Fig. 6

Regulation of VEGF/ERK/ERG/p300-dependent genes in vivo. (A) In situ hybridization using probes for dll4, hlx1, dusp5 and flt4 at 26 hpf. Embryos were treated with inhibitors of MEK (SL327), p300/CBP (c646) or VEGF (SU5416) starting at the 20-somite stage. Expression of each of these genes is MEK, p300 and VEGF dependent. Arrows indicate ISVs expressing the indicated genes. Representative images are shown. (B) Quantification of in situ hybridization experiments. The number of embryos analyzed is indicated. (C) pERK western blot in embryos pre-treated with SL327 for 1 h, followed by addition of PMA for 2 h. Coomassie staining was used to assess loading. Representative experiment of two. (D) qRT-PCR analysis of endothelial or non-endothelial cells isolated from kdrl:GFP embryos exposed to PMA for 2 h (starting at 24 hpf). All of these genes are induced in the endothelium in response to ectopic MEK activation (n=3). (E) qRT-PCR of whole individual embryos that were exposed to DMSO or c646 for 1 h, prior to stimulation with PMA for 2 h at 24 hpf. The induction of dll4, hlx and egr3 by PMA is p300 dependent (n=6).