PROJECT SUMMARY The clinical phenotypes associated with DiGeorge Syndrome, the most common microdeletion condition (22q11.2) in humans, illustrates the developmental link between cardiovascular and craniofacial morphogenesis. Recent fate mapping studies in mice and zebrafish further support this notion given the identification of a multipotent progenitor in the cardiopharyngeal field (CPF) that gives rise to the heart, branchiomeric muscles, and pharyngeal arch arteries, mediated through the pharyngeal arches (PAs). NKX2-5 and NKX2-6 are homeobox transcription factors frequently mutated in congenital heart defects. In zebrafish, we have previously shown that Nkx2.5 and Nkx2.7 play redundant roles in cardiac development. Using a novel loss-of-function nkx2.7 allele that is homozygous lethal, we demonstrate for the first time that nkx2.7-/- embryos fail to form craniofacial muscles and cartilage necessary for feeding. This developmental deficiency results from early disrupted genetic regulation in the PAs. We hypothesize that Nkx2.7 functions as an essential transcription factor during craniofacial development mediated through the pharyngeal arches. We will investigate our model by uncovering the tissue-specific roles of the nkx2.7+ CPF progenitors and by evaluating the transcriptional targets of nkx2.7 and assessing for associated human disease phenotypes. In Aim 1, we will characterize the developmental trajectories of the tissue-specific cell types in the PAs in nkx2.7-/- embryos, benefitting from an innovative, high speed, volumetric imaging platform, SCAPE microscopy. Moreover, we will determine cell- autonomous functions of nkx2.7+ progenitors through cell transplantation experiments employing tissue-specific transgenes. In Aim 2, we will investigate the temporal and molecular mechanisms mediated by Nkx2.7. Using a novel heat inducible transgene generated in our lab, we will overexpress nkx2.7 to dissect its requirement during various developmental windows. We will apply this knowledge to single cell RNA-sequencing in nkx2.7-/- embryos to generate a robust list of genetic targets in all pharyngeal tissues. We will also compare those putative effectors with genomic data from patients with congenital heart disease and craniofacial defects. To evaluate for direct or indirect binding of those targets, we will use chromatin immunoprecipitation and DNA sequencing (ChIP-seq). Altogether, this proposal will provide a comprehensive understanding of the role of Nkx2.7 in cardiopharyngeal development and will clarify the relationship between cardiac and craniofacial morphogenesis. Moreover, these studies have potential to ameliorate stem cell therapy strategies in patients with defects of the head musculature and cartilage.