Abstract The transcription factor HAND2 plays critical roles during cardiogenesis. Hand2 endocardial deletion ( H2CKO ) results in tricuspid atresia or double inlet left ventricle with accompanying intraventricular septum defects, hypo-trabeculated ventricles, and an increased density of coronary lumens. To understand the regulatory mechanisms of these phenotypes, single cell transcriptome analysis of E11.5 H2CKO hearts was performed revealing a number of disrupted endocardial regulatory pathways. Utilizing HAND2 DNA occupancy data, we identify several HAND2-dependent enhancers, including two endothelial enhancers for the sheer-stress master regulator, KLF2. A 1.8kb enhancer located 50kb upstream of the Klf2 transcriptional start site imparts specific endothelial/endocardial expression within the vasculature and endocardium. This enhancer is HAND2-dependent for ventricular endocardium expression but HAND2-independent for Klf2 vascular and valve expression. Deletion of this Klf2 enhancer reveals reduced Klf2 expression within ventricular endocardium. These data reveal that HAND2 functions within endocardial gene regulatory networks including sheer stress response.

Single cell evaluation of endocardial HAND2 gene regulatory networks reveals critical HAND2 dependent pathways impacting cardiac morphogenesis