SRP310930 PRJNA714949 GSE169039 Single cell RNA sequencing of P6 and P15 retinal endothelial cells Formation and maturation of a functional blood vascular system is required for the development and maintenance of all tissues in the body. During the process of blood vessel development, primordial endothelial cells are formed and become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues. Specification of arterial and venous endothelial cells occurs in conjunction with suppression of endothelial cell cycle progression, and endothelial cell hyperproliferation is associated with potentially lethal arterial-venous malformations. However, the mechanistic role that cell cycle state plays in arterial-venous specification is unknown. Herein, studying retinal vascular development in Cdh5-CreERT2;R26FUCCI2aR reporter mice, we found that venous and arterial endothelial cells are in distinct cell cycle states during development and in adulthood. That is, venous endothelial cells reside in early G1 state, while arterial endothelial cells reside in late G1 state. Single cell RNA sequencing of developing retinal endothelial cells revealed that BMP signaling and early G1 state are enriched in venous endothelial cells, while TGF-b signaling and late G1 state are enriched in arterial endothelial cells. Cultured endothelial cells in early vs. late G1 exhibited significant differences in gene expression and activity, especially among BMP/TGF-b signaling components. The early G1 state was found to be essential for BMP4-induced venous gene expression, whereas late G1 state is essential for TGF-b1-induced arterial gene expression. In a mouse model of endothelial cell hyperproliferation and disrupted arterial-venous specification, pharmacological inhibition of endothelial cell cycle prevented the vascular defects. Collectively, our results show that endothelial cell cycle control plays a key role in arterial-venous network formation, and distinct cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous specification. Overall design: Single Cell RNA Sequencing: Endothelial cells from P6 and P15 wild-type mice were isolated, and single cell suspensions of endothelial cells were selected through FACS of CD31+CD45- immunostaining. Single cell RNA sequencing was then performed by the Yale Center for Genomic Analysis, which used around 12,000 cells per sample through the 10x Genomics Drop-Seq platform to generate single cell barcoded cDNA libraries. Next-generation sequencing of single cell barcoded cDNA libraries was performed in collaboration with WuXi NextCODE (Boston, MA and Shanghai, China). Single cell RNA sequencing datasets were analyzed by Seurat v3.0 to filter out cells by high mitochondrial gene expression, low or high gene numbers, and contaminating cell types (Hbb.bt, Neurod1, Cd63). P6 and P15 datasets were integrated by IntegrateData function using SCT normalization. GSE169039 pubmed 36202789