Fig. S6

Endothelial cell behaviors in flt1 loss-of-function and vegfaa gain-of-function scenarios

(a)Tg(kdrl:nlskikGR)^hsc7 embryo injected with 1ng flt1 morpholino. (left panel) Selected area in PCV (pink dotted box) was UV-photoswitched at 30hpf. (middle panel) Upon photo-conversion cells start to express the red reporter. PCV-derived cells migrate to the dorsal aspect of vISVs and contribute to venous ectopic sprouting upon loss of flt1 at 3dpf (red cell in middle panel (arrowhead) contributes to ectopic sprouting - yellow-dotted box). (right panel) Merged image. (b-e) Confocal images of primary arterial segmental vessel sprouting in WT (b), flt1^ka601 (c), vhl^hu2114 (d), and flt1^ka601;vhl^hu2114 double mutants (e). (f,g) Quantification of filopodia characteristics for indicated genotypes. Note that there are no differences in sprouting or filopodia between indicated mutants and WT, mean ± s.e.m, n=27 (f), mean ± s.e.m, n=9 (g), t-test. (h-k) Time-lapse imaging of arterial sprouting in the developing zebrafish trunk in Tg(kdrl:EGFP)^s843, Tg(Xla.Tubb:DsRed)^zf148 double transgenic at indicated time points. Note sprouts develop in proximity to neural tube (j,k). (l) Characterization of intersegmental blood flow characteristics in flt1^ka601 (l, top panel) and flt1^ka601 injected with flt4 targeting morpholino (l, bottom panel). In flt1^ka601 mutant ISVs carry both arterial (red arrow up) and venous (blue arrow down) flow and the artery/vein ratio is about 1. Upon loss of flt4, almost all investigated ISVs carry arterial flow consistent with flt4 blocking remodeling of arteries into veins. hpf, hours post fertilization. Scale bar, 25μm in a, h-k; 10μm in b-e.