Supplementary Figure 2: Notch signalling activity in newly forming arteries.

(a) Living triple transgenic Tg(dll4:gal4)^mu106; Tg(UAS:GFP)^nkuasgfp1a; Tg(kdrl:Hsa.HRAS-mcherry)^s916zebrafish embryo at 50 hpf. Tg(dll4:gal4)^mu106; Tg(UAS:GFP)^nkuasgfp1a double transgenic embryos mark ECs with dll4 expression in green, while Tg(kdrl:Hsa.HRAS-mcherry)^s916 marks EC membranes in red. Note EGFP expression in NCA (n = 10 embryos, 3 independent experiments). (b) Living double transgenic Tg(TP1:GFP)^um14; Tg(kdrl:Hsa.HRAS-mcherry)^s916 zebrafish embryo. Tg(TP1:GFP)^um14 marks cells with Notch pathway activation in green (n = 10 embryos, 3 independent experiments). (c) Transplantation scheme. Kdrl knockdown cells (green) were transferred into wt hosts (red). (d) Representative embryo, showing sprouting NCA cells at 26 hpf time point. Note absence of green fluorescent donor ECs in sprouting NCA (n = 9 embryos, 3 independent experiments). (e) dll4 expression in sprouting NCA cells in control embryo at 30 hpf after DMSO treatment for 4 h and experimental embryo at 30 hpf after SU5416 (VEGF inhibitor) treatment for 4 h (n = 10 embryos, 2 independent experiments). (f) dll4 expression in sprouting NCA cells in control embryo at 32 hpf after DMSO treatment for 4 h and experimental embryo at 30 hpf after SU5416 (VEGF inhibitor) treatment for 4 h (n = 10 embryos, 2 independent experiments). Arrowheads indicate dll4 expressing ECs, marked by Tg(kdrl:EGFP)^s843. In experimental embryos, while ECs are present in the position of the NCA, these do not express dll4 (n = 10 embryos). (g,h) Still images of g video 2 and h video 3 at indicated time points of NCA sprouting in Tg(TP1:GFP)^um14; Tg(kdrl:NLS-mcherry)^is4 double transgenic embryos. White arrowheads with numbers indicate individual ECs. Note onset of GFP expression in cell number 1 at the 4:35 h time point, followed by GFP expression in cells 3 and 4 in video 2. Note onset of GFP expression in cell number 1 at the 3:28 h time point, followed by division of this cell, in video 3. GFP expression can be subsequently detected in cell 2. (i,j) Quantification of fluorescence intensity of green Tg(TP1:GFP)^um14 signal normalized to red Tg(kdrl:NLS-mcherry)^is4 signal over time in i video 2 and j video 3. (n = 3 embryos, 3 independent experiments). t₀ = 26 hpf. Scale bars, 50 μm. A.U.-arbitrary units; CrDI-Cranial division of the internal carotid artery; PMBC-primordial midbrain channel; NCA-nasal ciliary artery; h-hour; hpf-hours post fertilization.