Fig. 2

Active MAPK/ERK signaling regulates sprouting angiogenesis in zebrafish. (A) pERK staining (white) in embryos treated with vehicle (i.e. DMSO), MEK (i.e. SL327) or VEGFR2 (i.e. SU5416) inhibitors [treated from 18-20 hours post-fertilization (hpf) to ∼24 hpf]. See Fig. S2A for additional images and quantification. Yellow arrows indicate pERK-positive sprouting endothelial cells. (B) Inhibition of MEK activity by SL327 inhibits ISV sprout length. Inhibition of VEGFR2 signaling with SU5416 is included as a positive control. Quantification of ISV length at 28 hpf is shown. (C) dll4 expression in SL327-treated embryos at 28 hpf (treatment initiated at 18-20 hpf) as assessed by qRT-PCR (n=6 individual embryos). (D) Notch activity is reduced in the vasculature of SL327-treated Tg(kdrl:mCherry); Tg(Tp1bglob:Venus-PEST) embryos. Still images from time-lapse microscopy of a representative experiment are shown. Arrows indicate Notch signaling-positive ISVs, arrowheads indicate Notch signaling-positive endothelial cells in the dorsal aorta, asterisks indicate Notch-negative ISVs. See Fig. S3 (for additional still images) and Movies 1 and 2. (E) Similar experiment to that shown in D, but with Tg(kdrl:mCherry); Tg(Tp1bglob:EGFP) embryos. Scale bars: 50 μm (A,B,E); 20 μm (D).