Fig. 6. Evc is required for Ihh signalling. (A) Tissue in situ hybridisation analysis from E16.5 embryos showing indistinguishable Ihh expression between wild type and mutant, but markedly decreased hybridisation signals of Ptch1 and Gli1 in Evc^-/- embryos. Bright-field (left) and dark-field (right) hybridisation pictures from proximal tibia are shown. (B) RT-PCR analysis of Ptch1 expression in wild-type and two different Evc^-/- MEF cultures grown with (+) or without (-) purmorphamine. Hprt expression is used as a control. (C,D) Ptch1and Gli1 quantitative RT-PCR analysis in chondrocytes cultured with (+) or without (-) purmorphamine. ß-actin was used as control for both quantitative PCR experiments. (E) Western blot showing unchanged levels of full-length Gli3 (FL, 190 kDa) and Gli3R (R, 83 kDa) in limb protein extracts of E14.5 wild-type and Evc^-/- mice. Ratios of the ß-catenin control band:Gli3R band were 1.47 and 1.41 for wild-type and Evc^-/-, respectively.