Gata6 is an important regulator of mouse pancreas development.
ABSTRACT: Gata4, Gata5, and Gata6 represent a subfamily of zinc-finger transcriptional regulators that are important in the development and differentiation of numerous tissues, including many endodermally-derived organs. We demonstrate that Gata4 and Gata6 have overlapping expression patterns in the early pancreatic epithelium. Later, Gata4 becomes restricted to exocrine tissue and Gata6 becomes restricted to a subset of endocrine cells. In addition, we show Gata6, but not Gata4, physically interacts with Nkx2.2, an essential islet transcription factor. To begin determining the roles that Gata4 and Gata6 play during pancreatic development, we expressed Gata4-Engrailed and Gata6-Engrailed dominant repressor fusion proteins in the pancreatic epithelium and in the islet. At e17.5, transgenic Gata6-Engrailed embryos exhibit two distinct phenotypes: a complete absence of pancreas or a reduction in pancreatic tissue. In the embryos that do form pancreas, there is a significant reduction of all pancreatic cell types, with the few differentiated endocrine cells clustered within, or in close proximity to, enlarged ductal structures. Conversely, the majority of transgenic Gata4-Engrailed embryos do not have a pancreatic phenotype. This study suggests that Gata6 is an important regulator of pancreas specification.
Project description:Pancreatic agenesis is a human disorder caused by defects in pancreas development. To date, only a few genes have been linked to pancreatic agenesis in humans, with mutations in pancreatic and duodenal homeobox 1 (PDX1) and pancreas-specific transcription factor 1a (PTF1A) reported in only 5 families with described cases. Recently, mutations in GATA6 have been identified in a large percentage of human cases, and a GATA4 mutant allele has been implicated in a single case. In the mouse, Gata4 and Gata6 are expressed in several endoderm-derived tissues, including the pancreas. To analyze the functions of GATA4 and/or GATA6 during mouse pancreatic development, we generated pancreas-specific deletions of Gata4 and Gata6. Surprisingly, loss of either Gata4 or Gata6 in the pancreas resulted in only mild pancreatic defects, which resolved postnatally. However, simultaneous deletion of both Gata4 and Gata6 in the pancreas caused severe pancreatic agenesis due to disruption of pancreatic progenitor cell proliferation, defects in branching morphogenesis, and a subsequent failure to induce the differentiation of progenitor cells expressing carboxypeptidase A1 (CPA1) and neurogenin 3 (NEUROG3). These studies address the conserved and nonconserved mechanisms underlying GATA4 and GATA6 function during pancreas development and provide a new mouse model to characterize the underlying developmental defects associated with pancreatic agenesis.
Project description:Recently, heterozygous mutations in GATA6 have been found in neonatal diabetic patients with failed pancreatic organogenesis. To investigate the roles of GATA4 and GATA6 in mouse pancreas organogenesis, we conditionally inactivated these genes within the pancreas. Single inactivation of either gene did not have a major impact on pancreas formation, indicating functional redundancy. However, double Gata4/Gata6 mutant mice failed to develop pancreata, died shortly after birth, and displayed hyperglycemia. Morphological defects in Gata4/Gata6 mutant pancreata were apparent during embryonic development, and the epithelium failed to expand as a result of defects in cell proliferation and differentiation. The number of multipotent pancreatic progenitors, including PDX1+ cells, was reduced in the Gata4/Gata6 mutant pancreatic epithelium. Remarkably, deletion of only 1 Gata6 allele on a Gata4 conditional knockout background severely reduced pancreatic mass. In contrast, a single WT allele of Gata4 in Gata6 conditional knockout mice was sufficient for normal pancreatic development, indicating differential contributions of GATA factors to pancreas formation. Our results place GATA factors at the top of the transcriptional network hierarchy controlling pancreas organogenesis.
