Figure 4. Loss of Shh signaling is profound in CGNPs.

(A) X-gal staining for β-galactosidase in Gli1^lacZ and TNC^YFP-CreER;Smo^F/−;Gli1^lacZ mutants shows that X-gal is reduced in the PCL and EGL of mutants at P3 and P4. Rectangular region in P4 panel shows enlarged area in adjacent panel. (B) β-galactosidase immunohistochemistry in WT and mutants reveals a decrease in antibody staining in the mutant EGL at P4. GFP and Calbindin staining demonstrates location of BGs and PCs, respectively. Arrowheads indicate presence of β-gal+ cells in WT EGL, note that β-gal expression is absent in the mutant EGL. Arrows indicate GFP+ BGs that no longer express β-gal+, indicating a reduction in Gli1 expression in the mutant. (C) RT-PCR of Smo and Gli1 from purified TNC-YFP population shows reduced Shh signaling in Smo^cko mutants. (D) Western blotting of P5 WT and mutant CGNPs shows a decrease in Gli1 and Gli2 protein levels in the mutant. (E) Western blotting of P5 cerebellar lysates shows a reduction in Gli1 protein levels in the mutant. Abbreviations: EGL, external granular layer. PCL. Purkinje cell layer. CGNPs, cerebellar granular neuron precursors. CB, cerebellum. Scale bar: 100 µm and 20 µm.