PROJECT SUMMARY/ABSTRACT The process of vascular stabilization—which includes mural cell recruitment to the blood vessels—is critical for organism development and survival. The hemodynamic forces from blood flow itself play a part in these stabilization events; however, there are still many gaps in our understanding of how this mechanosensitive signaling occurs and how blood vessel integrity is influenced by disruptions in mechanosensing. Primary cilia on endothelial cells (ECs) have been proposed to be mechanosensitive structures that facilitate signaling in response to hemodynamic forces, though most of our current understanding of this process stems from in vitro work. Interestingly, genetic studies from patient cohorts with congenital heart defects (CHD) reveal an unexpectedly high association with cilia-related gene variants. Consequently, the study of hemodynamics, primary cilia, and congenital cardiovascular defects is a largely unexplored field that could have a meaningful impact on understanding and treating CHD. Thus, the objectives of this proposal are to determine if EC cilia respond to changes in hemodynamic forces during development, to define a mechanism for primary cilia in vessel stabilization, and to model novel cilia-related human CHD variants and assess the vascular phenotypes using the zebrafish animal model. The proposed studies are based on robust preliminary data showing EC primary cilia are evident—and unexpectedly, abluminal—during axial vasculature stabilization events, and that embryos with mutant cilia display phenotypic abnormalities associated with compromised vascular stability. Additional preliminary data suggests that both luminal and abluminal cilia can assemble or disassemble in response to changes in blood flow. Furthermore, embryos with mutant cilia display aberrant mural cell association. Together these data support the central hypothesis that endothelial primary cilia are mechanosensitive regulators of cardiovascular development in part due to their influence on mural cell function and vascular stability. My hypothesis will be tested with the three aims: 1) assess the orientation and localization of EC cilia in response to hemodynamic changes across vascular development, 2) determine the role of primary cilia in mural cell recruitment, differentiation, and vascular stabilization during development, and 3) utilize human CHD data to identify cilia-related defects and analyze the functional consequences of those gene mutations in zebrafish. This work is innovative because it will utilize the zebrafish model to explore mechanosensitive mechanisms linked to vascular stabilization events in vivo. The proposed research is significant in its aim to provide novel insight to the EC cilia field by identifying abluminal cilia and demonstrating that they are mechanosensitive structures in vivo, in addition to clarifying the impact of primary cilia on vascular stabilization and congenital cardiovascular defects.