Figure 6

Ca2+ signalling is required for rapid Erk activation in ablated vessels. (A) Still images from Video 8 demonstrating a pulse of Ca2+ signalling immediately adjacent to the wound (4 days post-fertilisation (dpf)). Left panels show actb2:GCaMP6f and kdrl:mCherry-CAAX, and right panels show actb2:GCaMP6f. Yellow arrows show ISV endothelial cells (ECs) with active Ca2+ signalling. Blue arrows show Ca2+ signalling in recruited immune cells. (B) Quantification of actb2:GCaMP6f intensity in unablated control ISVs (black, n = 4 larvae), ablated (red, n = 10 larvae) and adjacent (light blue, n = 10 larvae) ISVs following wounding. Intensity was normalised to actb2:GCaMP6f intensity in unablated tissue in the same larvae. (C) Ca2+ signalling is required for rapid activation of Erk-signalling in ablated ISV ECs. Quantification of post-/pre-ablation nuclear EKC intensity at 15 min post-ablation (mpa) in ECs of 1% dimethyl sulfoxide (DMSO)-treated non-ablated control ISVs (39 ECs, n = 13 larvae), and ISVs of larvae treated with either 1% DMSO (39 ablated/adjacent ISV ECs, n = 13 larvae) or 50 μM Nifedipine (36 ablated/adjacent ISV ECs, n = 12 larvae). (D) Quantification of post-/pre-ablation nuclear EKC intensity at 15 mpa in ECs of 1% DMSO-treated non-ablated control ISVs (18 ECs, n = 6 larvae), and ISVs of larvae treated with either 1% DMSO (27 ablated/adjacent ISV ECs, n = 9 larvae) or 100 μM Amplopidine (31 ablated ISV ECs and 33 adjacent ISV ECs, n = 11 larvae). (E) Ca2+ signalling is not required for sustaining Erk activity in ablated ISV ECs. Quantification of post-/pre-ablation nuclear EKC intensity at 3 hours post-ablation (hpa) in ECs of 1% DMSO-treated non-ablated control ISVs (24 ECs, n = 8 larvae), and ablated ISVs of larvae treated with either 1% DMSO (42 ECs, n = 14 larvae) or 50 μM Nifedipine (39 ECs, n = 13 larvae) for 30 min before 3 hpa (Figure 6—figure supplement 2A). (F) Quantification of post-/pre-ablation nuclear EKC intensity at 3 hpa in ECs of 1% DMSO-treated non-ablated control ISVs (21 ECs, n = 7 larvae), and ablated ISVs of larvae treated with either 1% DMSO (27 ECs, n = 9 larvae) or 50 μM Nifedipine (27 ECs, n = 9 larvae) for 30 min after vessel wounding (Figure 6—figure supplement 2H). (G, G’) Still images from Video 3 showing ablated ISV ECs of a 4 dpf EC-EKC larva after vessel wounding. Activation of Erk progresses from the wound to the vessel base. Image (G) shows fli1aep:EKC expression, and (G’) shows nuclear fli1aep:EKC intensity. Arrows indicate first (white), second (yellow), third (green), forth (red), and fifth (orange) ECs from the wounded site. (H) Quantification of nuclear EKC intensity (normalised to nuclear EKC intensity at 2 mpa) in ECs of ISVs in non-ablated control larvae (black, 24 ECs, n = 8 larvae), and the first (red, 9 ECs, n = 9 larvae), second (blue, 9 ECs, n = 9 larvae), third (green, 9 ECs, n = 9 larvae), fourth (orange, 8 ECs, n = 8 larvae), and fifth (purple, 5 ECs, n = 5 larvae) ablated ISV ECs from the wounded site following vessel wounding. ISV: intersegmental vessel. Statistical test: Kruskal-Wallis test was conducted for graphs (C-F). Two-sample Kolmogorov-Smirnov test was conducted for graph (H). Error bars represent standard deviation. Scale bars: 50 μm for image (A), 15 μm for image (G).