Notch signaling rapidly regulates the expression of the small GTPase RND1 and a diverse endothelial transcriptome

Endothelial Notch signaling regulates transcription of many downstream effectors controlling sprouting, migration, proliferation, barrier formation, and other phenotypes. However, endothelial-relevant Notch targets are largely uncharacterized. Few are studied in depth, and many transient, early response Notch targets are yet to be described. We therefore determined the Notch early response transcriptional profile in several experimentally relevant in vivo and in vitro contexts: ligand-specific or EGTA-induced Notch activation in different primary endothelial cells, and gamma secretase-mediated Notch inhibition in neonatal brain endothelium in a RiboTag mouse model. The intersection of these profiles reveals a core set of early Notch-responsive targets. Among these are effectors in GPCR signaling, not previously implicated downstream of Notch signaling. We demonstrate the Rho GTPase RND1 is a direct Notch transcriptional target and required for Notch control of sprouting angiogenesis, endothelial migration and Ras activity. Overall design: RNAseq screens from in-vitro activation of Endothelial Cells by tethered DLL4 (6h) or EGTA (1.5h) or EGTA in combination with CpE (1.5h) or in-vivo RiboTagEC mice treated with CpE (6h).