PROJECT SUMMARY The aberrant formation of the Pharyngeal Arch Arteries (PAAs) results in severe and lethal forms of congenital heart disease. The PAAs form through vasculogenesis, where an endothelial cell (EC) plexus invades the pharyngeal arches before coalescing into patent PAAs. Our lab has shown that a majority of the ECs that make up the PAAs derives from a population of mesodermal progenitor cells known as the Second Heart Field (SHF). Additionally, our lab has shown that Integrin ɑ5 (Itgɑ5) expression within the Isl1-lineages, encompassing the second heart field and pharyngeal endoderm and ectoderm, regulates the formation of the PAAs through unknown mechanisms. Integrins are cell surface receptors that organize the ECM and relay ECM signals to elicit cellular responses, including cell proliferation, survival, migration, and differentiation. Preliminary data suggests Itgɑ5 regulates the number of SHF-derived ECs that give rise to the PAAs. We hypothesize that integrin ɑ5 expression in the Isl1-lineages regulates the number of SHF-derived ECs in the pharyngeal arches by mediating SHF-progenitor cell survival and/or proliferation, migration, and/or differentiation. In Specific Aim I, we will investigate this hypothesis using Itgɑ5f/- ; Isl1Cre mutant and Itgɑ5f/+ ; Isl1Cre control embryos, which carry the Rosa26nTnG reporter. This reporter marks cells derived from the Isl1-lineages with the expression of nuclear GFP. Utilizing this conditional knockout mouse model and immunohistochemistry, we will investigate the proliferation and apoptosis frequencies, migration patterns, and differentiation capacity, of the Isl1-derived cells that give rise to the ECs of the PAAs. My preliminary data additionally shows SHF-derived ECs crossing the midline in the developing pharyngeal region within our mutants. During vertebrate development, cells in general do not cross the midline ensuring proper left-right patterning. The ECM organization and programmed cell death response at the midline create a barrier that stops cells from migrating across the midline. Because we know Itgɑ5 regulates ECM organization we hypothesize that Itgɑ5 expression in the Isl1-lineages regulates SHF-derived EC patterning through mediating the ECM organization and the programmed cell death response at the midline. We will investigate this hypothesis in Specific Aim II, utilizing our conditional mouse model and immunohistochemistry. We will compare the ECM organization and apoptosis frequency at the midline in control and mutant embryos. Additionally, we will investigate the distribution of Isl1-derived cells at the midline during early embryogenesis because if Isl1-derived cells are able to cross the midline in our mutants, the Isl1-derived cells that give rise to the PAAs could be directed to the wrong side, resulting in aberrant PAA formation. The completion of the experiments outlined in this proposal will provide insights into mechanisms involved in the development of the PAA...