BrdU-immunohistochemistry was performed on paraffin-em- bedded sections as described [27]. Cell cycle length and exit studies were carried out as described [28] with minor modifica- tions. Deparaffinised, rehydrated paraffin-embedded sections were boiled 3 times for 15 minutes each in antigen-retrieval buffer (Vector Laboratories, Burlingame, CA). Sections were blocked in 4% horse serum, 0.2% Triton X-100, 1% BSA in PBS for 60 minutes followed by primary antibody incubation overnight at 4uC with mouse anti-BrdU (1:150, BD Biosciences, San Jose, CA) or rabbit anti-Ki67 (1:200, Novocastra Laboratories, Newcastle upon Tyne, UK) followed by incubation for 1 hour at room temperature with fluorescent labelled secondary antibodies: goat anti-mouse Alexa Fluor 488 (1:200) or goat anti-rabbit Cy3 (1:250). Percentages of two fields of randomly selected 50 Ki67+/ BrdU+ or 50 BrdU+/Ki672 cells from each neocortex (n = 3) was compared by unpaired Student’s t-test. Neurosphere cultures Neocortices were dissected from E14.5 Ptc1 or Rbpj mutant mice, filtered through a 40 mm nylon cell strainer (Stem Cell Technologies Inc. Vancouver, BC) and plated directly. For the SFUA (Sphere forming unit assay), cells were plated at a density of 26104 cells/well in 96 well plates containing Neuro Cult complete medium (Stem Cell Technologies) with EGF (20 ng/ml) [29]. For each individual sample, the assay was set up in triplicates. The numbers of spheres per well were counted 7 days post plating and were plotted as the percentage of the number of spheres obtained per 20,000 cells plated per well. Statistical analysis was performed using GraphPad Prism 4 for unpaired Student’s t-test with Welch’s correction. For passaging, neurospheres were harvested and dissociated in 0.05% trypsin (Gibco) followed by addition of trypsin inhibitor. Resuspended cell pellets were then replated at a density of 26105 cells/ml. Cell counts at every passage were used for growth curve analysis. For analysis of neurosphere replating assays in GraphPad Prism 4, expansion numbers obtained per passage were transformed into LOG and linear regression analysis was performed on resulting curves. Student’s t-test analysis was used to determine significant differences between slopes. Pair cell assay The Pair Cell Assay was performed as described [30,31]. Briefly, the neocortices from E14.5 embryos were dissected into HBSS (Gibco, Invitrogen, Carlsbad, CA) and enzymatically dissociated using 0.125% trypsin (Gibco) at 37uC for 10 minutes and titrated with a fire polished glass Pasteur pipette to generate single cell suspension. The cell suspension was passed through a 40 mm cell strainer (BD Biosciences), rinsed 3 times in HBSS followed by gentle centrifugation. The cell pellet was resuspended in serum-free pair cell medium [32]. Single cells were plated on poly-d-lysine (Sigma Aldrich) coated coverslips at a cell density of about 50,000 cells per well and cultures were maintained for 24 hours. For immunofluorescence studies, the cells were fixed in 4% PFA at 37uC for 30 minutes, before blocking for 30 minutes at room temperature in 0.5% BSA in PBS. The fixed cells were then incubated with rabbit monoclonal anti-TuJ1 (1:1000; Covance) and guinea pig polyclonal anti-GLAST (1:200; Chemicon) primary antibodies at room temperature for 1 hour, followed by incubation with secondary antibodies for 1 hour: chicken anti- rabbit Alexa Fluor 488 and goat anti-guinea pig Alexa Fluor 594 (1:250, Molecular Probes) and DNA was counterstained with DAPI (Sigma, St. Louis, MO). Coverslips were mounted on slides and imaged using laser confocal microscope (LSM Meta, Carl Zeiss). The number of progenitor pairs undergoing proliferative or neurogenic/differentiative cell divisions was determined by Control of Neural Stem Cells counting 100 pair of the cells per mutant and wild type samples (n = atleast 3) and the data was analysed using GraphPad Prism 4 software. VZ microdissection and Real-Time PCR E14.5 embryos were dissected and embedded immediately in OCT medium. 