PROJECT SUMMARY Lymphatic valves are essential for the proper lymph flow occurring during tissue fluid drainage and transport, immune cell trafficking and nutrient absorption in the intestine. Despite their critical roles in vascular function and health, relatively little is known about the molecular mechanism that controls lymphatic valve development and maintenance. In this proposal, we aim to elucidate the mechanotransduction that incorporates the fluid flow-driven external signals into the internal genetic programs governing the valve formation and function in the lymphatic system. We have recently identified a unique fluid flow condition that could induce the signatures of the early stages of lymphatic valve development, including Prox1 upregulation, an initial hallmark of valvular endothelial cell specification that was not attainable by other reported flow conditions. Imposing mechanical cell stretching, this experimental setup induces p65 nuclear translocation, Prox1 upregulation and dephosphorylation, and their functional collaboration through both protein-protein interaction and binding site sharing. These observations have led us to hypothesize that Prox1 and p65 cooperatively control a yet uncharacterized valve-forming mechanotransduction pathway in lymphatic compartment, and possibly conserved in venous vessels as well. We propose that our experimental flow condition will allow us to address this hypothesis by offering a unique opportunity to dissect the mechanotransduction. In addition, we will confirm the regulation and function of the essential physical, molecular and genetic constituents of the mechanotransduction using various animal models. The outcome will not only advance our current understanding of lymphatic valve formation and function, but also provide fundamental insights into the mechanisms underlying the flow-regulated vascular development and function.