Crk/Crklregulates early angiogenesis in mouse embryos by accelerating endothelial cell maturation

Abstract

AbstractRationaleUbiquitously expressed cytoplasmic adaptors CRK and CRKL mediate multiple signaling pathways in mammalian embryogenesis. They are also associated with cardiovascular defects occurring in Miller-Dieker syndrome and 22q11.2 deletion syndrome, respectively. The embryonic mesoderm contributes to the formation of the cardiovascular system, yet the roles thatCrkandCrklplay there are not understood on a single cell level.ObjectivesTo determine functions ofCrkandCrklin the embryonic mesoderm during early mouse vascular development. Secondly, we will examine the molecular mechanisms responsible for early embryonic endothelial cell (EC) defects by performing single cell RNA-sequencing (scRNA-seq) andin vivovalidation experiments.Methods and ResultsInactivation of bothCrkandCrkltogether usingMesp1Creresulted embryonic lethality with severe vascular defects. Although vasculogenesis appeared normal, angiogenesis was disrupted both in the yolk sac and embryo proper, leading to disorganized vascular networks. We performed scRNA-seq of theMesp1Cremesodermal lineage and found that there was upregulation of a great number of angiogenesis and cell migration related genes in ECs in the mutants, including NOTCH signaling genes such asDll4andHey1. Further bioinformatic analysis of EC subpopulations identified a relative increase in the number of more differentiated angiogenic ECs and decrease in EC progenitors. Consistent with this, we identified an expansion ofDll4expressing cells within abnormal arteries,in vivo. Also, our bioinformatic data indicates that there is dysregulated expression of lineage genes that promote EC differentiation causing accelerated cell fate progression during EC differentiation.ConclusionsOur results show thatCrkandCrklare crucial for regulating early embryonic angiogenesis. Combined inactivation ofCrk/Crklcaused precocious EC maturation with an increase of atypical differentiated angiogenic ECs and failed vascular remodeling. This is in part due to increased NOTCH signaling and altered expression of cell migration genes.