GATA4 is essential for formation of the proepicardium and regulates cardiogenesis.
ABSTRACT: The role of GATA4 during the earliest stages of cardiogenesis has not been defined because Gata4 knockout embryos suffer an early developmental arrest caused by deficiencies in extraembryonic visceral endoderm function. We have used tetraploid embryo complementation to rescue these defects and generated clonal embryonic day 9.5 Gata4(-/-) embryos directly from embryonic stem cells. GATA4-null embryos display heart defects characterized by disrupted looping morphogenesis, septation, and a hypoplastic ventricular myocardium. We find that myocardial gene expression is relatively normal in GATA4-null hearts including expression of GATA6. Moreover, GATA4 expression in the endocardium is dispensable for trabeculae formation. Remarkably, the proepicardium is absent in GATA4-null embryos, blocking formation of the epicardium. Therefore, we propose that the observed myocardial defects may be a secondary consequence of loss of the proepicardium. These findings definitively demonstrate a requirement for GATA4 during early cardiac development and identify an essential factor for generation of the proepicardium.
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:Mutations in developmental regulatory genes have been found to be responsible for some cases of congenital heart defects. One such regulatory gene is Gata4, a zinc finger transcription factor. In order to circumvent the early embryonic lethality of Gata4-null embryos and to investigate the role of myocardial Gata4 expression in cardiac development, we used Cre/loxP technology to conditionally delete Gata4 in the myocardium of mice at an early and a late time point in cardiac morphogenesis. Early deletion of Gata4 by Nkx2-5Cre resulted in hearts with striking myocardial thinning, absence of mesenchymal cells within the endocardial cushions, and selective hypoplasia of the RV. RV hypoplasia was associated with downregulation of Hand2, a transcription factor previously shown to regulate formation of the RV. Cardiomyocyte proliferation was reduced, with a greater degree of reduction in the RV than in the LV. Late deletion of Gata4 by Cre recombinase driven by the alpha myosin heavy chain promoter did not selectively affect RV development or generation of endocardial cushion mesenchyme but did result in marked myocardial thinning with decreased cardiomyocyte proliferation, as well as double-outlet RV. Our results demonstrate a general role of myocardial Gata4 in regulating cardiomyocyte proliferation and a specific, stage-dependent role in regulating the morphogenesis of the RV and the atrioventricular canal.
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:Mutations in GATA4 and TBX5 are associated with congenital heart defects in humans. Interaction between GATA4 and TBX5 is important for normal cardiac septation, but the underlying molecular mechanisms are not well understood. Here, we show that Gata4 and Tbx5 are co-expressed in the embryonic atria and ventricle, but after E15.5, ventricular expression of Tbx5 decreases. Co-localization and co-immunoprecipitation studies demonstrate an interaction of Gata4 and Tbx5 in the developing atria and ventricles, but the ventricular interaction declines after E14.5. Gata4(+/-);Tbx5(+/-) mouse embryos display decreased atrial and ventricular myocardial thickness at E11.5, prior to cardiac septation. To determine the cell lineage in which the interaction was functionally significant in vivo, mice heterozygous for Gata4 in the myocardium or endocardium and heterozygous for Tbx5 (Gata4(MyoDel/wt);Tbx5(+/-) and Gata4(EndoDel/wt);Tbx5(+/-), respectively) were generated. Gata4(MyoDel/wt);Tbx5(+/-) mice displayed embryonic lethality, thin myocardium with reduced cell proliferation, and atrioventricular septation defects similar to Gata4;Tbx5 compound heterozygotes while Gata4(EndoDel/wt);Tbx5(+/-) embryos were normal. Cdk4 and Cdk2, cyclin-dependent kinases required for myocardial development and septation were reduced in Gata4(+/-);Tbx5(+/-) hearts. Cdk4 is a known direct target of Gata4 and the regulation of Cdk2 in the developing heart has not been studied. Chromatin immunoprecipitation and transactivation studies demonstrate that Gata4 and Tbx5 directly regulate Cdk4 while only Tbx5 activates Cdk2 expression. These findings highlight the mechanisms by which disruption of the Gata4 and Tbx5 interaction in the myocardium contributes to cardiac septation defects in humans.
Project description:Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans.
