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. 2021 May 18;10:e62196. doi: 10.7554/eLife.62196

Figure 4. Wounded but not adjacent vessels maintain high Erk activity as the regenerative response proceeds.

(A–B’) Lateral spinning disc confocal images of ablated (A) and adjacent ISVs (B) of a 4 days post-fertilisation (dpf) EC-EKC larva before and following vessel wounding at indicated timepoints. Erk activity is progressively lost in the adjacent but retained in the wounded ISV endothelial cells (ECs). Images (A) and (B) show fli1aep:EKC expression, and images (A’) and (B’) show nuclear fli1aep:EKC intensity. White dotted lines show the wounded site. (C,D) Quantification of post-/pre-ablation nuclear EKC intensity of ECs in non-ablated control ISVs (black, 24 ECs, n = 8 larvae), ablated ISVs (red, 30 ECs, n = 10 larvae), and adjacent ISVs (light blue, 30 ECs, n = 10 larvae) before and after vessel wounding at indicated timepoints. Graph (C) shows the quantification of individual ECs and graph (D) shows the average of all biological replicates. At 1 hour post-ablation (hpa): control vs ablated ISV ECs: p>0.001; control vs adjacent ISV ECs: p=0.108 (Kruskal-Wallis test). ISV: intersegmental vessel. Statistical test: permutation test was conducted for graph (C). Error bars represent standard deviation. Scale bar: 20 μm.

Figure 4—source data 1. Post-/pre-ablation nuclear EKC measurements in control, ablated, and adjacent ISV ECs from pre-ablation to 3 hpa.

Figure 4.

Figure 4—figure supplement 1. Distinct Erk activity between ablated and adjacent ISV ECs 3 hpa.

Figure 4—figure supplement 1.

(A–I’) Lateral spinning disc confocal images of ISV endothelial cells (ECs) in 4 days post-fertilisation (dpf) EC-EKC larvae at indicated timepoints. Images (A-I) show fli1aep:EKC expression, while images (A’-I’) show the nuclear fli1aep:EKC intensity. (J–M’) Erk-signalling is activated in ablated, but not adjacent ISV endothelial cells (ECs) at 3 hours post-ablation (hpa). Lateral spinning disc confocal images of ablated and adjacent ISV ECs in 4 dpf EC-EKC larvae before (J–K’) and 3 hr following vessel wounding (L–M’). Images (J-M) show fli1aep:EKC expression, while images (J’-M’) show the nuclear fli1aep:EKC intensity. Images (K) and (M) are higher magnification images of the yellow boxes in images (J) and (L). White circle in image (L) shows the wounded site. Arrows indicate the first (white), second (yellow), third (green), fourth (red), and fifth (orange) ECs from the wounded site. (N) Quantification of post-/pre-ablation nuclear EKC intensity of ECs in non-ablated control ISVs (27 ECs, n = 9 larvae), ablated ISVs (42 ECs, n = 14 larvae), and adjacent ISVs (42 ECs, n = 14 larvae) 3 hpa. ISV: intersegmental vessel. Statistical test: Kruskal-Wallis test was conducted for graph (N). Error bars represent standard deviation. Scale bars: 20 μm for image (A), 20 μm for images (J) and (K).
Figure 4—figure supplement 1—source data 1. Post-/pre-ablation nuclear EKC measurements in control, ablated, and adjacent ISV ECs at 3 hpa.
Figure 4—figure supplement 1—source data 2. Post-/pre-ablation nuclear EKC measurements in control and control ablated ISV ECs at 15 mpa and 3 hpa.
Figure 4—figure supplement 2. Vessel wounding is required for sustained Erk activity in ablated ISV ECs.

Figure 4—figure supplement 2.

(A,B) Vessels that are not wounded do not sustain Erk activity. Lateral spinning disc confocal images of ISV endothelial cells (ECs) in 4 days post-fertilisation (dpf) EC-EKC larvae at 0 min/pre-ablation (left), 15 min/15 min post-ablation (mpa) (middle), or 3 hr/3 hours post-ablation (hpa) (right). Image (A) shows an ISV in a non-ablated control larva and image (B) shows an ISV in a larva with tissue ablated in between two ISVs (control ablation). Images (A) and (B) show fli1aep:EKC expression, while images (A’) and (B’) show the nuclear fli1aep:EKC intensity. White dotted lines show the wounded site. (C) Quantification of post-/pre-ablation nuclear EKC intensity of ECs in either non-ablated control ISVs or control ablation ISVs at 15 mpa (control, 39 ECs, n = 13 larvae; control ablation, 48 ECs, n = 16 larvae) or 3 hpa (control, 18 ECs, n = 6 larvae; control ablation, 24 ECs, n = 8 larvae). ISV: intersegmental vessel. Statistical test: Kruskal-Wallis test was conducted for graph (C). Error bars represent standard deviation. Scale bar: 15 μm.