Project Summary The anterior second heart field (SHF) cells are a population of late differentiating cardiovascular progenitor cells that contribute to myocardial cells, smooth muscle cells, and endothelial cells in the heart. It was not discovered until recently that these SHF progenitors can also contribute to cardiac lymphatic endothelial cells (LECs) as well as the lymphatics of the craniofacial area in mice. Lymphatics play essential roles in regulating interstitial fluid homeostasis and immune cell modulation. It is unclear whether lymphatic defects can cause congenital cardiac and craniofacial malformations. Mutation in CHD7, a gene encoding Chromodomain Helicase DNA Binding Protein 7 that functions as an ATP-dependent chromatin remodeler, cause craniofacial defects and cardiovascular malformations. It has been reported that CHD7 plays a cell-autonomous role in SHF progenitors to regulate downstream gene expression. Our preliminary data further demonstrated that CHD7 is highly expressed in cardiac lymphatic progenitors and chd7 mutant zebrafish show defects in lymphatics in the craniofacial areas, on the out flow tract and the heart ventricle. We will test the hypothesis that CHD7 regulates gene expression and chromatin accessibility in cardiac and craniofacial LECs derived from SHF progenitors. We propose two independent aims to 1) perform confocal and live imaging and lineage tracing to analyze the chd7 mutant phenotypes and SHF contributions to craniofacial and cardiac LECs; 2) perform single nuclei Multiomic amalysis, spatial transcriptomics and CUT&RUN to determine the molecular mechanisms by which CHD7 regulates downstream gene expression and chromatin landscapes. Our proposed experiments will reveal previously unappreciated roles of CHD7 in LECs derived from SHF progenitors and help identify CHD7 downstream target genes that can be potential therapeutic targets for patients with CHD and lymphatic malformations.