Fig 2: Generation of TRE-hShh transgenic mouse and verification of forebrain and cerebellum specific driver lines. A Taqman RT-PCR of MEFs co-transfected with CMV-rtTA and TRE-hShh vectors that were treated with or without Dox. Relative levels of hShh mRNA were compared, and beta (b)-actin was used for RT-PCR normalization. B Western blot of MEFs co-transfected with CMV-rtTA and TRE-hShh vectors that were treated with or without Dox. C9C5 (anti-Shh N-terminus antibody) and Ab53281 (anti-Shh C-terminus antibody) were used, and their targeting sites were shown in the top panel. C Immunostaining of MEFs co-transfected with CMV-rtTA and TRE-hShh vectors that treated with or without Dox. The left panel, representative confocal images (DAPI (blue), Zsgreen1 (green), and anti-hShh (red)).The right panel, the correlation between Zsgreen1 and anti-Shh. D Scheme for generating TRE-hShh transgenic mice through site-specific integration. ϕC31mRNA and the targeting vector were co-injected into the pronucleus of TARGATT mouse ES cells. Transgene insertions of three transgenic lines were analyzed by PCR using site-specific primer sets, SSL and SSR: SSL identified the left junction of attP, and 136 bp, 206 bp, and 282 bp PCR products indicated 5’ insertion at attP1, attP2, and attP3, respectively; SSR identified the right junction of attP, and 225 bp and155bp PCR products indicated 3’ insertion at attP2 and attP3, respectively. Line 2 and 3 had identical attP1/attP3 insertions with gene contents flanked by attB (without BB). Line 4 had attP3/attP3 insertion with the whole targeting vector (see Additional file 11: Figure S1C for details). E The scheme to predict southern blot products from TRE-hShh mice (line 2 and line 3). HindIII digestion producing two fragments: a 5193 bp left fragment (detectable with L probe that made of PCR products using Tsh3prF2/Tsh3prR3 primers) and a 2857 bp right fragment (detectable with R probe that made of PCR products using Tsh5prF/Tsh5prR primers). F Southern blots of WT and TRE-hShh (line2) using radiolabeled L and R probes. G The strategy of generating TRE-LacZ, Camk2a-rtTA;TRE-LacZ, and Pcp2-rtTA;TRE-LacZ mice to test rtTA driver lines. Mice were treated Dox from conception (E0) or P0 with various Dox administrations. All brain slices were stained with X-gal at P30, and n ≥ 5 mice per group. H Representative images of X-gal stained coronal brain sections from P30 Camk2a-rtTA;TRE-LacZ mice with Dox treatment (625 mg/Kg food pellets and 3.5 mg/ml in drinking water) from E0. Hippocampus was shown. Negative controls, including TRE-LacZ(+ Dox) and Camk2a-rtTA;TRE-LacZ (-Dox), were shown in Additional file 11: Figure S1F. I Representative images of X-gal stained sagittal brain sections from Pcp2-rtTA;TRE-LacZ mice with Dox treatment from E0 (625 mg/Kg food pellets and 3.5 mg/ml in drinking water). Cerebellum was shown. J Representative images of X-gal stained coronal brain sections from P30 Camk2a-tTA;TRE-LacZ mice without Dox treatment. Hippocampus was shown, and see Additional file 11: Figure S1G for the full sagittal section, and n ≥ 3. K Representative images of X-gal-stained sagittal brain sections from P14 Pcp2-tTA;TRE-LacZ mice without Dox treatment. Cerebellum was shown, and see Additional file 11: Figure S1H for the full sagittal section, and n ≥ 3. All brain slices in (H–K) were counter-stained with nuclear fast red. See also Additional file 11: Figure S1.

Fig 3: Pcp2-tTA;TRE-hShh mice show PC-specific hShh overexpression and enhance Shh signaling in cerebellum. A X-gal staining of sagittal brain sections of TRE-LacZ and Pcp2-tTA;TRE-LacZ at P0, P6, and P14. Brain sections were counter-stained with nuclear fast red, and cerebellum was shown. B Scheme of generating B6.Pcp2-tTA;TRE-hShh double transgenic mice. C Representative tile scan confocal images of a sagittal brain section of a 2-month-old Pcp2-hShh mouse. GFP channel was shown. D Immunostaining of Pcp2-hShh sagittal brain sections for Calbindin (red). Granular layer (GL), PC, and molecular layer (ML) were shown, and see Additional file 3: Video S3 for details. E FACS of cerebellums from TRE-hShh, Camk2a-hShh, and Pcp2-hShh mice. F Taqman RT-PCR for hShh, mGli1, and mPTCH1 from TRE-hShh and Pcp2-hShh cerebellum at P6 and P60. Data were represented as mean ± SEM and analyzed by repeated two-way ANOVA and Sidak’s multiple comparisons tests, and n = 5 per group. G Sagittal sections of P6 brains of TRE-LacZ, Camk2a-tTA;TRE-LacZ, Pcp2-tTA;TRE-LacZ, and Camk2a-tTA;Pcp2-tTA;TRE-LacZ mice were analyzed by X-gal staining. The sections were counter-stained with nuclear fast red. H P6 hippocampus from 5 pairs of “TRE-hShh;Gli1-LacZ and Camk2a-hShh;Gli1-LacZ” littermates and P6 cerebellum from 5 pairs of “TRE-hShh;Gli1-LacZ and Pcp2-hShh;Gli1-LacZ” littermates analyzed by Beta-Glo assay. Data were represented as mean ± SEM and analyzed by paired t-tests. See also Additional files 3 (Video S3), 11 (Figure S3).