Dll4^+/− mutant mice show defective increase in vascular proliferation. (A) The vasculature of wild-type and Dll4^+/− embryos were examined using PECAM whole-mount immunostaining. Dorsal aorta and cardinal vein are labeled as a and v, respectively. Absence of large vessels and an increase in vessel branching and density was seen in Dll4^+/− embryos at E10.5 compared to wild type. (B) Vascular response in Dll4^+/− adult mice was examined as in panel A after tumor implantation. Wild-type mice showed organized vascular proliferation in the tumor (left half), while mutant mice showed markedly increased vascular response that lacks organization and vascular hierarchy. (C) Expression of Dll4 in tumor and normal regions in Dll4^+/− mutant mice was examined by β-gal staining. Dll4 expression was observed (arrows) in a few discrete vessels in the normal tissue, while the tumor region showed many β-gal–positive vessels of similar appearance indicative of Dll4 induction in tumor vessels. (D) Pericyte coverage around newly forming vessels was examined by α-SMA localization. In wild-type mice, the vessels showed colocalization of PECAM and α-SMA (left panel). In Dll4^+/− mice tumor vessels, however, the number of α-SMA–positive cells lining the endothelial cells was profoundly reduced (right panel). Images in panel A were viewed under an Olympus SZX12 stereomicroscope (Tokyo, Japan) with Leica PL Fluotar 0, 5, 20×/0.5 NA dry objective (Wetzlar, Germany), captured with an Olympus C4040 camera, and processed with Olympus DP-Soft 3.2. Images in panels B-D were viewed under a Leica DMRA2 fluorescence microscope with Leica HC PL Fluotar 0, 5, 20×/0.5 NA dry objective, captured using Photometrics CoolSNAP HQ, (Photometrics, Friedland, Denmark), and processed with Metamorph 4.6-5 (Molecular Devices, Sunnyvale, CA).