Project Summary: The blood vascular circulatory system is composed of arterial, venous, and capillary vessels, which work together to facilitate the efficient exchange of gasses, nutrients, and waste from all tissues, while regulating blood pressure and distribution of immune cells. The endothelial cells that line these vessels must acquire specialized characteristics to enable specialized arterial, venous and capillary functions, a process known as endothelial cell specification. Disruption of this process during development can lead to embryonic lethality, while postnatally it causes arterial-venous malformations and contributes to pathological conditions, including dysfunctional tumor vasculature. Understanding the mechanisms that control arterial-venous endothelial cell specification and network formation is essential for advancing our ability to improve therapies for vascular pathologies, revascularize injured tissues, and optimize the differentiation of pluripotent human stem cells for vascular tissue engineering and regenerative medicine therapies. Our previous studies, in the postnatal murine retinal vascularization model of sprouting angiogenesis, revealed a critical role for blood flow-regulated endothelial cell cycle state in their fate specification. We found that arterial/arteriolar shear stress levels promote Notch signaling, and downstream p27-induced late G1 state, which enables TGF-β1-induced arterial gene expression (Fang 2017; Chavkin 2022). Conversely, flow magnitudes typical of veins/venules induce early G1, which enables BMP4-induced upregulation of venous genes (Chavkin 2022). However, blood flow is not required for the initiation of arterial-venous specification during embryonic vascular development (Chong 2011), and whether endothelial cell cycle control is required for this process is unknown. I hypothesize that endothelial cell cycle control is required for embryonic arterial and venous specification and its dysregulation causes arterial-venous malformations. In support of this, my preliminary data show that, during embryonic vascular development, even before flow, arterial endothelial cells are enriched in late G1, while venous endothelial cells are enriched in early G1. Additionally, we identified target genes that may regulate endothelial cell cycle state and/or specification. Using the Fucci2 cell cycle reporter mice, we will rigorously define the cell cycle state of arterial and venous endothelial cells in the developing embryo and determine the role of endothelial cell cycle control in fate specification. We will also identify novel key regulators of endothelial cell cycle state and arterial-venous fate that can provide new targets for the treatment of diseases associated with endothelial cell hyperproliferation and loss of identity, including vascular malformations and tumor angiogenesis.