Endothelial Subpopulations in Heart Valve Development and Congenital Disease Project Summary Normal heart valve structure and composition are established during development, and congenital valve malformations, such as those arising from mutations in structural protein genes as in Marfan syndrome (MFS), have progressive disorganization and dysfunction over time. Myxomatous valve disease (MVD) is characterized by thickening and progressive degeneration of valve leaflets, leading to valvular regurgitation and reduced heart function. While fluctuating mechanical forces related to blood flow act on the valve leaflets in development and disease, it is not known how these forces affect valve structure and function at the molecular level. Single cell RNA sequencing (scRNAseq) demonstrated valve endothelial cell (VEC) subpopulations localized in regions of heart valve leaflets with distinct blood flow profiles. One of these VEC subpopulations expresses Prox1, a mechanosensitive transcription factor, but its roles in heart valve development, homeostasis, and disease are unknown. Moreover, Prox1 expression is expanded to the laminar flow side of myxomatous valves in a mouse model of MFS with reported valve regurgitation. We hypothesize that Prox1 expression in heart VECs, subject to Wnt/β-catenin activation, is critical for valve leaflet organization during development and for maintenance of valve structure and ECM organization in adults. The Aims are: 1) Determine the requirements for Prox1 in heart valve development and homeostasis. 2) Determine if Prox1 mislocalization in myxomatous valve leaflets contributes to macrophage infiltration and MVD progression. 3) Determine if Wnt/beta-catenin signaling in valve endothelial cells is required for localized Prox1 expression and endothelial junction maturation. The long-term goals of these studies are to define mechanosensitive regulatory mechanisms of heart valve development and pathologic remodeling, thus leading to the development of new nonsurgical therapeutic approaches for myxomatous valve disease.