Unbiased Expression Profiling Identifies a Novel Notch Signaling Target RND1 as Regulator of Angiogenesis

Notch signaling controls normal and pathological angiogenesis through transcriptional regulation of a wide network of target genes. Despite intensive studies of the endothelial Notch function, a comprehensive list of Notch-regulated genes, especially direct transcriptional targets, has not been assembled in endothelial cells (ECs). Here we uncovered novel EC Notch targets that are rapidly regulated by Notch signaling using several unbiased in vivo and in vitro screening approaches that captured genes regulated within 6 hours or less of Notch signal activation. We used a gamma-secretase inhibitor in neonates to profile Notch targets in the brain endothelium using the RiboTag technique, allowing for isolation of endothelial specific mRNA from a complex tissue without disrupting cell-cell contact. We used two types of primary cultured endothelial cells to define ligand-specific Notch targets by tethered-ligand stimulation. The identified Notch targets were validated by determining their regulation within one to two hours of EGTA-mediated Notch activation. By comparing significantly regulated genes in each of the screens, we assembled a comprehensive database of potential Notch targets