PKCζ is required for developmental angiogenesis in zebrafish and for endothelial cell sprouting. (A) Images from whole mount Tg(fli1a:EGFP) fixed embryos at 30 hpf. Uninjected embryo (CT; left) or injected with a morpholino targeting prkcz (PKCζ-MO1; right) are presented. (B) Confocal images of the vasculature in the trunk of Tg(fli1a:EGFP) embryos. The uninjected control embryos (CT) showed complete ISVs sprouting from the dorsal aorta (DA) to DLAV at 30 hpf, whereas in PKCζ-MO1–injected embryos, ISV sprouting was disturbed mostly at the horizontal myoseptum with increased filopodia extensions from tip cells. (C) Percentage of defective ISVs in control and PKCζ-MO1 or PKCζ-MO2–injected embryos. Data are represented as mean ± SEM (*P < .05 compared with CT embryos). (D) Representative images from time-lapse in vivo imaging of Tg(fli1a:EGFP) embryos showing ISV sprouts from 18 to 28 hpf in CT (top) and PKCζ-MO (bottom)–injected embryos. The arrow indicates, in the PKCζ-ΜΟ–injected embryo, the detachment of tip cells from the DA. (E) Representative images from spheroid based angiogenesis assay generated from CT-, PKCζ-, or β-catenin-siRNA–transfected cells and stimulated with Ang-1, VEGF, or left untreated (CT). Pictures are taken 24 hours after embedding in collagen gel. Higher magnification views of the boxed region are shown. The arrow indicates, in the PKCζ or β-catenin-siRNAs, the detachment of tip cells from the sprouts. The arrowhead indicates bifurcation at end of the sprouts. One representative experiment of 3 performed is shown. (F) Quantification of capillary-like sprouting from spheroids was measured in every condition as displayed. Data are shown as mean of sprout length (*P < .05 vs nonstimulated control condition; †P < .05 compared with stimulated CT-siRNA). (G) Number of sprouts per spheroid. Results are displayed as mean values ± SEM of sprout lengths observed in at least 10 spheroids per experiment in 3 independent spheroid assays. Scale bar represents 100 μm.