Conserved roles of GATA4 and its target gene TBX2 in regulation of human cardiogenesis

The transcription factor (TF) GATA4 is a key mediator of cardiogenesis. GATA4 regulates cardiogenesis through the expression of its target genes, only some of which have been identified. We have used a gain of function model based on pluripotent embryonic ectoderm explants from Xenopus embryos expressing GATA4, to identify a set of downstream targets of GATA4 which are also regulated by Nodal, a known cardiogenic signal. GATA4 was shown to be required for the expression of target genes Tbx2 and Prdm1 in vivo, likely acting in a direct fashion by interacting with their regulatory regions. In addition, Tbx2 and Prdm1 are shown to have roles of their own in vivo, as downregulation of Tbx2, a positive target, and overexpression of Prdm1, a negative target, interferes with cardiac development in Xenopus embryos. The conservation of the GATA4-TBX2-PRDM1 regulatory relationship was shown in human iPSC-derived cardiomyocytes. Loss of function of GATA4 lead to downregulation of TBX2, upregulation of PRDM1 expression and failure of cardiogenesis. GATA4 deficient cells failed to form normal cardiomyocytes, with most cells likely adopting alternative fates such as smooth muscle cells and fibroblasts, and only a small minority expressing an aberrant cardiomyocyte phenotype. Genome wide transcriptomic analysis documented severe reduction of cardiomyocyte and endothelial cell transcriptomes and upregulation of transcriptional profiles of smooth muscle cells and fibroblasts. Disruption of TBX2 function did not alter cardiomyocyte differentiation efficiency but led to the formation of abnormal, hypertrophic cardiomyocytes, characterised by defective sarcomeres and upregulation of cardiomyocyte Gene Ontology terms. In addition, we show that PRDM1 is not essential for formation of cardiomyocytes, implying suppression of alternative fates and fine-tuning the expression of the cardiomyocyte program. The results presented establish a conserved regulatory relationship between GATA4 and its target genes TBX2 and PRDM1, expanding the cardiac GRN and providing further insight into how cardiogenesis proceeds