NRP1 expression depends on Notch activity. (A) Down-regulation of Neuropilin1 at E14.5 precedes up-regulation of COUP-TFII. At E14, Notch activation (a), and NRP1 and COUP-TFII expression (b,c) appeared normal in mutant embryos. At E14.5, Notch activity (d) and expression of Neuropilin1 (NRP1) was reduced (e), although COUP-TFII was not yet up-regulated in the arterial endothelium of E14.5 embryos (f). Green indicates α-smooth muscle actin; red indicates antigen. Nuclei are counterstained with Draq5 (blue). (B) Arrangement of RBPJκ and COUP-TFII binding sites in 5 kb of the Nrp1 promoter region. (C) The Neuropilin1 promoter responds to Notch1 activation in vitro. Coculture of CHO cells stably expressing Dll1 with HeLa-N1 cells activated an RBP-luc reporter (set as 100%) and less strongly the Nrp1-luc reporter. (D) Effective activation of the Nrp1 promoter fragment by Notch requires all RBPJκ binding sites. M1-M12345 refers to mutations in the binding sites indicated in panel B. Data are the results of 3 independent experiments. Bars indicate SD.