Gγ₂ modulated VEGF signaling pathway in vivo. (A-C) vegf₁₂₁ mRNA (20 ng/μL) overexpression can increase level of flk1 transcripts in the axial vasculature and the intersomitic vessels in zebrafish embryos (compare to WT control in Figures 3A,D). (D-F) vegf₁₂₁ mRNA (20 ng/μL) overexpression followed by targeted knockdown of G protein (gng2-MO, 100 μM) specifically inhibited sprouting of intersomitic vessels from the dorsal aorta (D-E), and strikingly, the yolk common cardinal vein was completely inhibited (F, arrow). (G-I) vegf₁₂₁ mRNA (50 ng/μL) overexpression can activate PLCγ₁ as detected by anti-phospho-PLCγ₁ antibody (H, arrow) compared with WT control (G). Knockdown of gng2 using gng2-MO splicing morpholino specifically inhibited vegf activation of PLCγ₁ (I, arrow). (J-L) vegf₁₂₁ mRNA (50 ng/μL) overexpression can also activate AKT as detected by anti-phospho-AKT antibody (K, arrow) compared with WT control (J). Knockdown of gng2 specifically inhibited vegf activation of AKT (L, arrow). vegf₁₂₁ mRNA and gng2-MO splicing morpholino (100 μM) were injected at about 1 nL volume at 1-cell stage embryo and followed by whole-mount immunohistochemical staining at 1 dpf. WT control (G) and (J), vegf mRNA overexpression (A-C,H,K), and coinjection of vegf₁₂₁ mRNA and gng2-MO (D-F,I,L). (M) Summary of results from 3 coinjection experiments. vegf₁₂₁ RNA (20 ng/μL) overexpression followed by morpholino knockdown of gng2 specifically inhibited sprouting of intersomitic vessels and common cardinal veins from the dorsal aorta. (N-O) Results from 3 injection experiments. Overexpression of vegf₁₂₁ mRNA (50 ng/μL) induced activation of PLCγ₁ (N) and AKT (O) in the axial vasculature. Knockdown of gng2 abolished the vegf-induced phosphorylation of both PLCγ₁ and AKT. Error bars are SEM (M-O). All scale bars are 100 μm (A-L).