Project Summary Mechanotransduction describes the cellular processes that translate mechanical stimuli into biochemical signals, thus enabling cells to adapt to their dynamic physical surroundings. Mechanosensing pathway is essential to development and homeostasis, and impaired mechanotransduction is implicated in a wide spectrum of diseases. However it is unclear which cells are mechanosensitive, how mechanosensing is regulated and what mechanisms link mechanical forces to intracellular signaling. Examining the role of mechanosensitive ion channel complex in embryonic development will provide key insights into those important questions. In this proposal, I aim to understand how Ano1/Tmem16A, a calcium-activated chloride channel, and Piezo1, a machanosensitive channel, can transduce mechanical cues into intracellular biochemical signaling. My preliminary analyses show that inactivation of Ano1 during mouse embryonic development leads to sternum defect, cardiovascular anomalies, tracheomalacia and esophagus stenosis, as well as renal dysplasia, all of which resemble the phenotypes observed in VACTERL association that affect multiple organs in humans. The data indicate that cellular defects seen in Ano1 mutants may arise from impaired mechanosensing and suggest a model in which Ano1 and Piezo1 act synergistically in the mechanotransduction pathway to control morphogenesis. I hypothesize that the action of Ano1 may be modulated through Piezo1-mediated calcium increase, and in turn regulates intracellular machinery to adjust cell volume, number, geometry and proliferation. In Specific Aim 1, I will characterize the roles of Ano1 and Piezo1 during embryogenesis. In Specific Aim 2, I will determine the functional and physiological coupling of Ano1 and Piezo1 in regulating mechanosensitive current. In Specific Aim 3, I will use in vivo and in vitro models to investigate possible mechanisms that link Ano1 and Piezo1 in mechanosensing during embryogenesis and homeostasis. The results will provide the first indication that Ano1-mediated CaCC acts in concert with Piezo1 to control morphogenesis, a finding that is crucial for our understanding of how mechanical force integrates with channel function and calcium signaling in mammalian development. I anticipate that my proposed study will open the way to eventual treatment strategies for mechanosensing associated diseases, including congenital birth defects and polycystic kidney disorders.