nkxgenes establish SHF cardiomyocyte progenitors at the arterial pole and pattern the venous pole through Isl1 repression

NKX2-5 is the most commonly mutated gene associated with human congenital heart defects (CHDs) with a predilection for cardiac pole abnormalities. This homeodomain transcription factor is a central regulator of cardiac development and is expressed in both the first and second heart fields (FHF and SHF). We have previously revealed essential functions of nkx2.5 and nkx2.7, two Nkx2-5 homologues expressed in zebrafish cardiomyocytes, in maintaining ventricular identity. However, the differential roles of these genes in the specific subpopulations of the anterior (aSHF) and posterior (pSHF) SHFs have yet to be fully defined. Here, we show that nkx genes regulate aSHF and pSHF progenitors through independent mechanisms. We demonstrate that nkx genes restrict proliferation of aSHF progenitors in the outflow tract, delimit the number of pSHF progenitors at the venous pole, and pattern the sinoatrial node acting through Isl1 repression. Moreover, optical mapping highlights the requirement for nkx gene dosage in establishing electrophysiological chamber identity and integrating FHF and SHF cardiomyocyte physiologic connectivity. Ultimately, our results may shed light on the discrete errors responsible for NKX2-5-dependent human CHD of the cardiac outflow and inflow tracts.