Project description:Congenital heart disease is the most common type of birth defect with an incidence of 1%. Previously, we described a point mutation in GATA4 that segregated with cardiac defects in a family with autosomal dominant disease. The mutation (G296S) exhibited biochemical deficits and disrupted a novel interaction between Gata4 and Tbx5. To determine if Gata4 and Tbx5 genetically interact in vivo, we generated mice heterozygous for both alleles. We found that nearly 100% of mice heterozygous for Gata4 and Tbx5 were embryonic or neonatal lethal and had complete atrioventricular (AV) septal defects with a single AV valve and myocardial thinning. Consistent with this phenotype, Gata4 and Tbx5 are co-expressed in the developing endocardial cushions and myocardium. In mutant embryos, cardiomyocyte proliferation deficits were identified compatible with the myocardial hypoplasia. Similar to Gata4, Gata6 and Tbx5 are co-expressed in the embryonic heart, and the transcription factors synergistically activate the atrial natiuretic factor promoter. We demonstrate a genetic interaction between Gata6 and Tbx5 with an incompletely penetrant phenotype of neonatal lethality and thin myocardium. Gene expression analyses were performed on both sets of compound heterozygotes and demonstrated downregulation of alpha-myosin heavy chain only in Gata4/Tbx5 heterozygotes. These findings highlight the unique genetic interactions of Gata4 and Gata6 with Tbx5 for normal cardiac morphogenesis in vivo.
Project description:The G1 cyclins play a pivotal role in regulation of cell differentiation and proliferation. The mechanisms underlying their cell-specific roles are incompletely understood. Here, we show that a G1 cyclin, cyclin D2 (CycD2), enhances the activity of transcription factor GATA4, a key regulator of cardiomyocyte growth and differentiation. GATA4 recruits CycD2 to its target promoters, and their interaction results in synergistic activation of GATA-dependent transcription. This effect is specific to CycD2 because CycD1 is unable to potentiate activity of GATA4 and is CDK-independent. GATA4 physically interacts with CycD2 through a discreet N-terminal activation domain that is essential for the cardiogenic activity of GATA4. Human mutations in this domain that are linked to congenital heart disease interfere with CycD2-GATA4 synergy. Cardiogenesis assays in Xenopus embryos indicate that CycD2 enhances the cardiogenic function of GATA4. Together, our data uncover a role for CycD2 as a cardiogenic coactivator of GATA4 and suggest a paradigm for cell-specific effects of cyclin Ds.
Project description:Mutations of Fog2 in mice result in a phenotype that includes pulmonary lobar defects. To determine whether formation of the accessory lobe bronchus is mediated by a Gata family cofactor, we evaluated embryonic lungs from mice carrying missense mutations that cause loss of FOG-GATA protein interaction. Lungs from embryos carrying a missense mutation in Gata6 were structurally normal, while lungs from embryos carrying mutations of either Gata4 or of both Gata4 and Gata6 had a structural phenotype that matched the Fog2 mutant phenotype. Expression analysis showed that Gata4 and Fog2 are expressed in the ventral and medial pulmonary mesenchyme during secondary budding. Although Gata4 has not previously been suspected as playing a role in lung development, we have found that a Fog2-Gata4 interaction is critical for the development of normal pulmonary lobar structure, and this phenotype is not influenced by the additional loss of Gata6 interaction. Fog2 and Gata4 in the early pulmonary mesenchyme participate in patterning the secondary bronchus of the accessory lobe.
Project description:Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions, leading to gene network dysregulation and human disease. Human mutations in GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling, and metabolic activity. In human cardiomyocytes, GATA4 broadly co-occupied cardiac enhancers with TBX5, another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment, particularly to cardiac super-enhancers, concomitant with dysregulation of genes related to the phenotypic abnormalities, including cardiac septation. Conversely, the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity, leading to aberrant chromatin states and cellular dysfunction, including those related to morphogenetic defects.
Project description:Using the zebrafish model we describe a previously unrecognized requirement for the transcription factor gata4 controlling embryonic angiogenesis. The development of a vascular plexus in the embryonic tail, the caudal hematopoietic tissue (CHT), fails in embryos depleted of gata4. Rather than forming a normal vascular plexus, the CHT of gata4 morphants remains fused, and cells in the CHT express high levels of osteogenic markers ssp1 and runx1. Definitive progenitors emerge from the hemogenic aortic endothelium, but fail to colonize the poorly vascularized CHT. We also found abnormal patterns and levels for the chemokine sdf1a in gata4 morphants, which was found to be functionally relevant, since the embryos also show defects in development of the lateral line, a mechano-sensory organ system highly dependent on a gradient of sdf1a levels. Reduction of sdf1a levels was sufficient to rescue lateral line development, circulation, and CHT morphology. The result was surprising since neither gata4 nor sdf1a is obviously expressed in the CHT. Therefore, we generated transgenic fish that conditionally express a dominant-negative gata4 isoform, and determined that gata4 function is required during gastrulation, when it is co-expressed with sdf1a in lateral mesoderm. Our study shows that the gata4 gene regulates sdf1a levels during early embryogenesis, which impacts embryonic patterning and subsequently the development of the caudal vascular plexus.