Fig. S6

Figure S6 (Related to Figure 6 and Figure 7): The F2 enhancer and endogenous Dll4 expression are regulated by Vegf/MAP kinase signaling. (A) From the experiment shown in Figure 6C,D, induction of Vegfa-inducible efnb2 was also inhibited in embryos injected with erg/fli1a morpholinos. Shown is qRT-PCR from 5 individual embryos per group (mean ± SEM). * indicates a significant difference by 1-way ANOVA. ns, not significant. (B) F2-luciferase activity was enhanced upon inhibition of PI3 kinase signaling (LY29004 treatment) in BAEC. The activity of F2-luciferase was decreased, and the responsiveness to PI3K inhibition was lost, when the ETS-B site was mutated. Shown is a representative experiment, triplicate determinations (mean ± SEM). (C) F2:GFP or kdrl:GFP embyros were treated the MAP kinase inhibitor, U0126, starting at the 10-somite stage, and imaging was performed at 26 or 28 hpf. F2:GFP expression was decreased in the presence of U0126, but kdrl:GFP was unaffected. (D) Quantification of (C) is shown (n=4 per group; mean ± SEM). (E) Expression of endogenous dll4, efnb2a, hey2 and kdr was assessed by qRT-PCR (n=5-10 individual embryos; mean ± SEM). (F) Embryos were injected with a construct that drives expression of constitutively active MEKK in the endothelium (Fli1-mCherry-ActMEKK). In embryos with expression of the construct in the vein, F2:GFP expression was also observed in venous cells (arrows).