Emergence of Endothelial Cell Subtypes and Role of Cell Cycle Control in Arterial-Venous Specification During Embryonic Vascular Development [bulk RNA-seq]

During embryonic vascular development, endothelial cells that line all blood vessels undergo specification into arterial, capillary, and venous subtypes to form a circulatory network. Regulation of endothelial cell cycle state has been shown to play a critical role in enabling arterial-venous specification and remodeling in a postnatal, blood flow-mediated, and tissue specific manner; however, if a similar cell cycle-mediated mechanism is a common mechanism that regulates embryonic vascular development, even prior to blood flow, is unknown. To investigate this, we first defined the emergence of distinct subtypes by isolating endothelial cells from wild type embryos at embryonic day (E)8.0, prior to blood flow; E8.5, just after flow begins; and E9.5. We performed single cell RNA sequencing and analyses, and found increased specification of arterial, venous and hemogenic subtypes over time, concomitant with decreased primordial and capillary endothelial cells. Gene Ontology analysis revealed that cell cycle control was significantly enriched over time, and we found arterial identity highly correlated with growth arrest. To gain more insight into the potential role of cell cycle control in specification, we isolated similar endothelial cells from Fucci-expressing embryos, sorted them into distinct cell cycle states (early G1, late G1 and S/G2/M), performed bulk RNA sequencing, and bioinformatically correlated endothelial cell cycle states with subtype identities. We found venous endothelial cells are highly enriched in early G1 and arterial endothelial cells highly enriched in late G1, which was corroborated with fluorescent imaging of Fucci embryos. Furthermore, we showed that endothelial cell hyperproliferation, induced by deletion of cell cycle inhibitor Cdkn1b (p27), impaired arterial-venous specification and vascular development. These results support that at the earliest stage of vascular development, endothelial cells in arterial and venous vessels reside in different cell cycle states and endothelial cell cycle control is required for their specification.