50 mm sagittal sections were cut and mounted onto PALM Membrane Slides (Carl Zeiss Pty Ltd). Slides were then fixed and stained with Cresyl Violet according to the LCM Staining Kit (Ambion, Austin, TX). The neocortical ventricular zones were microdissected under a dissecting microscope using a sterile scalpel blade and pooled. Total RNA was extracted using an RNeasy Mini Kit (Qiagen) followed by DNase treatment using RNase Free DNase set (Qiagen, Germantown, MD). For real time RT-PCR analysis, first strand cDNA was synthesized from 200 ng total RNA using Superscript III Reverse Transcriptase (Invitrogen). Real-time PCR reactions were pre- pared in triplicate on ABI Prism SDS 7000 using a 20 ml reaction mixture with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). The raw data for Ptc1 SYBR Green RT-PCR was normalised based on the expression of Hprt. Primers used in this analysis were: Ptc1 (Forward: 59 GGC AAG TTT TTG GTT GTG GGT C 39 and Reverse: 59 CCT CTT CTC CTA TCT TCT GAC GGG 39). mHprt (Forward: 59GCA GTA CAG CCC CAA AAT GG 39 and Reverse: 59 AAC AAA GTC TGG CCT GTA TCC AA 39). Inventoried assay on demand was used for Gli1 (Mm00494645_m1, Applied Biosystems), Ptc1 (Mm00436029_m1, Applied Biosystems; for detection of total Ptc1 transcript), Hes5 (Mm00439311_g1, Applied Biosystems), Blbp (Mm00445225_m1, Applied Biosystems) and Hes1 (Mm01342805_m1, Applied Biosystems) RT PCR reactions. Reactions were prepared in duplicates on ABI Prism SDS 7500 using a 20 ml reaction mixture with Taqman Master Mix (Applied Biosystems). The raw data was normalised based on the expression of mouse Gapdh (VIC-labelled assay on demand, Applied Biosystems). Microdissected VZ genotyping To confirm for the recombined Ptc1 exon 3 deleted transcript exon 2/exon 6 genotyping PCR was used. Cre-mediated recombination results in a functionally Ptc1 protein truncated 13 amino acids after the first transmembrane domain [22]. Primers used for this analysis were: Ptc1 exon 2 (Forward: 59 CAC CGT AAA GGA GCG TTA CCT A 39) and Ptc1 exon 6 (Reverse: 59 TGG TTG TGG GTC TCC TCA TAT T 39). Genotyping of the wildtype Ptc1 transcript would produce a 450 bp band, upon Cre-mediated recombination exon3 is excised and the resulting transcript is 250 bp. Results Activation of Hedgehog signaling in Nestin+ cells by Patched1 deletion leads to altered neocortical patterning To demonstrate activation of Hh signaling in mice homozygous for Nestin+ cell specific inactivation of the Ptc1 gene (Ptc1Lox/Lox;NestinCre ), the expression of Ptc1 and Gli1 transcripts [33] was analysed. Both Ptc1 and Gli1 are universal Hh targets and upon Ptc1 inactivation in Ptc1Lox/Lox;NestinCre mice an upregulation of both Ptc1 and Gli1 transcripts in the VZ of the developing E14.5 neocortex was observed (Fig. 1B, 1D, 1Ai–Di). Although we were unable to detect Ptc1 and Gli1 expression in the neocortex of Ptc1Lox/Lox mice by section in situ hybridisation (Fig. 1A, 1C), total Ptc1 transcript was detected by Taqman RT-PCR in microdissected VZ (Fig. 1E) and the deleted or mutant transcript was detected by SYBR Green RT-PCR (Fig. S1B). These results demonstrate that Ptc1 is normally expressed in the PLoS ONE | www.plosone.org 3 February 2011 | Volume 6 | Issue 2 | e14680