Supplementary Figure 1: Validation and activation of dll4 transgenic reporter line.

(a) Double FISH of dll4 mRNA together with GFP mRNA and an antibody staining to detect GFP protein at different time points in Tg(dll4:gal4)^mu106; Tg(UAS:GFP)^nkuasgfp1a double transgenic embryos to mark ECs with dll4 expression. Inset indicates zoomed in NCA region below (n = 8 embryos for each time point, 2 independent experiments). (b) Double FISH of dll4 mRNA together with GFP mRNA and an antibody staining to detect GFP protein in the trunk region of a zebrafish embryo at 24 hpf. Insets mark zoomed in region. Arrowheads mark dll4 mRNA (red), GFP mRNA (green), GFP protein (white) or overlay of channels (n = 8 embryos, 2 independent experiments). (c) Still images of video 2 at indicated time points of NCA sprouting from the PMBC and connecting to the CrDI in Tg(dll4:gal4)^mu106; Tg(UAS:GFP)^nkuasgfp1a, marking cells expressing dll4 in green, and Tg(kdrl:NLS-mcherry)^is4, which marks all EC nuclei in red, triple transgenic embryos. White arrowheads with numbers mark individual ECs. Note onset of GFP expression in cell number 1 at the 2:36 h time point, followed by GFP expression in cells 2 and 3. (d) Still images of video 3 at indicated time points of NCA sprouting from the PMBC and connecting to the CrDI in Tg(dll4:gal4)^mu106; Tg(UAS:GFP)^nkuasgfp1a; Tg(kdrl:NLS-mcherry)^is4 triple transgenic embryos. White arrowheads with numbers mark individual ECs. Note onset of GFP expression in cell number 1 and 2 at the 5:00 h time point, followed by GFP expression in cell number 3. (e) Quantification of fluorescence intensity of the green Tg(dll4:gal4^)mu106; Tg(UAS:GFP)^nkuasgfp1a signal normalized to red Tg(kdrl:NLS-mcherry)^is4 signal over time in video 2. (f) Quantification of fluorescence intensity of the green Tg(dll4:gal4)^mu106; Tg(UAS:GFP)^nkuasgfp1a signal normalized to red Tg(kdrl:NLS-mcherry)^is4 signal over time in 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; EC-endothelial cell; h-hour; hpf-hours post fertilization; ISV-intersegmental blood vessel; NCA-nasal ciliary artery; PMBC-primordial midbrain channel; FISH-Fluorescent in situ hybridization.