Heart formation, development, and differentiation in the human embryo must result from the ordered expression of specific gene products. Unfortunately, little is currently known about the genes and regulatory factors involved in cardiac morphogenesis in humans and other mammals. For this reason there is virtually no scientific foundation for understanding the causes of congenital heart disease. Our laboratory has recently identified a novel mouse transcription factor, termed GATA-4. This factor belongs to a group of related zinc finger proteins that recognize the consensus DNA sequence (a/T)GATA(A?G), known as the """"""""GATA"""""""" motif. Expression of GATA-4 is restricted to heart, with some expression also found in gonads, small intestinal epithelium, and yolk sac endoderm. Based on DNA-binding and co-transfection studies, we propose that GATA-4 functions as a transcriptional activator in heart and other tissues. The goal of this project is to elucidate the role of GATA-4 in cardiac gene expression and development. RNA analysis, in situ hybridization, and immunohistochemistry will be used to determine the pattern of GATA-4 expression in rodent heart during development and in response to hemodynamic overload and hormonal manipulation. The cis-elements regulating cardiac-specific expression of GATA-4 will be defined through deletional and mutational analysis of the promoter using several assay systems including transient transfection of cultured cardiocytes, stable transfection of pluripotential cell lines followed by differentiation into cardiac tissue, direct injection of rodent ventricle, and transgenic mice. To assess the role of GATA-4 in differentiation and development, targeted mutagenesis followed by gene conversion will be used to disrupt the GATA-4 gene in embryonic stem (ES) cells. The consequences of this mutation on in vitro differentiation of ES cell into myocardial and endodermal tissues will be determined. Chimeric mice will be derived by injecting blastocysts with ES cells containing a disrupted GATA-4 allele, and the subsequent breeding of these founder mice will produce GATA-4 homozygous deficient mice. Analysis of these mice will provide insight into the function of GATA-4 in the developing heart. Future studies will then build upon these results and provide a framework for detailed analyses of GATA-4 and its target genes in human hart during normal and pathological development and differentiation.
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