Project description:The GATA family zinc finger transcription factors GATA4 and GATA6 are known to play important roles in the development of the pancreas. In mice, both Gata4 and Gata6 are required for pancreatic development. In humans, GATA6 haploinsufficiency can cause pancreatic agenesis and heart defects. Congenital heart defects also are common in patients with GATA4 mutations and deletions, but the role of GATA4 in the developing human pancreas is unproven. We report five patients with deletions (n = 4) or mutations of the GATA4 gene who have diabetes and a variable exocrine phenotype. In four cases, diabetes presented in the neonatal period (age at diagnosis 1-7 days). A de novo GATA4 missense mutation (p.N273K) was identified in a patient with complete absence of the pancreas confirmed at postmortem. This mutation affects a highly conserved residue located in the second zinc finger domain of the GATA4 protein. In vitro studies showed reduced DNA binding and transactivational activity of the mutant protein. We show that GATA4 mutations/deletions are a cause of neonatal or childhood-onset diabetes with or without exocrine insufficiency. These results confirm a role for GATA4 in normal development of the human pancreas.
Project description:The factors necessary for normal pancreatic islet morphogenesis have not been well characterized. Here we report that connective tissue growth factor (CTGF) is involved in the establishment of normal islet endocrine cell ratio and architecture. CTGF is a secreted protein known to modulate several growth factor-signaling pathways including TGF-beta, BMP, and Wnt. Although its role in pancreatic diseases such as pancreatitis and pancreatic cancer are well documented, a role for CTGF in normal pancreas development and function has heretofore not been examined. Using a lacZ-tagged CTGF allele, we describe for the first time the expression pattern of CTGF in the developing pancreas and the requirement of CTGF for normal islet morphogenesis and embryonic beta-cell proliferation. CTGF is highly expressed in pancreatic ductal epithelium and vascular endothelium, as well as at lower levels in developing insulin(+) cells, but becomes down-regulated in beta-cells soon after birth. Pancreata from CTGF null embryos have an increase in glucagon(+) cells with a concomitant decrease in insulin(+) cells, and show defects in islet morphogenesis. Loss of CTGF also results in a dramatic decrease in beta-cell proliferation at late gestation. Unlike CTGF null embryos, CTGF heterozygotes survive past birth and exhibit a range of islet phenotypes, including an intermingling of islet cell types, increased number of glucagon(+) cells, and beta-cell hypertrophy.
Project description:Gata4 and Gata6 are closely related transcription factors that are essential for the development of a number of embryonic tissues. While they have nearly identical DNA-binding domains and similar patterns of expression, Gata4 and Gata6 null embryos have strikingly different embryonic lethal phenotypes. To determine whether the lack of redundancy is due to differences in protein function or Gata4 and Gata6 expression domains, we generated mice that contained the Gata6 cDNA in place of the Gata4 genomic locus. Gata4(Gata6/Gata6) embryos survived through embryonic day (E)12.5 and successfully underwent ventral folding morphogenesis, demonstrating that Gata6 is able to replace Gata4 function in extraembryonic tissues. Surprisingly, Gata6 is unable to replace Gata4 function in the septum transversum mesenchyme or the epicardium, leading to liver agenesis and lethal heart defects in Gata4(Gata6/Gata6) embryos. These studies suggest that Gata4 has evolved distinct functions in the development of these tissues that cannot be performed by Gata6, even when it is provided in the identical expression domain. Our work has important implications for the respective mechanisms of Gata function during development, as well as the functional evolution of these essential transcription factors.
Project description:The islet of Langerhans is a unique micro-organ within the exocrine pancreas, which is composed of insulin-secreting beta-cells, glucagon-secreting alpha-cells, somatostatin-secreting delta-cells, pancreatic polypeptide-secreting PP cells and ghrelin-secreting epsilon-cells. Islets also contain non-endocrine cell types such as endothelial cells. However, the mechanism(s) of islet formation is poorly understood due to technical difficulties in capturing this dynamic event in situ. We have developed a method to monitor beta-cell proliferation and islet formation in the intact pancreas using transgenic mice in which the beta-cells are specifically tagged with a fluorescent protein. Endocrine cells proliferate contiguously, forming branched cord-like structures in both embryos and neonates. Our study has revealed long stretches of interconnected islets located along large blood vessels in the neonatal pancreas. Alpha-cells span the elongated islet-like structures, which we hypothesize represent sites of fission and facilitate the eventual formation of discrete islets. We propose that islet formation occurs by a process of fission following contiguous endocrine cell proliferation, rather than by local aggregation or fusion of isolated beta-cells and islets. Mathematical modeling of the fission process in the neonatal islet formation is also presented.
