Fig. 5
Vegf Regulates the Dll4 Enhancer through an ETS Element (A) Expression of F2:GFP (arterial) and kdrl:GFP (pan-endothelial) in control or vegfa morphant embryos. Lateral dorsal aorta (arrows); dorsal aorta (DA). Scale bar represents 50 μm. (B) Quantification of GFP intensity in the DA (n e 10 per group). (C) Expression (qRT-PCR) of dll4 (arterial), kdr, and kdrl (pan-endothelial) in control and vegfa morphant embryos (n e 3 per group). (D) Overexpression of Vegfa enhanced expression of F2:GFP in arterial cells and expanded expression to venous cells. CA, caudal artery; CVP, caudal vein plexus. Quantification is shown (n = 6–8 embryos per group). Scale bar represents 50 μm. (E) Expression of DLL4 (qRT-PCR) in BAECs treated with VEGF or VEGF receptor inhibitor (SU5416) for 24 hr (n = 3). (F) Relative F2:luciferase activity in BAEC treated with VEGF or SU5416. A representative experiment with three technical replicates is shown. Asterisk indicates a significant difference in luciferase activity compared to all other constructs. Number sign (#) indicates a significant difference between RBPJk MT and ETS-B MT, 6x-ETS MT and RBPJk/ETS-B MT. (G) ChIP assays for RNA Pol II and ERG in BAEC treated with VEGF or SU5416 for 24 hr (n = 3). (H) Western blotting demonstrating equal expression of ERG protein in BAECs treated with VEGF or SU5416 for 24 hr. (I) Intracellular localization of ERG was unchanged by VEGF treatment. Scale bar represents 20 μm. (J) Expression of ERG was elevated in arterial cells (HUAEC) compared to venous cells (HUVEC). All graphical data are mean ± SEM. See also Figure S5. |
Reprinted from Developmental Cell, 26(1), Wythe, J.D., Dang, L.T., Devine, W.P., Boudreau, E., Artap, S.T., He, D., Schachterle, W., Stainier, D.Y., Oettgen, P., Black, B.L., Bruneau, B.G., and Fish, J.E., ETS Factors Regulate Vegf-Dependent Arterial Specification, 45-58, Copyright (2013) with permission from Elsevier. Full text @ Dev. Cell