Actomyosin contractility is a central regulator of cell polarity, cell shape and tissue mechanics in development, physiology and disease. Dysregulation of actomyosin organization can lead to cancer and birth defects, such as neural tube defects (NTD), heart defects and hereditary deafness. Thus, understanding how actomyosin contractility generates both the physical forces and mechanosensitive signaling to coordinate cell behaviors is of both fundamental and medical importance. The conserved Planar Cell Polarity (PCP) pathway has been implicated in mechano-sensing and orientation of cell polarity within the tissue plane during vertebrate tissue morphogenesis. In addition, our work has identified an essential, parallel PCP signaling pathway mediated by Ptk7, a pseudo-receptor tyrosine kinase (RTK) that lacks kinase activity. Live imaging of neural convergent extension movements in mouse embryos has revealed that, while core PCP pathway components regulate apical junction remodeling, Ptk7 is required for planar polarization of neural plate cells during neural tube morphogenesis. Ptk7 signals to Src family kinases (SFKs) to modulate actomyosin contractility at epithelial cell contacts. However, the signal transduction mechanisms by which Ptk7 controls planar polarity are poorly understood. To fill this significant knowledge gap, we will use multidisciplinary approaches combining biochemistry, mouse genetics, including a CRISPR-generated Ptk7 allelic series, and fluorescence imaging of cytoskeletal, junction and Rho GTPase signaling dynamics in neural plate cells in intact live mouse embryos. Based on a strong foundation of preliminary data, Aim 1 will test the hypothesis that Ptk7 imparts planar polarity through RTK-like conformational changes, using conformation-specific anti-Ptk7 antibodies, PCP signaling assays, and Ptk7 knockin mouse mutants carrying human NTD-associated variants or predicted allosteric mutations. Aims 2 and 3 will test whether the cell polarity protein Par3, a junction-associated Angiomotin-Merlin complex, and the transcription factors Yap1/Taz mediate mechanotransduction in the developing neural tube downstream of Ptk7, using conditional mouse mutants, PCP signaling assays and live imaging of cellular and cytoskeletal dynamics. By correlating neural tube, cytoskeletal and cell behavior defects in these mutants, we will be able to tease apart the contributions of distinct cellular and molecular mechanisms, including apical junction remodeling, polarized basal protrusive activities and apical constriction of neural epithelial cells, to the complex process of neural tube formation. A long-term objective of this work is to gain a mechanistic understanding of how cells integrate actomyosin contractility and intra- and inter-cellular signaling in space and time during neural tube morphogenesis. In addition, the proposed work will generate new insights into mechanisms of action of pseudo-RTKs and the etiology of human neural tube defe...