The mammalian heart forms from multiple embryonic lineages whose specification and contributions to the developing heart are exquisitely controlled by transcription factors and signaling molecules. GATA4 is a zinc finger transcription factor that is expressed in multiple cardiovascular lineages and is required for embryonic viability. GATA4 controls heart development by regulating gene expression in multiple embryonic lineages, including the endocardium, which is an inner lining of specialized cardiac endothelium, and the myocardium, which is the muscular layer of the heart. In spite of its importance in heart development, the transcriptional pathways upstream of Gata4 in the developing heart were previously unknown.
Here, we describe several enhancers from the mouse Gata4 locus that are active in the embryo and focus on a distal enhancer that is active in multiple cardiac lineages early in heart development. Detailed expression and lineage analyses show that the cardiac Gata4 enhancer is active in both myocardial and endocardial lineages when the heart first forms but restricts to the endocardium as the heart undergoes looping and further development. This pattern of activity requires GATA-binding sites, which appear to play a role in the repression of enhancer activity in extracardiac tissues in the embryo. The activation of this enhancer in transgenic embryos and in cultured aortic endothelial cells is dependent on four ETS sites. To identify which ETS transcription factors might be involved in Gata4 regulation via the ETS sites in the enhancer, we determined the expression profile of 24 distinct ETS factors in embryonic mouse hearts. Among multiple ETS transcripts present, ETS1, FLI1, ETV1, ETV5, ERG, and ETV6 were the most abundant in the early embryonic heart. We found that ETS1, FLI1, and ERG were strongly expressed in the heart at embryonic day 8.5 and that ETS1 and ERG bound to the endogenous Gata4 enhancer in cultured endothelial cells. FLI1 and ERG activated the enhancer in a fibroblast cell line. Thus, these studies define the ETS expression profile in the early embryonic heart and identify an ETS-dependent cardiac enhancer from the Gata4 locus.