Emergence of endothelial subtypes and role of cell cycle control in arterial-venous specification during embryonic vascular development

During vascular development, endothelial cells (ECs) specify into arterial, capillary, and venous subtypes to form a circulatory network. While the cell cycle state enables postnatal arterial-venous specification in a flow- and tissue-specific manner, its role during embryogenesis remains unclear. To investigate this, we isolated ECs at embryonic day (E)8.0 (pre-flow), E8.5 (post-flow), and E9.5 and performed single-cell RNA sequencing. Arterial, venous, and hemogenic subtypes emerged with significant enrichment of cell-cycle-related pathways. Using Fucci embryos, ECs were sorted into early G1, late G1, and S/G2/M states and profiled by bulk RNA sequencing. Integration with our single-cell data showed that venous ECs aligned with early G1 and arterial ECs aligned with late G1 transcriptional profiles, consistent with imaging of Fucci embryos. Deleting cell cycle inhibitor Cdkn1b (p27) in embryonic ECs disrupted arterial-venous development, demonstrating that cell cycle control plays a critical role in embryonic arterial-venous specification at the earliest stages of vascular development.