Vegfab/Vegfr2 signaling is required for VTA formation. (A–D) 152 hpf Tg(kdrl:ras-mcherry) trunk vasculature after treatment with DMSO (A), Sunitinib (B), SKLB1002 (C), or LY294002 (D) starting at 80 hpf. Sunitinib, SKLB1002, and LY294002 treatments significantly inhibit VTA formation (yellow arrows). (E–H) 178 hpf Tg(hsp70l:sflt1) (E and F), control (G), and Tg(hsp70l:sflt4) (H) trunk ECs visualized by Tg(kdrl:nls-EGFP) expression. Larvae were subjected to no heat shock (HS) (E) or multiple heat shocks (F–H). High-magnification images of ECs in the position of the VTAs are shown in E′–H′. Overexpression of sFlt-1 leads to a significantly reduced number of ECs in the position of the VTAs (yellow arrows, F). (I) Quantification of the average number of somites with VTAs for the experiments A–D (20 somites examined per animal). (J) Quantification of nls-EGFP+ EC number in the position of the VTAs within 10 somites per animal for the experiments E–H. (K and L) 8 dpf TgBAC(etv2:EGFP) vegfab+/+ (K) and vegfab−/− (L) trunk vasculature. All vegfab−/− larvae examined (n = 20) lacked VTAs (yellow arrows). (M) Percentage of larvae without VTAs, as judged by TgBAC(etv2:EGFP) expression. Values represent means ± SEM (I and J), **P < 0.01 and ***P < 0.001. (Scale bar, 100 µm.)
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