Perturbation of hedgehog signaling associated with developmental defects in the <i>Ift140</i>^{<i>null1/null1</i>} and <i>Ift140</i>^{<i>220/220</i>} mutant embryos.

(A) To assess hedgehog signaling in cultured MEFs, RNA was isolated from cells that were left untreated or treated with 400 nM SAG for 24 h. Gli1 and Gapdh gene expression was measured by quantitative real-time PCR. For Ift140220 cells, n = 1 control and 2 mutant lines analyzed 3 times. For Ift140null1 cells, n = 3 control and 3 mutant lines each analyzed 1 time. **p ≤ 0.01; ***p ≤ 0.001; ns, not significant, assessed by one-way ANOVA. (B) Immunostaining for differentiation markers in the neural tube of E10.5 embryos revealed disturbance in Shh regulated dorsoventral patterning of the neural tube in the Ift140220/220 mutants. Ventralization is indicated with dorsal shift in expression of ventral markers OLIG2, and NKX6.1, and dorsal retraction in expression of PAX6, a dorsal marker. Arrows denote the boundaries of antibody staining. (C, D) Sagittal and Frontal views of E14.5 wild-type and Ift140220/220 mutant embryos carrying a Gli-LacZ reporter, delineating regions of hedgehog signaling. (E) In situ hybridization of limb buds from E10.5 wild-type and Ift140220/220 mutant embryos showed perturbation of Shh signaling, with expanded expression of Gremlin indicating polydactyly. (F) Ift140220/220 (left), Ptch1LacZ/LacZ (middle), and Ift140220/220:Ptc1LacZ/LacZ (right) E10.5 embryos carrying the Ptc1-LacZ knockout allele were X-gal stained to delineate regions of hedgehog signaling. The severe phenotype of the Ptc1LacZ/LacZ mutant embryo is partially rescued in the double homozygous Ift140220/220:Ptc1LacZ/LacZ mutant embryo. Scales bars: B = 100 μm, C, D, F = 1 mm, E = 0.25 mm. The data underlying this figure can be found in supplemental file S1 Data. MEF, mouse embryonic fibroblast.