Supplementary Figure 7: The chemokine receptor cxcr4a affects tip cell migration, while dll4 knockdown causes aberrant blow flow patterns.

(a) Wt ctr video 1 showing sprouting of the NCA from the PMBC at indicated time points. Note migratory behaviours of cells 1 and 2 towards the CrDI during the 3:30 h time course. Tg(kdrl:EGFP)^s843 marks ECs in green. (b) Wt ctr video 2 showing sprouting of the NCA from the PMBC at indicated time points. Note migratory behaviours of cells 1 and 2 towards the CrDI during the 3:30 h time course. (c) NCA sprouting in cxcr4a mutant embryo 1. Note normal initiation of NCA sprouting, but subsequent stalling of EC 1. Even at the 8:14 h time point, no connection between CrDI and NCA can be observed. At 16:47 h time point, a small sprout extends from the NCA towards the CrDI (arrowhead). (d) NCA sprouting in cxcr4a mutant embryo 2. Note stalling of ECs 1 and 2. At the 6:46 h time point, a sprout emanates from the CrDI (arrow) and subsequently connects to the NCA, but without generating a proper lumen. (n = 3 embryos, 3 independent experiments). (e) Quantification of blood flow patterns at 54 hpf and 72 hpf in ctr MO injected embryos (n = 19 embryos, 2 independent experiments). Note regular flow patterns and absence of ectopic sprouts. (f) Quantification of blood flow patterns at 54 hpf and 72 hpf in dll4 MO injected embryos (n = 19 embryos, 2 independent experiments). Note lack of blood flow, increase in venous ISVs and ectopic sprouts (green bars). (g) Aberrant blood flow patterns correlate with increased ectopic blood vessel sprouting in dll4 knockdown embryos (n = 19, 2 independent experiments). White arrows indicate ectopic blood vessel sprouts. These are more frequently found in areas containing ISVs without blood flow. (h) Blocking blood flow from 48 to 52 hpf leads to upregulation of cxcr4a expression in the DA and DLAV (arrows). ‘n/N’ reports the ‘number of embryos with staining pattern in image’/‘total embryos from three experiments’ (N = 20 embryos for DMSO and N = 15 embryos for Nifedipine treatment). ^∗∗∗P < 0.0001, Fischer’s exact test. Scale bars, 50 μm. CrDI-Cranial division of the internal carotid artery; EC-endothelial cell; PMBC-primordial midbrain channel; NCA-nasal ciliary artery; h-hour; DA-dorsal aorta; PCV-posterior cardinal vein.