Endothelial Cell Heterogeneity And Arteriovenous Lineage Relationship During Early Vascular Development In Mammals

During mammalian embryogenesis,the cardiovascular system is the first functional organ system to form.Vascular endothelial cells（VECs）undergo de novo differentiation from mesodermal precursors and assemble into a primordial vascular network through vasculogenesis.During the establishment of the vasculature,Vascular endothelial cells are needed to grow new vessels from pre-existing ones via sprouting angiogenesis,while the immature vascular plexus undergoes further remodeling to form recognizable arteries and veins and build a hierarchically organized vascular network.In mouse embryos the arteriovenous features appear to remain ambiguous in the initially formed vascular endothelial cells.Recent studies have suggested that arterial-and venous-fated vascular endothelial cells may initially reside in the same vasculature,emphasizing the importance of molecular identification of vascular endothelial cells.The growth of arteries but not capillaries has been demonstrated to be the key to restoring effective circulation to compromised tissues.Unfortunately,due to the insufficient understanding of arterial specification and arterial conduit formation,the field of therapeutic angiogenesis has not made significant progress in the past decade.Studies of cellular and molecular events underlying the specification of arterial vascular endothelial cells during embryonic development will help to understand adult arteriogenesis.Recently,single-cell RNA sequencing（sc RNA-seq）has been applied to investigate the heterogeneity of endothelial cells in distinct tissues from adult mice.sc RNA-seq has also been used to study gastrulation and organogenesis in mouse embryos.Nevertheless,a high-precision and genome-scale gene expression landscape of early embryonic vascular endothelial cells is still lacking.Knowledge of the cellular evolutions and molecular programs underlying the stepwise arteriovenous fate settling will have important implications for developing new approaches for both regenerative and therapeutic purpose.To construct the single-cell transcriptomic landscape of vascular endothelial cells,single-cell RNA sequencing including STRT-seq and 10×Genomics of vascular endothelial cells during the time window for the occurrence of key vasculogenic and angiogenic events in both mid-gestational mouse and human embryos were performed,and a further integrative transcriptomic analysis of human and mouse embryonic vascular endothelial cells was conducted.The sequencing data revealed that the vascular endothelial cells can be categorized into three main groups:early vascular endothelial cells,major arterial vascular endothelial cells,and vein&plexus vascular endothelial cells.To delineate the exact spatiotemporal distribution of the transcriptomically identified vascular endothelial cell populations,two pan-arterial vascular endothelial cell reporter mouse lines(Unc5b^{td Tomato} and Dll4^{td Tomato})and a set of marker combinations were developed,with most of the in-silico identified vascular endothelial cell populations here immunophenotypically and anatomically determined and in turn transcriptomically validated by further sc RNA-seq.The results showed that morphologically distinct cardinal vein and venous plexus exhibit molecularly similar transcriptional profiles,whereas major artery and arterial plexus possess distinct transcriptional profiles,indicated that vascular beds of embryos present asymmetric characteristics.We demonstrated the evolutionary conservation of principal vascular endothelial cell types during development and revealed a set of evolutionarily conserved genes of distinct arterial and venous vascular endothelial cell populations.Unexpectedly,vascular plexus with similar morphology in the embryo proper showed different arteriovenous characteristics,and two pathways for arterial specification from primordial vascular endothelial cells were predicted by computational analysis.One was toward mature arterial vascular endothelial cells,experiencing early arterial vascular endothelial cells and the subsequent maturing arterial vascular endothelial cells.The other was toward early plexus vascular endothelial cells and further vein&venous plexus vascular endothelial cells,with the final fate as arterial plexus vascular endothelial cells.Finally,to validate arterial plexus vascular endothelial cells arise from arterialization of venous plexus vascular endothelial cells,inducible genetic lineage tracing using a newly developed Nr2f2^{Crex ER} mouse model was conducted.And also a dual recombinase-mediated intersectional genetic approach using Tie2-Dre;Nr2f2-Crex ER mouse line was performed to specifically target venous vascular endothelial cells.Using both constitutive and conditional lineage tracing methods,our results showed early and widespread arterialization from the capillaries with a considerable venous property.Together,our findings provide comprehensive details of endothelial heterogeneity and lineage relationships during embryogenesis,and establish a previously unresolved model regarding the expansion behaviors of early intra-embryonic vasculatures.The comprehensive understanding of hierarchy and intrinsic heterogeneity of the vascular endothelial cells during embryogenesis in vivo will provide important cues to facilitate future investigations directing regeneration biology.The cellular evolution and molecular programs underlying the capillary arterialization at early angiogenesis stages will shed new light on the understanding of adult arteriogenesis,which has obvious diagnostic and therapeutic impacts in multiple important disease conditions.