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Heterogeneous endothelial requirements for Vegfs-dependent angiogenesis in the ventral brain around the neurohypophysis (NH)/organum vasculosum of the lamina terminalis (OVLT). (A–H) Dorsal views of 120 hours post fertilization (hpf) wild-type (WT) (A), vegfab-/- (B), vegfd-/- (C), vegfc-/- (D), vegfab-/-;vegfd-/- (E), vegfc-/-;vegfd-/- (F), vegfab-/-;vegfc-/- (G), and vegfab-/-;vegfc-/-;vegfd-/- (H) ventral brain vasculature visualized by Tg(kdrl:EGFP) expression. A significant fraction of vegfab-/-;vegfc-/- (G) and vegfab-/-;vegfc-/-;vegfd-/- (H) larvae exhibited a partial formation of the hypophyseal artery (HyA), resulting in HyA stalling toward the palatocerebral arteries (PLA) (arrows, G, H). (I) Quantification of HyA, Hy loop, and hypophyseal vein (HyV) formation at 120 hpf (the number of animals examined per genotype is listed in the panel). vegfab-/-;vegfc-/- and vegfab-/-;vegfc-/-;vegfd-/- larvae displayed a specific and partially penetrant defect in HyA formation. (J–O) Dorsal views of 72 hpf WT (J), vegfc-/- (K), vegfab-/- (L), vegfaa-/- (M), vegfc-/-;vegfaa-/- (N), and vegfab-/-;vegfaa-/- (O) ventral brain vasculature visualized by Tg(kdrl:EGFP) expression. Similar to vegfab-/-;vegfc-/- larvae, vegfaa-/- fish exhibited a specific and partially penetrant defect in HyA formation, leading to HyA stalling toward the PLA (arrow, M). The severity of this phenotype was exacerbated by the simultaneous deletion of vegfab (O), but not of vegfc (arrow, N), showing a genetic interaction between vegfaa and vegfab, but not between vegfaa and vegfc. Intriguingly, phenotypes in vegfab-/-;vegfaa-/- larvae were restricted to the fenestrated HyA and Hy loop (asterisk, O) with no significant defect in HyV formation. (P) Schematic representations of the severe vascular phenotypes observed in 72 hpf various vegf mutants. (Q) Quantification of HyA, Hy loop, and HyV formation at 72 hpf. In panels (I and Q), each data point shown in magenta represents individual animal’s vessel formation score, and values represent means ± SD (*** and **** indicate p<0.001 and p<0.0001, respectively, by one-way analysis of variance [ANOVA] followed by Tukey’s HSD test). Scale bars: 50 µm in (H) for (A–H) and in (O) for (J–O).
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