Supplementary Figure 5: Absence of Notch activation in dll4 knockdown embryos during NCA formation.

(a,b) Still images of a video 2 and b video 3 at indicated time points of NCA sprouting in Tg(TP1:GFP)^um14; Tg(kdrl:NLS-mcherry)^is4 double transgenic embryo injected with dll4 MO. Tg(TP1:GFP)^um14 marks cells with Notch pathway activation in green, while Tg(kdrl:NLS-mcherry)^is4 marks EC nuclei in red. White arrowheads with numbers indicate individual ECs. (c,d) Quantification of fluorescence intensity of green Tg(TP1:GFP)^um14 signal normalized to red Tg(kdrl:NLS-mcherry)^is4 signal over time in c video 2 and d video 3. t₀ = 26 hpf. (n = 3 embryos, 3 independent experiments). (e) Transplantation scheme. Transferring dll4 knockdown cells from Tg(kdrl:Hsa.HRAS-mcherry)^s916; Tg(TP1:GFP)^um14 into wt hosts allows to distinguish trans- from cis-activation of Notch signalling in ECs. Tg(kdrl:Hsa.HRAS-mcherry)^s916 marks donor EC membranes in red, while Tg(TP1:GFP)^um14 marks donor cells with Notch signalling pathway activation in green. (f–h) Schematic detailing different outcomes of cell transplantations. (i–o) Embryo 2. (p–v) Embryo 3 (i,p) NCA cells stained with a Fli1b antibody detecting EC nuclei (white). (j,q) Transplanted dll4 knockdown ECs. Arrows mark tip cells. (k,r) Cells with activated Notch signalling identified via GFP expression (green arrows). (l,s) Overlay of transplanted NCA cells with host NCA cells, showing that only cell 1 is donor-derived, while cells 2, 3, 4 and 5 are host-derived. (m,t) Overlay of transplanted cells showing Notch pathway activation (green arrows) with host cells. Only the tip cell (number 1) shows GFP expression. (n,u) Overlay of all three channels, identifying transplanted dll4 knockdown tip cell being GFP positive. (o) Zoom in of boxed area in n. Note tip cell (number 1) being next to a wt host cell (number 2). (v) Zoom in of boxed area in u. Note tip cell (number 1) being next to a wt host cell (number 2). Green arrows denote Notch pathway activation (n = 3 mosaic embryos with single donor cell at tip position, 3 independent experiments). Scale bars, 50 μm. Ab-antibody; EC-endothelial cell; hpf-hours post fertilization; CrDI-Cranial division of the internal carotid artery; PMBC-primordial midbrain channel; NCA-nasal ciliary artery.