Project description:Damaging GATA6 variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to GATA6 loss of function (LoF) and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating SMYD1 that activates HAND2, and KDR that with HAND2 orchestrates outflow tract formation. LoF variants perturbed cardiac genes and also endoderm lineage genes that direct PDX1 expression and pancreatic development. Remarkably, an exon 4 GATA6 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing GATA4, FOXA1/2, and PDX1 expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct GATA6 variants.
Project description:BACKGROUND:The homeodomain containing transcription factor Nkx2.2 is essential for the differentiation of pancreatic endocrine cells. Deletion of Nkx2.2 in mice leads to misspecification of islet cell types; insulin-expressing beta cells and glucagon-expressing alpha cells are replaced by ghrelin-expressing cells. Additional studies have suggested that Nkx2.2 functions both as a transcriptional repressor and activator to regulate islet cell formation and function. To identify genes that are potentially regulated by Nkx2.2 during the major wave of endocrine and exocrine cell differentiation, we assessed gene expression changes that occur in the absence of Nkx2.2 at the onset of the secondary transition in the developing pancreas. RESULTS:Microarray analysis identified 80 genes that were differentially expressed in e12.5 and/or e13.5 Nkx2.2-/- embryos. Some of these genes encode transcription factors that have been previously identified in the pancreas, clarifying the position of Nkx2.2 within the islet transcriptional regulatory pathway. We also identified signaling factors and transmembrane proteins that function downstream of Nkx2.2, including several that have not previously been described in the pancreas. Interestingly, a number of known exocrine genes are also misexpressed in the Nkx2.2-/- pancreas. CONCLUSIONS:Expression profiling of Nkx2.2-/- mice during embryogenesis has allowed us to identify known and novel pancreatic genes that function downstream of Nkx2.2 to regulate pancreas development. Several of the newly identified signaling factors and transmembrane proteins may function to influence islet cell fate decisions. These studies have also revealed a novel function for Nkx2.2 in maintaining appropriate exocrine gene expression. Most importantly, Nkx2.2 appears to function within a complex regulatory loop with Ngn3 at a key endocrine differentiation step.
Project description:The zinc-finger transcription factors GATA4 and GATA6 play critical roles in embryonic development. Mouse embryos lacking GATA4 die at embryonic day (E) 8.5 because of failure of ventral foregut closure and cardiac bifida, whereas GATA6 is essential for development of the visceral endoderm. Although mice that are heterozygous for either a GATA4 or GATA6 null allele are normal, we show that compound heterozygosity of GATA4 and GATA6 results in embryonic lethality by E13.5 accompanied by a spectrum of cardiovascular defects, including thin-walled myocardium, ventricular and aortopulmonary septal defects, and abnormal smooth muscle development. Myocardial hypoplasia in GATA4/GATA6 double heterozygous mutant embryos is associated with reduced proliferation of cardiomyocytes, diminished expression of the myogenic transcription factor MEF2C (myocyte enhancer factor 2C), and down-regulation of beta-myosin heavy chain expression, a key determinant of cardiac contractility. These findings reveal a threshold of GATA4 and GATA6 activity that is required for gene expression in the developing cardiovascular system and underscore the potential of recessive mutations to perturb the delicate regulation of cardiovascular development.
Project description:The derivation of the primitive endoderm layer from the pluripotent cells of the inner cell mass is one of the earliest differentiation and morphogenic events in embryonic development. GATA4 and GATA6 are the key transcription factors in the formation of extraembryonic endoderms, but their specific contribution to the derivation of each endoderm lineage needs clarification. We further analyzed the dynamic expression and mutant phenotypes of GATA6 in early mouse embryos. GATA6 and GATA4 are both expressed in primitive endoderm cells initially. At embryonic day (E) 5.0, parietal endoderm cells continue to express both GATA4 and GATA6; however, visceral endoderm cells express GATA4 but exhibit a reduced expression of GATA6. By and after E5.5, visceral endoderm cells no longer express GATA6. We also found that GATA6 null embryos did not form a morphologically recognizable primitive endoderm layer, and subsequently failed to form visceral and parietal endoderms. Thus, the current study establishes that GATA6 is essential for the formation of primitive endoderm, at a much earlier stage then previously recognized, and expression of GATA6 discriminates parietal endoderm from visceral endoderm lineages.