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Figure 3 Wounded vessels rapidly activate Erk independent of macrophages or Vegfr-signalling. (A,B) Lateral confocal images of a 4 days post-fertilisation (dpf) Tg(kdrl:EGFP) larva following vessel wounding (post-ablation). Image (A) shows the kdrl:EGFP expression and image (B) shows the trans-light image of image (A). Ablated ISV, adjacent ISVs, and the wounded site are indicated with white arrows. (C) Schematic representation of imaging schedule for larvae in images (D-G) and Videos 3–5. (D–G’) Still images from Video 4 (D–E’) and Video 5 (F–G’) showing ISV endothelial cells (ECs) before (pre-ablation) and after vessel wounding. Ablated and adjacent ISV ECs rapidly activate Erk-signalling. (D-G) fli1aep:EKC expression; (D’-G’) nuclear intensity. (H,I) Quantification of post-/pre-ablation nuclear EKC intensity of ECs in non-ablated control ISVs (black, 24 ECs, n = 8 larvae), ablated ISVs (red, 27 ECs, n = 9 larvae), and adjacent ISVs (light blue, 27 ECs, n = 9 larvae). (H) shows quantification of individual ECs and (I) shows the average of all biological replicates. Green dotted line indicates 15 min post-ablation (mpa). (J) Quantification of post-/pre-ablation nuclear EKC intensity 15 mpa in ECs of non-ablated control ISVs (103 ECs, n = 34 larvae), ablated venous ISVs (75 ECs, n = 25 larvae), and ablated arterial ISVs (57 ECs, n = 19 larvae). Both venous and arterial ISV ECs activate Erk-signalling. (K) Quantification of post-/pre-ablation nuclear EKC intensity 15 mpa in ECs of non-ablated uninjected control ISVs (45 ECs, n = 15 larvae), non-ablated spi1/csf3r morphant ISVs (42 ECs, n = 14 larvae), uninjected control ISVs (45 ablated/adjacent ISV ECs, n = 15 larvae), and spi1/csf3r morphant ISVs (56 ablated ISV ECs and 57 adjacent ISV ECs, n = 19 larvae). Macrophages are not required to rapidly activate Erk-signalling in ablated or adjacent ISV ECs. (L) Quantification of post-/pre-ablation nuclear EKC intensity 15 mpa in ECs of 0.5% dimethyl sulfoxide (DMSO)-treated non-ablated control ISVs (33 ECs, n = 11 larvae) and ISVs of larvae treated with either 0.5% DMSO (42 ablated/adjacent ISV ECs, n = 14 larvae), 15 μM SL327 (39 ablated/adjacent ISV ECs, n = 13 larvae), 4 μM SU5416 (36 ablated/adjacent ISV ECs, n = 12 larvae), 10 μM SU5416 (42 ablated/adjacent ISV ECs, n = 14 larvae), or 500 nM AV951 (42 ablated/adjacent ISV ECs, n = 14 larvae). Vegfr-signalling is not required to rapidly activate Erk-signalling in ablated or adjacent ISV ECs. ISV: intersegmental vessel. Statistics: permutation test was conducted for graph (H). Kruskal-Wallis test was conducted for graphs (J-L). Error bars represent standard deviation. White dotted lines/circle shows the wounded sites of each larva. Scale bar: 100 μm for image (A), 20 μm for image (D).