Figure 3.3

(A–C) Lateral spinning disc confocal images of 4 days post-fertilisation (dpf) Tg(kdrl:EGFP);Tg(mpeg1:mCherry) larvae pre-ablation (A), 15 min post-ablation (mpa) (B), or 3 hours post-ablation (hpa) (C). Macrophages are recruited to the wounded site by 3 hpa but not by 15 mpa. (D) Quantification of macrophage number recruited to the wounded site pre-ablation (n = 25 embryos), 15 mpa (n = 14 embryos), or 3 hpa (n = 24 embryos). (E, F) Lateral spinning disc confocal images of 3 dpf Tg(mpeg1:mCherry) uninjected control (E) or spi1/csf3r morphants (F). (G) Quantification of macrophage number within the trunk spanning three somites length in 3 dpf Tg(mpeg1:mCherry) uninjected control (n = 29 embryos) or spi1/csf3r morphants (n = 25 embryos). (H, I) Macrophages are required for vessel regeneration. Lateral confocal images of 24 hpa, 4 dpf, Tg(fli1aep:EKC) uninjected control (H) or spi1/csf3r morphants (I). (J) Quantification of ISV horizontal length (as percentage of control) for ablated ISVs in 24 hpa, 4 dpf, EC-EKC uninjected control (n = 11 larvae) or spi1/csf3r morphants (n = 13 larvae). (K–V’) Lateral spinning disc confocal images of ISV endothelial cells (ECs) in 3 dpf EC-EKC uninjected control (K–L’, O–P’, S–T’) and spi/csf3r morphants (M–N’, Q–R’, U–V’). Erk-signalling is rapidly activated in both ablated and adjacent ISV ECs in larvae with a reduced macrophage number. Images (K-N’) show non-ablated control ISV ECs, images (O-R’) show ablated ISV ECs, and images (S-V’) show adjacent ISV ECs. Images (O, Q, S, U) were taken pre-ablation and images (P, R, T, V) were taken 15 mpa. Images (K-V) show the fli1aep:EKC expression and images (K’-V’) show the nuclear fli1aep:EKC intensity. ISV: intersegmental vessel. Statistical test: Kruskal-Wallis test was conducted for graph (D) and Mann-Whitney test was conducted for graphs (G and J). Error bars represent standard deviation. White dotted lines/circles show the wounded sites of each embryo/larva. Scale bars: 20 μm for image (A), 50 μm for images (E) and (H), and 15 μm for image (K).