Ptch1 vascular phenotype is independent of VEGF and Notch. (A) Whole-mount in situ hybridization for Vegf mRNA in E8.5 Ptch1 mutants. Representative embryos from 8-somite stage Ptch1^+/+ (n = 4), Ptch1^+/− (n = 5), and Ptch1^−/− (n = 3) littermate embryos are shown. (B) Q-RT-PCR for Vegf mRNA in Ptch1^+/+ (n = 2), Ptch1^+/− (n = 5), and Ptch1^−/− (n = 5) embryos. Q-RT-PCR was performed on 8-somite stage littermates from which the yolk sac, allantois, and amnion had been removed. Data are presented as gene expression relative to the loading control, Polr2a. (C) Q-RT-PCR for Ptch1 and Gli1 mRNA in Flt1^+/− (n = 4) and Flt1^−/− (n = 4) embryos. Q-RT-PCR was performed on 7- to 8-somite stage littermates from which the yolk sac, allantois, and amnion had been removed. Data are presented as gene expression relative to the loading control, Polr2a. (D) Confocal images for whole-mount PECAM1-stained Ptch;Dll4 compound mutants. Representative embryos from 8-somite stage Ptch^+/−;Dll4^+/+ (n = 2), Ptch^+/+;Dll4^+/− (n = 2), Ptch^−/−;Dll4^+/+ (n = 2), Ptch^−/−;Dll4^+/− (n = 3) littermates are shown. Top panel for each embryo represents projected PECAM1 stain (red) with DRAQ5 nuclear stain (gray). Bottom panels represent single confocal section (xy plane) from each embryo with yz (left) and xz (top) stacks shown (PECAM1, red; DRAQ5 nuclear, gray). Arrows indicate the abnormal, hollow vascular structures in Ptch1^−/−;Dll4^+/+, and Ptch1^−/−;Dll4^+/− embryos. Scale bars represent 100 μm.