Defective vascular patterning in Ptch1 mutant embryos. (A) Lateral view of whole-mount PECAM1 staining of E8.5 (8-somite stage) and E9.5 Ptch1 mutant and littermate control embryos. Representative images from 8-somite stage Ptch1^+/+ (n = 3) and Ptch1^−/− (n = 3) embryos are shown. Arrowheads indicate cranial plexus region. Scale bar represents 500 μm. (B) Whole-mount in situ hybridization for Dll4 mRNA in E8.5 (8-somite stage) Ptch1 mutant embryos. Representative embryos from Ptch1^+/+ (n = 2) and Ptch1^−/− (n = 2) littermate embryos are shown. Arrowheads indicate the region in which Dll4 is ectopically expressed in Ptch1 mutants versus controls. Scale bar represents 500 μm. (C) Q-RT-PCR for Dll4 mRNA in Ptch1^+/+ (n = 2), Ptch1^+/− (n = 5), and Ptch1^−/− (n = 5) embryos. Q-RT-PCR was performed on 8-somite stage embryos from which the yolk sac, allantois, and amnion had been removed. Data are presented as Dll4 expression relative to the loading control, Polr2a. Single asterisk indicates statistical significance (t test) between Ptch1^+/+ and Ptch1^−/− sample groups (P < .05). Double asterisk indicates statistical significance (t test) between Ptch1^+/− and Ptch1^−/− sample groups, (P < .005). (D) FDR-OPT images of an E9.5 Ptch1^−/− embryo. Top left side is a surface-rendered image of the 3D reconstructed autofluoresence signal with locations of digital slices shown in right side panels indicated. Bottom left side is a 3D reconstruction of PECAM1-Cy3 signal alone. Right side images are digital slices from 3D reconstructed FDR-OPT data, top are PECAM1-Cy3 signal overlaid on autofluoresence signal, bottom are PECAM1-Cy3 signal from the same slice alone. Arrows indicate PECAM1⁺ structures surrounding other tissue (autofluoresence signal present), arrowheads indicate PECAM1⁺ signal encompassing hollow space (autofluoresence signal not present).