FIG. 5.

Loss of Fused does not impact Hh signal transduction in vivo or in vitro. (A) Loss of Fused does not impact Hh target gene activation in vivo. Quantitative PCR from various p7 tissues is shown for the ptch1 gene. No effects were observed on ptch1 levels in any tissue. Similar results were observed for the gli1 gene. KO, knockout; dH₂O, distilled water. (B) Loss of Fused does not inhibit Hh target gene expression in the cerebellum, as measured by ³²P-labeled in situ hybridization to ptch1 and gli1. Cerebellum sections (magnification, ×10) from wild-type (WT) and fu^−/− neonates are shown stained with H&E with the comparable radiolabeled signal in the EGL for both ptch1 (left) and gli1 (right). (C) Loss of Fused does not impact the ability of cerebellar granule cells to respond to Shh. Cerebellar granule cells from wild-type, fu^+/−, and fu^−/− mice were treated in vitro for either 24 h (left image) or 48 h (right image) with octyl-modified Shh at 0, 5, 50, or 500 ng/ml. Cells were pulsed with [³H]thymidine 5 h prior to harvesting, and results are plotted as the percent response over unstimulated cells. (D) Loss of Fused does not alter ptch1 expression in vivo. High expression of ptch1 was observed in the brain (star), branchial arches (arrow), ventral CNS somites (arrowheads), and posterior limb buds of both Ptch^D11 fu^+/+ and Ptch^D11 fu^−/− mice. A cross section through the neural tube of these embryos shows comparable levels of ptch1 expression. (E) siRNA knockdown of mFu does not disrupt Shh-mediated signaling in C3H/10T1/2 S12 cells. Expression of mFu and mSmo was knocked down to approximately 25% and 12% of the normal levels, respectively (inset), as measured by quantitative PCR, and cells were treated with (black bars) and without (white bars) 200 ng/ml of octyl-modified Shh. No effect upon Shh-mediated activation of the 9x-Gli-BS-Luciferase reporter is observed when mFu is knocked down, while similar knockdown of mSmo totally abolishes Shh-mediated signaling. GFP, green fluorescent protein.