Figure 5. E177 is required for Shh signaling in vivo.

A. Shh^E177A/− embryos display gross morphological defects compared to Shh^+/+ at E12.5. Schematic showing homologous recombination in embryonic stem cells. The mouse bacterial artificial chromosome 429m20 contains a 16 kb fragment spanning the Shh genomic region. A glutamate to alanine point mutation at amino acid 177 was introduced in exon 2, and a conditional triple polyadenylation signal was inserted in the 5′ UTR. Schematic representation shows homologous recombination between the BAC targeting vector and the mouse Shh (mShh) genomic locus to generate the floxed transcription stop E177A targeted allele (Shh^TS-E177A). Shh^TS-E177A/+ mice were bred with EllaCre mice that express Cre recombinase under the control of the adenovirus Ella promoter to excise the floxed transcription stop. The resulting allele contains only one loxP site and the E177A mutation (Shh^E177A). Black rectangles represent exons, lines represent introns. Correct targeting in ES cells is shown by Southern blotting. ApaLI and HindIII restriction sites are indicated. Stars represent the location of probes. B. RNA isolated from Shh^+/−, Shh^E177A/+ and Shh^E177A/− embryos is reverse transcribed and cDNA is amplified by PCR. The E177A point mutation results in the generation of an MluI restriction enzyme site. MluI digest of an intron-spanning PCR on cDNA shows that the E177A mutation is expressed and the transcript is correctly spliced. Sequencing of cDNAs isolated from E9.5 Shh^+/+ and Shh^E177A/− embryos confirms introduction of E177A, and Mlu1 restriction site. C. X-galstaining for Ptc-lacZ expression in whole E9.5 embryos shows Ptc1 target gene expression is lost in Shh^E177A/−Ptc^lacZ/+ and Shh^−/−Ptc^lacZ/+ embryos. D–L. RNA in situ hybridization analysis using Ptc1 (D–F), Gli1 (G–I) and Shh (J–L) probes on E9.5 lumbar spinal cord sections in Shh^+/+ (D,G,J), Shh^E177A/− (E,H,K), and Shh^−/− (F,I,L). M–a. Immunofluorescence staining of spinal cord progenitor markers HNF3β (M–O), Nkx2.2 (P–R), Nkx6.1 (S–U), Pax6 (V–X) and Pax7 (Y–a) on Shh^+/−, Shh^E177A/−, and Shh^−/− E9.5 lumbar spinal cord sections, as indicated. (b–d) Nkx2.1 RNA situ hybridization of E12.5 forebrain sections from Shh^+/+ (b), and anterior sections from Shh^E177A/−(c) and Shh^−/− (d). Scale bars: 500 μm (A,C) and 50 μm (D–a).