Fig. 4

Fig. 4

Dll4 Is Regulated by ETS Factors (A) Representative images (left) and quantification (right) of wild-type, ETS-B, and ETS-6x mutant F2:GFP embryos at 48 hpf. ISV, intersomitic vessel; DA, dorsal aorta; PCV, posterior cardinal vein. Asterisk indicates a significant difference in arterial expression compared to wild-type (χ² test). (B) Expression of endogenous DLL4 in HUVEC electroporated with ETV2 or ERG expression constructs (n = 3, ETV2; n = 5, ERG). (C) V5-ERG expression in electroporated HUVEC (left). ChIP in V5-ERG-electroporated HUVEC. Fold enrichment (V5 versus IgG) was measured at the Dll4 enhancer (intron 3) and Dll4 exon 11 (n = 2). (D) ChIP in BAEC for RNA Polymerase II (RNA Pol II), ERG or the Notch intracellular domain (NICD) (n = 3). Asterisk indicates significant enrichment over IgG control. (E) Expression of F2:GFP (arterial) and kdrl:GFP (pan-endothelial) in erg;fli1a morphants. Representative images (left) and quantification (n = 5–10) (right). (F) Endogenous dll4, ephrinb2a, hey2, and kdr in control and erg;fli1a morphants assessed by qPCR (n = 5–10 individual embryos). (G) Notch activity (Tp1:GFP) was diminished in the dorsal aorta of <30% of erg/fli1a morphants. (H) Levels of arterial markers and a pan-endothelial marker (Pecam1) were quantified by qRT-PCR in E8.5 embryos (n = 3 for wild-type, n = 2 for Erg^{ΔEx4/ ΔEx4} embryos). (I) Endogenous Dll4 mRNA is downregulated in the DA of Erg^{ΔEx4/ ΔEx4} embryos (arrows; n = 3 embryos per genotype) at E9.5. (J) Efnb2 section in situ hybridization at 9.5 shows downregulation of Efnb2 in the DA (arrows) of Erg^{ΔEx4/ ΔEx4} embryos (Erg^+/+, n = 3; Erg^{ΔEx4/ ΔEx4} n = 2). Scale bars represent 50 μm (A, E, and G), 500 μm (I), 100 μm (J). All graphical data are mean ± SEM. See also Figures S3 and S4.