Figure 5.

Endothelial cell-autonomous and cell non-autonomous requirements of Vegfc for vascularization of the diencephalic choroid plexus (dCP)/pineal gland (PG) interface.

(A–E) Dorsal views of 72 hours post fertilization (hpf) wild-type (WT) (A), vegfc^um18/um18 (B), vegfab^-/- (C), and vegfab^-/-;vegfc^um18/um18 (D, E) cranial vasculature visualized by Tg(kdrl:EGFP) expression. Yellow arrows point to the prosencephalic artery (PrA), blue arrows to the PG vessel (PGV), and orange arrows to the anterior cerebral vein (ACeV). Although none of vegfc^um18/um18 and vegfab^-/- fish exhibited a defect in PrA formation (B, C), approximately 21% of vegfab^-/-;vegfc^um18/um18 larvae lacked the PrA at either or both sides (E). (F) Quantification of PrA, PGV, and ACeV formation at 72 hpf (the number of animals examined per genotype is listed in the panel). Specific defect was observed in PrA formation in vegfab^-/-;vegfc^um18/um18 larvae compared to other three genotypes. (G, H) Dorsal views of 72 hpf vegfaa^-/- (G) and vegfaa^-/-;vegfc^um18/um18 (H) cranial vasculature visualized by Tg(kdrl:EGFP) expression. Yellow arrows point to the PrA, blue arrows to the PGV, and orange arrows to the ACeV. Although vegfaa^-/- or vegfc^um18/um18 larvae fully formed vasculature at the dCP/PG interface, most of vegfaa^-/-;vegfc^um18/um18 larvae failed to form the PrA and PGV at either or both sides. (I) Quantification of PrA formation at 72 hpf (the number of animals examined per genotype is listed in the panel). The quantitative results of several genotypes were presented again or integrated in this graph for comparison purposes. Previously presented results are the vegfc^-/- (hu6410 allele) data from Figure 4R, and the data in (F) were either re-presented or combined with the quantitative results shown in Figure 5G and H. Paracrine activity-deficient vegfc^um18/um18 larvae in the vegfab^-/- background displayed a significantly milder defect in PrA formation than that observed in vegfab^-/-;vegfc^-/- (hu6410 allele) animals that lack both endothelial cell-autonomous and cell non-autonomous Vegfc function. (J) Quantification of mesencephalic vein (MsV) formation at 72 hpf (the number of animals examined per genotype is listed in the panel). Severe defects in MsV formation in vegfaa^-/- larvae were further exacerbated by genetic deletions of vegfc (um18 allele) or vegfab. (K) Schematic representations of the severe vascular phenotypes observed in 72 hpf various vegf mutants at the dCP/PG interface. Genetic results indicate highly heterogeneous molecular requirements for angiogenesis around the dCP/PG interface. In panels (F), (I), and (J), each data point shown in magenta represents individual animal’s vessel formation score, and values represent means ± SD (*, **, and **** indicate p<0.05, p<0.01, and p<0.0001, respectively, by one-way analysis of variance [ANOVA] followed by Tukey’s HSD test). Scale bars: 50 µm in (A) for (A–E) and in (G) for (G–H).