Project Summary Craniofacial anomalies account for a third of all human congenital birth defects and significantly impact national health care budgets. Affected individuals typically undergo multiple surgeries throughout their lifetime, which are rarely fully corrective. Therefore, it is critical to develop therapies for improved prognosis and prevention, but this can only come from a better understanding of the genetic and cellular mechanisms governing craniofacial development and the etiology and pathogenesis of individual disorders. Disruptions in neural crest cell (NCC) development are considered the underlying cause of many craniofacial birth defects. Therefore, understanding the genetic and cellular mechanisms that regulate NCC development and their ultimate generation of craniofacial tissue is crucial for developing preventative therapies and improved surgical prognosis. Delamination from the neuroepithelium is a critical step in the formation of migrating NCC. However, the molecular and cellular mechanisms governing NCC delamination in mammalian embryos are poorly understood. Delamination is a biophysical process by which a cell departs its tissue environment, and my extensive preliminary data indicates that cell extrusion may be a novel mechanism facilitating cranial NCC delamination. This proposal focuses on cell extrusion and will broaden our understanding of NCC delamination by elucidating the cellular and genetic systems regulating NCC delamination via cell extrusion. Aim1 will visualize and capture the dynamic cytoarchitectural and morphological changes that drive cranial NCC delamination in mouse embryos. Aim2 will investigate the role of Piezo1, a mechanosensitive ion channel in cranial NCC delamination and craniofacial development. The impact of Piezo1 loss-of-function on NCC delamination and craniofacial development will be analyzed through null and conditional genetic knock out of Piezo1 mechanosensitive ion channels in mouse embryos. The downstream regulatory network and signaling pathways will then be elucidated through transcriptomic comparisons measuring the differences in candidate downstream regulators between wild type and knock out embryos. Completion of the study will advance fundamental knowledge and further our understanding of neural crest cell and craniofacial development and of congenital birth defects.