by RACE (rapid amplification of cDNA ends) using RNA from MIN6 cells, a mouse insulinoma cell line. The full length of cDNA was 1.38 kb, consistent with the estimated size of the transcript by Northern blot. The deduced mouse PAX4 protein was 349 amino acids and had the predicted molecular weight of 38 kDa. Two DNA binding motifs, a 128-amino acid paired domain and a 61-amino acid paired-type homeodomain exhibit the highest amino acid homology with PAX6 (71.2%, 65.0%, respectively), another member of the PAX gene family. However, the sequence of the C-terminal segment of PAX4 diverged and showed no significant homology with any other known PAX genes. As to the genomic DNA, the coding region of the mouse PAX4 gene spanned approximately 5.5 kb and was composed of 10 exons. In the public DNA database, a human cosmid (g1572c264), which was localized on human chromosome 7q31.3, was found to contain a gene homologous to PAX4. The nucleotide and protein sequence homologies between mouse PAX4 and its human homologue were 83.1% and 80.0%, respectively. Interestingly, the ARP5 (ADP-ribosylation factor 5) gene was also found in the same cosmid g1572c264, suggesting the ARP5 gene to be adjacent to the human PAX4 homologue. The human cosmid g1572c264 contains at least four SSRPs (simple sequence repeat polymorphism), which could be used for genetic linkage studies of the locus. The results of this study, i.e. isolation of the full-length cDNA sequence of PAX4 and identification of the homologous human gene, will facilitate further functional and genetic studies of the PAX4 gene. PMID: 9480859 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: Proc Assoc Am Physicians. 1998 Jan-Feb;110(1):12-21. A newly discovered role of transcription factors involved in pancreas development and the pathogenesis of diabetes mellitus. Habener JF, Stoffers DA. Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Boston 02114-2696, USA. The prevalence of diabetes mellitus is increasing worldwide, averaging 5% to 15% in various population groups. Diabetes predisposes to premature morbidity and death. The underlying metabolic cause of diabetes is a failure of the beta-cells of the pancreas to provide insulin in amounts sufficient to meet the body's needs, leading to hyperglycemia. Juvenile (type 1) diabetes results from immune destruction of the beta-cells. Adult onset (type 2) diabetes, which accounts for 90% of all forms of diabetes, is a complex polygenic disease manifested in a dysregulation of insulin secretion. Environmental influences and complex genetic traits contribute to the pathogenesis of both types of diabetes. However, a subpopulation of type 2 diabetes is monogenic and due to inactivating mutations in genes that are critical for normal beta-cell function. Heterozygous carriers of the mutant genes develop early-onset diabetes known as MODY (mature onset diabetes of the young). Notably, three MODY genes encode transcription factors implicated in the regulation of insulin gene transcription: hepatocyte nuclear factors 1 alpha and 4 alpha, and islet duodenum homeobox-1 (IDX-1, also known as IPF-1). The fourth gene encodes glucokinase, the rate-limiting enzyme required for glucose metabolism in beta-cells. Further, an individual born without a pancreas (agenesis) is homozygous for an inactivating mutation of the IDX-1 gene, recapitulating the phenotype of the IDX-1 knockout mouse and demonstrating that expression of IDX-1 is critical for pancreas development. Recently, mouse knockouts of the transcription factors Pax4, Pax6, beta 2/neuroD, and Isl-1 result in severe anomalies in the development of the endocrine pancreas. Gene mutations for these factors are possible candidates for additional MODY genes. Publication Types: Review Review, Tutorial PMID: 9460079 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Nature. 1997 May 22;387(6631):406-9. Pax6 is required for differentiation of glucagon-producing alpha-cells in mouse pancreas. St-Onge L, Sosa-Pineda B, Chowdhury K, Mansouri A, Gruss P. Max-Planck-Institut fur Biophysikalische Chemie, Gottingen, Germany. The functional unit of the endocrine pancreas is the islet of Langerhans. Islets are nested within the exocrine tissue of the pancreas and are composed of alpha-, beta-, delta- and gamma-cells. beta-Cells produce insulin and form the core of the islet, whereas alpha-, delta- and gamma-cells are arranged at the periphery of the islet and secrete glucagon, somatostatin and a pancreatic polypeptide, respectively. Little is known about the molecular and genetic factors regulating the lineage of the different endocrine cells. Pancreas development is known to be abolished in Pdx1-mutant mice and Pax4 mutants lack insulin-producing beta-cells. Here we show that the paired-box gene Pax6 is expressed during the early stages of pancreatic development and in mature endocrine cells. The pancreas of Pax6 homozygous mutant mice lack glucagon-producing cells, suggesting that Pax6 is essential for the differentiation of alpha-cells. As mice lacking Pax4 and Pax6 fail to develop any mature endocrine cells, we conclude that both Pax genes are required for endocrine fate in the pancreas. PMID: 9163426 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Gene. 1995 Sep 11;162(2):267-70. Identification of DNA recognition sequences for the Pax3 paired domain. Chalepakis G, Gruss P. Max-Planck Institute for Biophysical Chemistry, Gottingen, Germany. The Pax gene family, encoding transcription factors, has been classified into four subfamilies according to their genomic organization, the sequences of the paired domains (PD) and the expression pattern. Pax1 and Pax9 constitute one subfamily, Pax2, Pax5 and Pax8 another, Pax3 and Pax7 another one and Pax4 and Pax6 the fourth subfamily. The PD exhibits DNA-binding activity, and is the most conserved functional motif in all Pax proteins. A high-resolution analysis of a PD structure has been performed [Xu et al., Cell 80 (1995) 639-650] and the DNA-binding characteristics of members of two Pax subfamilies (Pax2, Pax5 and Pax6) have been determined. Here, we have utilized a PCR-based selection approach to identify the DNA-binding sequences of the Pax3/PD, a member of a subfamily which has not yet been characterized. Comparison of the Pax3/PD-binding sequences with those of other PD proteins revealed both similarities and differences in the DNA-recognition sequence. This suggests that different Pax proteins can regulate the expression of the same target gene, but they can also regulate the expression of completely unrelated genes by binding to their DNA regulatory regions. PMID: 7557441 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------