The endothelial-specific KO of Dll1 resembles the hypomorphic arterial phenotype and can be rescued by constitutive overexpression of activated Notch. (A) Regimen of Tamoxifen or DAPT application. Tamoxifen or the γ-secretase inhibitor DAPT was injected into pregnant mice on 3 consecutive days (E11.5-E13.5). Embryos were analyzed at E15.5. (B) Microphotographs of vessels stained with antibodies. Antibody staining for Dll1 shows that is successfully removed from the endothelium in tamoxifen-treated Dll1^loxp; VE-Cadherin-Cre-ERT embryos (a,b). Notch activation is disturbed in these embryos (e, f) and the expression of the arterial markers Neuropilin1 (i,j), VEGFR2 (n,m) and EphrinB2 (q,r) is down-regulated. Concomitant endothelial-specific overexpression of constitutively active Notch (g) restored the expression of arterial markers (k,o,s). DLL1 expression is not affected by constitutive Notch activity (c). Administration of the γ-secretase inhibitor DAPT blocked Notch activation (h) and reduced arterial makers (l,p,t) similar to loss of DLL1-mediated Notch activation. DLL1 expression is not affected (d). Green indicates α-smooth muscle actin; red indicates antigen. a′ to t′ show magnifications of the boxed areas indicated in a through t. (C) Western blot analysis with protein lysates of the heart outflow tract confirmed the results obtained by immunohistochemistry. Actin was used as a loading control. (D) Increased capillary branching in the skin of E17.5 Dll1 hypomorphic embryos indicated by antibody staining for collagen typeIV (red) and endomucin (green). Data are mean values of 5 embryos per genotype. Bars indicate SD.