Exogenous PIP2 restores angiogenesis in CDS2 morphants. (A) Simplified schematic diagram of phosphoinositide recycling and its relation to VEGF signal transduction and angiogenesis. VEGF/phospholipase C γ (PLCG) signaling converts phosphatidylinositol-4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol triphosphate (IP3). CDP-DAG synthetase (CDS) activity is required for resynthesis of CDP-DAG and PIP2. (B-F) Exogenous PIP2 rescues intersegmental vessel (ISV) growth in cds2-deficient zebrafish in vivo. Tg(fli-EGFP)^y1 zebrafish, injected with control (1.8 pg) cds2 (1.8 pg), low-dose plcg1 (3 pg), or low-dose VEGFaa (1.5 pg) morpholino at 1- to 2-cell stage, received an additional intermyotomal injection of liposome without (−) or with (+) PIP2 at 18 hours after fertilization (hpf), as noted. (B-E) Shown are representative confocal images of zebrafish trunk vasculature, which were obtained at 30 hpf. (F) Quantitation of the percentage of ISVs reaching dorsally to form the dorsal longitudinal anastomotic vessel (DLAV). Twenty embryos were counted for each group, with 11 trunk segments counted per embryo. (G) Exogenous PIP2 rescues ERK activation in cds2-deficient zebrafish in vivo. Trunk tissues were collected at 30 hpf and then assayed by immunoblotting with anti-ERK1/2 and anti–phospho-ERK1/2 antibodies. Normalization was performed as described in Figure 3D. Scale bars = 100 μm (B-E). Ctrl indicates control; MO, morpholino oligonucleotide; plcg1, phospholipase C γ1.