Single-cell Transcriptomics Of Murine Endothelial Cells And Mural Cells Reveals Cellular Heterogeneity

ObjectThe wall of the vascular was composed of endothelial cells(ECs)and mural cells(MCs),which maintain the vascular homeostasis.With the development of sequencing methods,the heterogeneity of ECs in transcriptome was widely accepted.However,systematic studies of endothelial transcriptome at embryonic stage was rare.The heterogeneity of mural cells was focused on their functions,not transcriptome.In this article,we try to(1)reveal the heterogeneity of ECs at embryonic stage;(2)explore the function of EC-enriched mi R-126 at embryonic stage;(3)reveal the heterogeneity of MCs at transcriptional level.Methods and Results4 embryos at embryonic day 14.5 without gene edition were harvested and digested,followed by fluorescence-activated cell sorting(FACS)to collect single ECs(CD31+CD44+CD45-).Single-cell RNA sequencing(sc RNA-seq)was applied to up to?1.2X10⁴ ECs.11 different clusters were defined,with 9clusters defined as specific tissue or organ derived.The diversity of ECs in the same organ or tissue was explored at spatio-temporal level.Furthermore,mi R126^fl/fl;Tie2-Cre mice line was constructed as ECs-specific knockout of mi R126.5208 ECs from 2 mi R126^fl/fl;Tie2-Cre embryos at E14.5 were obtained,and integrated with their littermate controls.Hypoxia-related genes were upregulated in mi R126^fl/fl;Tie2-Cre embryos,which indicated increased intussusceptive angiogenesis.Besides,in mi R126^fl/fl;Tie2-Cre embryos,ECs in pulmonary microvessels were decreased evenly,which indicated impaired blood-air barrier.Re-clustering analysis of hepatic ECs indicated abnormal blood sugar level and arterial development in the liver.Cartilage and neuron related genes were increased in ECs of mesenchymal transited,which indicated increased vascular calcification and abnormal neurogenensis.8 tissues with isolated cluster of MC from published sc-RNA seq dataset GSE109774 were used for the heterogeneity analysis of MCs.We found MCs were heterogenous with 3 perspectives:(1)the transcriptomic of MCs comparing with others and ligand-receptor interactions of MCs and other cell-types were different;(2)MCs were divided into vascular smooth muscle cells,pericyte,transitional mural cell between VSMC and PC,endothelial-like mural cells,and pulmonary mural cells;(3)the coverage and distribution of each cluster of MCs were different in each tissues.ConclusionTranscriptionally heterogenous ECs at embryonic stage implies diverse functions of EC in different microenviroment.mi R-126 regulates blood vessels at embryonic stage by both sprouting and intussusceptive angiogenesis,of which blood vessel-enriched liver and lung were affected most.the heterogeneity of MCs in transcriptome implies their various functions.