Essential function of Gγ2 for angiogenesis in vivo. (A) and (D) flk1 expression in WT zebrafish embryos at 1 dpf, in axial vasculature and intersomitic vessels (IS) (D, arrow) by whole-mount in situ hybridization. (B-C) and (E-F) gng2-splicing knockdown (gng2-MO, 100μM) inhibited the formation of intersomitic vessel, a process of angiogenesis, (B) and (E) were complete loss of IS, (C) and (F) were partial loss of IS. (G) Summary of in situ analysis of flk-1 expression in the intersomitic vessel (IS) of embryos injected with gng2 splicing morpholino (11 experiments) and RNA rescue (4 experiments) experiments. (H-I) gng2 RNA, which is resistance to the splicing morpholino, can significantly rescue gng2 splicing knockdown. (J-K) Targeting both G protein (gng2-MO, 40μM) and vegf (vegf-MO, 25 μM) using antisense morpholinos dramatically increased the efficacy of antiangiogenesis as visualized by loss of sprouting of intersomitic vessels from the dorsal aorta at 1-dpf zebrafish embryos (K). vegf knockdown alone (vegf-MO, 25μM) at the sub-effective dose showed no effect (J). Inserts in (J-K) are higher magnification of IS (J-K, arrows). (L) Summary of embryos coinjected with gng2-MO and vegf-MO significantly inhibited flk-1 expression in the IS. Results from 4 injection experiments. Error bars are SEM (G,L). Injection volume was about 1 nL. All scale bars are 100 μm (A-F, H-K).
