Fig. S6

Ectopic ISV sprouting and VTA formation defects in larvae lacking flt1, vegfab, and/or radial glia. (A–D) 7 dpf WT (A), vegfab^bns92/bns92 (B), flt1^bns29/bns29 (C), and vegfab^bns92/bns92;flt1^bns29/bns29 (D) larval trunk vasculature visualized by TgBAC(etv2:EGFP) expression. VTAs are indicated by white arrows, ectopic vascular sprouts by yellow arrows. (E) Quantification of average number of somites that showed ectopic blood vessels in WT, vegfab^bns92/bns92, flt1^bns29/bns29, and vegfab^bns92/bns92;flt1^bns29/bns29 larvae (10 somites examined per animal; 10 animals examined per genotype). Values represent means ± SEM (*** indicates P < 0.001 by ANOVA followed by Tukey's HSD test). (F) Percentage of larvae without VTAs, as judged by TgBAC(etv2:EGFP) expression. All of the vegfab^−/− and vegfab^−/−;flt1^−/− larvae lack VTAs. (G–J) 7 dpf Tg(gfap:NTR);Tg(kdrl:EGFP) animals that were treated with DMSO or Mtz starting at different developmental stages. Fish were treated with DMSO from 30 hpf to 7 dpf (G) or Mtz from 30 hpf to 7 dpf (H), 54 hpf to 7 dpf (I), or 62 hpf to 7 dpf (J). VTAs were completely absent in radial glia-ablated animals treated with Mtz from 54 hpf to 7 dpf (I) and 62 hpf to 7 dpf (J). VTAs were not correctly formed in radial glia-ablated animals treated with Mtz from 30 hpf to 7 dpf (H), yet vessels (white arrows) were occasionally observed near where VTAs usually form, in addition to ectopically sprouting vessels (yellow arrows) between the dorsal ISVs. (K and L) 7 dpf Tg(gfap:NTR);Tg(kdrl:EGFP) trunk vasculature after treatment with DMSO from 30 hpf to 7 dpf (K) or Mtz from 30 to 78 hpf followed by treatment with freshly prepared Mtz from 78 hpf to 7 dpf (L). Under these Mtz treatment conditions, a more complete ablation of radial glia was achieved (L) compared with the Mtz treatment shown in H. Although ectopic ISV sprouting was observed (yellow arrows, L), VTAs were absent under these Mtz treatment conditions (white arrows, L). (M) 7 dpf Tg(gfap:NTR);Tg(kdrl:EGFP) vegfab^−/− trunk vasculature after treatment with Mtz from 30 hpf to 7 dpf. Although ectopic ISV sprouting was observed (yellow arrows), VTAs were absent in the radial glia-ablated vegfab^−/− animal (M). This phenotype is similar to that observed in vegfab^bns92/bns92;flt1^bns29/bns29 larvae (D). (N–P) 7 dpf Tg(elavl3:NTR);Tg(kdrl:EGFP) animals that were treated with DMSO or Mtz starting at different developmental stages. Fish were treated with DMSO from 30 hpf to 7 dpf (N), or Mtz from 54 hpf to 7 dpf (O), or 62 hpf and 7 dpf (P). Unlike radial glia-ablated animals, Mtz-induced neuronal ablation starting at different developmental stages did not lead to a defect in trunk vascular patterning. (Scale bar, 100 µm.)