Figure 8.

Figure 8. Temporal inhibition of Vegfa signaling by sFlt1 overexpression is sufficient to cause impaired formation of the hypophyseal artery (HyA) and Hy loop.

(A–C) Dorsal views of 72 hours post fertilization (hpf) wild-type (WT) (A), vegfc^um18/um18 (B), and vegfc^um18/um18;vegfab^-/- (C) ventral brain vasculature visualized by Tg(kdrl:EGFP) expression. vegfc^um18/um18;vegfab^-/- larvae exhibited partial formation of the HyA, resulting in HyA stalling toward the palatocerebral arteries (PLA) (arrow, C). (D) Quantification of HyA formation at 120 hpf (the number of animals examined per genotype is listed in the panel). vegfc^um18/um18;vegfab^-/- larvae exhibited a significantly increased defect in HyA formation at a comparable level to that observed in vegfab^-/-;vegfc^-/- (hu6410 allele) animals. The HyA quantitative results of vegfab^-/-;vegfc^-/- (hu6410 allele) larvae from Figure 7I were integrated in this graph for comparison purposes. (E) Time course of the heatshock (HS) experiments for panels (F–I). (F–I) Dorsal views of ventral brain vasculature in 144 hpf Tg(hsp70l:sflt1) (F, H), Tg(hsp70l:sflt4) (I), and their control sibling (G) larvae that carried both the Tg(kdrl:EGFP) and Tg(kdrl:NLS-mCherry) transgenes after treatment with (G–I) and without (F) multiple HS. HS-induced overexpression of sFlt1 caused severe defects in HyA formation, leading to HyA stalling toward the PLA (arrow, H). (J, K) Quantification of the number of vascular endothelial cells (vECs) that comprise the HyA, Hy loop, and hypophyseal vein (HyV) at 72 (J) and 144 (K) hpf. HS-induced overexpression of sFlt1 led to a drastic reduction in the number of vECs that comprise the HyA and milder reduction of vEC numbers in Hy loop. In contrast, sFlt4 overexpression displayed no effect on the number of vECs that comprise the Hy loop and HyV, but caused a significant reduction in HyA vEC numbers at 144 hpf (K). The number of animals examined per treatment at 72 hpf: without HS treatment, n=11 for control, n=11 for sflt1, and n=10 for sflt4; with HS treatment, n=20 for control, n=25 for sflt1, and n=20 for sflt4. The number of animals examined at 144 hpf: without HS treatment, n=25 for control, n=11 for sflt1, and n=18 for sflt4; with HS treatment, n=15 for control, n=22 for sflt1, and n=21 for sflt4. In panels (D), (J), and (K), each data point shown in magenta represents individual animal’s quantification, and values represent means ± SD (*, **, ***, and **** indicate p<0.05, p<0.01, p<0.001, and p<0.0001, respectively, by one-way analysis of variance [ANOVA] followed by Tukey’s HSD test). Scale bar: 50 µm in (C) for (A–C) and in (I) for (F–I).