Supplementary Figure 8: dll4 and cxcr4a are not expressed in endothelial cells during venous plexus blood vessel sprouting.

(a) Expression of the dll4 reporter Tg(dll4:gal4)^mu106; Tg(UAS:GFP)^nkuasgfp1a(green) cannot be detected in sprouting venous plexus ECs (arrowheads) marked with Tg(kdrl:Hsa.HRAS-mcherry)^s916 (red) at 28 hpf, 32 hpf or 38 hpf (n = 10 for each time point). (b) Expression of cxcr4a (red) cannot be detected in sprouting venous plexus ECs marked with an anti-GFP antibody staining in Tg(kdrl:EGFP)^s843 (green) embryos (n = 10 for each time point). Visible red staining comes from overlying fin fold tissue. Images are representative of three independent experiments. (c) Current model of tip/stalk cell specification through lateral inhibition. VEGF signalling induces the Notch ligand dll4 in tip cells. High dll4 expression in tip cells activates Notch signalling in neighbouring stalk cells thereby preventing them from acquiring a tip cell phenotype. (d) Model integrating artery formation and Notch pathway activation during angiogenesis. Initially, Notch negative tip cells sprout from veins. These tip cells subsequently express dll4. Stalk cells also start expressing dll4. This leads to Notch activation via trans-activation first in tip and then also in stalk cells. Notch activation induces expression of the chemokine receptor cxcr4a. This allows proper tip cell migration towards the existing artery, ultimately allowing blood flow from arteries to veins. Scale bars, 50 μm. CVP-caudal vein plexus; DA-dorsal aorta; EC-endothelial cell; hpf-hours post fertilization.