Fig. 4. Blocking Nrp1 function reproduces the Nrp1^-/- yolk sac vessel phenotype. Wild-type embryos from a CD1 background were treated with various proteins by intracardiac injection followed by whole-embryo culture. (A-D) Subsequent to culture, the vasculature was fixed and stained for Pecam1. (E-G) The images were quantified for vascular morphology. Untreated embryos showed remodeling, typical of E9.5 embryos (A). VEGF₁₂₀ injection caused an increase in overall vascular coverage (B). Injection of antibodies against Nrp1, which are reported to block Sema3A binding (C) and VEGF binding (D) to Nrp1 (Pan et al., 2007b), were both effective at decreasing the number of branchpoints and increasing vessel segment length as seen in the Nrp-1^-/- embryos (E,F). Injection of both antibodies did not show a synergistic effect on the number of branchpoints. Only antibodies against the VEGF-binding domain of Nrp1 were capable of increasing average area of avascular space, as seen in Nrp1^-/- embryos (G). Injection of Sem3A and Nrp1 had no statistically significant effect on capillary geometry (E-G). ^*P<0.05, ^**P<0.01, ^***P<0.005; Student's two-tailed t-test. Scale bars: 100 µm.