Early and extensive venous arterialization during mammalian embryogenesis

The cellular evolutions and molecular programs underlying the arteriovenous fate settling of embryonic vascular endothelial cells (ECs) are critical for understanding arteriogenesis and inspiring new approaches for regenerative biology. Using different strategies of single-cell RNA sequencing, we constructed the transcriptional landscape of early arteriovenous EC development in both mouse and human embryos, demonstrating the evolutionary conservation of principal vascular EC types and providing a series of conserved arteriovenous genes. We showed an unexpected diversity of arteriovenous characteristics in morphologically alike vascular plexus and further uncovered two transcriptomically distinct arterial EC types, whereas most of heterologous ligand-receptor pairs were shared by different arterial vasculatures. By computational predicting and further genetic lineage tracing, we revealed the widespread venous arterialization in the mid-gestational mouse embryo proper. Interestingly, we demonstrated at transcriptomic level that Notch1 was dispensable for venous arterialization but required subsequently for the arterial feature strengthening in the arterial plexus ECs. Altogether, our findings unprecedentedly detail the comprehensive single-cell mapping of early embryonic vascular ECs in vivo, decipher an asymmetric arteriovenous characteristics different than that in adults, and reveal an extensive venous-to-arterial fate conversion in the vascular plexus. Overall design: Here, we performed both well-based single-cell RNA-sequencing (scRNA-seq) of 2,213 cells in mouse embryos and 966 cells in human embryos and droplet-based scRNA-seq of 10,465 cells in mouse embryos to firstly construct a molecular landscape of embryonic vascular endothelium at single cell resolution. Moreover, we also performed well-based single-cell RNA-sequencing of 144 cells from mouse Dll4-tdTomato reporter model, 240 cells from mouse Unc5b-tdTomato reporter model, 192 cells from one mouse Nr2f2-related lineage tracing model and 112 cells from another mouse Nr2f2-related lineage tracing model, to validate our findings. Finally, we performed single-cell RNA-sequencing of 339 cells by well-based method and 25,147 cells by 10x method from mouse Notch1-knockout model, to decode the role of Notch signaling in arterialization.