PROJECT SUMMARY The homeodomain transcription factor, NKX2-5, is the most commonly mutated gene associated with congenital heart defects (CHDs), accounting for 1-4% of specific malformations with a predilection for abnormalities at the arterial and venous poles of the heart. The underlying molecular mechanisms responsible for cardiac malformations found in patients with NKX2-5 mutations remain poorly understood. To improve diagnostic and therapeutic measures in these cases, a more precise understanding of early cardiac developmental processes at the outflow (OFT) and inflow (IFT) tracts is critical. The embryonic heart begins as a linear tube derived from first heart field (FHF) progenitors, with expansion occurring through accretion of late-differentiating cells of the second heart field (SHF) to the arterial and venous poles. Nkx2-5 is expressed in both the FHF and SHF and, while vital functions of Nkx genes in the FHF have been implicated in cardiac specification and morphogenesis, little is known about the distinct mechanisms regulated by Nkx genes in the anterior (aSHF) and posterior (pSHF) SHFs. By exploiting benefits of the zebrafish model, we recently published evidence demonstrating that nkx2.5 and nkx2.7, two NKX2-5 homologs expressed in the zebrafish heart, play essential roles in maintaining ventricular identity and display similar chamber-specific functions in SHF cardiomyocytes. Furthermore, our preliminary data provide new evidence that nkx genes exhibit previously unappreciated, crucial functions in regulating SHF progenitor populations through discrete mechanisms at OFT and IFT. We find that Nkx genes promote aSHF progenitor augmentation at the arterial pole and restrict isl1, a LIM homeodomain transcription factor, to the sinus venosus. In this proposal, we test the novel hypothesis that nkx genes are required to recruit aSHF progenitors to the OFT and to restrict pSHF progenitors via isl1 to the IFT. In Aim 1, we will dissect the cellular and temporal roles of Nkx genes in specification, accretion, proliferation, and identity maintenance in the aSHF and pSHF employing heat-shock inducible overexpression of nkx2.5, time series analysis, EdU incorporation studies, developmental timing assays, and state-of-the-art microscopy. In Aim 2, we will utilize the zebrafish model along with CRISPR and ChIP methodologies to examine previously unrecognized direct and indirect downstream effectors of Nkx genes and also new candidates associated with CHD in humans. Combining the tools available in zebrafish and human genomics data, our research will uncover the developmental mechanisms regulated by Nkx genes that are responsible for SHF-derived CHDs, some of the most severe and lethal malformations associated with NKX2-5 mutations.