We hypothesized that identification of genes rapidly regulated by endothelial Notch signaling in a timeframe similar to that of established direct targets would indicate novel direct Notch targets and reveal novel Notch effectors that function in angiogenesis. In studies conducted in primary endothelial cells, microarray analysis of constitutively activated Notch signaling have identified downstream genes, such as VEGFR1, but have not been able to capture the range of rapidly regulated genes and did not distinguish between direct and indirect targets 22, 23. Paradoxically, examination of genes regulated by constitutive Notch ICD expression may not optimally detect direct Notch targets, due to rapid upregulation of transcriptional repressors of the Notch pathway 1, 7, 8, 9. Prior screens to identify Notch targets have generally utilized constitutive signaling via activating mutations or overexpression of Notch ICD. These screens have demonstrated that Notch targets vary widely by cell type, suggesting that targets identified in immortalized cells are not broadly applicable in endothelial cells where, for instance, Notch regulates the VEGF Receptor genes (VEGFR1, VEGFR2, and VEGFR3). Genome-wide screens for Notch/CSL binding sites or direct Notch targets have been conducted in a variety of immortalized cell types, including T-lymphoblastic leukemia cells, myoblasts, and mouse kidney cells 14, 15, 16, 17, 18, 19, 20, 21. Many Notch targets have been identified, but only in select cases has an effector function been delineated, for example NRARP controls vessel regression 10, 11 and EFNB2 promotes migration and proliferation 12, 13. Angiogenic control by Notch is driven by gene transcription and of both direct and secondary targets that control cellular behaviors, termed Notch effectors. In endothelial cells, Notch transcriptional targets include those that are directly regulated, such as VEGFR3/ FLT4 6, or those that respond to further transcriptional regulation, such as the transcriptional repressors Hairy and Enhancer-of-split family- HES and HEY genes 1, 7, 8, 9. While Notch acts as a receptor at the cell surface, activation by Notch ligands from the Delta like ( DLL) or Jagged ( JAG) families permit γ-secretase cleavage 2, 3 of the Notch Intracellular domain (ICD) which then translocates to the nucleus, forms a transcriptional complex with CSL/RBP-J and MAML, and drives transcription of genes containing RBP-J binding sites 4, 5. To control sprouting angiogenesis, Notch signaling must integrate these cellular steps in a temporal and context dependent manner 1. Notch signaling regulates angiogenesis by controlling diverse endothelial cell behaviors, including migration, proliferation, metabolism, sprout formation, and barrier integrity. We conclude that RND1 is directly regulated by endothelial Notch signaling in a rapid fashion in order to suppress endothelial migration. In endothelial cells, RND1 was shown to be a novel direct Notch transcriptional target and required for Notch control of sprouting angiogenesis, endothelial migration, and Ras activity. Among the novel Notch-regulated signaling pathways identified were effectors in GPCR signaling, notably, the constitutively active GTPase RND1. In each combination of endothelial type and Notch manipulation, transcriptomic analysis identified distinct but overlapping sets of rapidly regulated genes and revealed many novel Notch target genes. We examined transcriptional changes from 1.5 to 6 h after Notch signal activation via ligand-specific or EGTA induction in cultured primary human endothelial cells and neonatal mouse brain. In contrast to previous screens that focus on genes regulated by constitutively active Notch, we characterized the dynamic response to Notch. Notch activity is highly dynamic in sprouting endothelial cells, while constitutive Notch signaling drives homeostatic endothelial polarization, indicating the need for both rapid and constitutive Notch targets. Each of these responses may be regulated by distinct Notch-regulated effectors. To control sprouting angiogenesis, endothelial Notch signaling suppresses tip cell formation, migration, and proliferation while promoting barrier formation.
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