neocortices from E12.5 MF1 mouse embryos were used for each ChIP. For these and subsequent studies, all animal work was approved by local ethics review committees and, where relevant, carried out according to UK Home Office national guidelines. ChIP was carried out with specific rabbit polyclonal Pax6 antibodies generated against C-terminal peptides that recognise both splice variants of Pax6: C-term 1 -Chemicon, Cat no. AB5409 and C-term 2 - Covance PRB-278P. For array analysis, Pax6-bound genomic DNA was purified by ChIP using the Chemicon antibody, and ChIP material and whole cell extract DNA were globally amplified by ligation-mediated PCR [62]. To validate ChIP-on-chip data, unamplified ChIP material prepared with both Pax6 antibodies and from control samples lacking the primary antibody were used for gene-specific, quantitative PCR. Primers to amplify 100–250 bp target regions surrounding the predicted genomic binding locations for Pax6 were designed using Primer3 (http://frodo.wi.mit.edu/) [63] or Primer Express (Applied Biosystems), and checked for specificity in the genome using the BLAT algorithm (http://genome.ucsc.edu/). Quantita- tive PCR was carried out using the Roche Lightcycler system or the Applied Biosystems 7300 system. The amount of target regions was quantified in Pax6 and control chromatin immunoprecipita- tions lacking the primary antibody (no-antibody controls, NoAb). The enrichment for each gene was calculated by normalising the Pax6/NoAb ratio against the Pax6/NoAb ratio for a promoter region that was not found to be bound by Pax6 (non-bound control region from the Syt8 gene). Promoter Microarray Analysis Amplified Pax6 ChIP and whole cell extract DNA were labelled by indirect incorporation of Cy3 or Cy5-labelled nucleotides and hybridised to Agilent mouse promoter 244 K arrays according to the manufacturer’s instructions. Slides were scanned in an Agilent scanner and data extracted and normalised using Agilent Feature Extractor. Data were analysed by neighbour analysis [62] implemented in ChipAnalytics (Agilent) to calculate probability scores of Pax6 binding for each array oligonucleotide (p[Xbar] scores). Binding events were identified by sliding a 1000 bp window across the genomic space covered by the promoter array. Within a window, the best p[Xbar] score from each of the three ChIP-chip experiments was identified. A binding event was called if: (1) 2 or more scores were , = 0.015, and (2) the product of three scores was , = 0.000025. Gene ontology analysis of Pax6-bound genes was performed using GOToolBox (http://crfb.univ-mrs.fr/ GOToolBox/home.php). D6-Pax6 Transgenic Mice To generate D6-Pax6 transgenic mice, the 5.7 Kb D6 promoter fragment was cloned upstream of the canonical Pax6 open reading frame [35–37] and injected into the pronucleus of fertilized (C57BL/ 66BALB/c) F1 mouse oocytes to generate founder mice. In situ RNA hybridization using digoxigenin (DIG)-labeled RNA probes was performed according to methods described at the Rubenstein lab website (http://physio.ucsf.edu/rubenstein/protocols/index.asp). Sections from the different genotypes (WT and D6-Pax6) were processed in parallel. Basal ganglia expression was used an internal control to compare results between different experiments and between experimental and WT samples. Probes used: Pax6 (P. Gruss), Neurog2 (F. Guillemot), Tbr1 and Eomes/Tbr2 (Rubenstein lab). Immunohistochemistry was performed on frozen sections (10 or 20 mm). Antibodies used: monoclonal anti-bIII-tubulin antibody (clone TUJ1; Covance), 1:1000; anti-phospho-Histone H3 (Ser10) (Upstate) 1:200; anti Pax6 (Developmental Studies Hybridoma Bank) 1:1000; anti-Eomes/Tbr2 (gift from Robert Hevner, University of Pax6 Levels in Neural Stem Cell Self-Renewal Washington School of Medicine, Seattle). Pax6 levels were quantified in 9 wildtype and 14 D6-Pax6 tissue sections by the intensity of the immunofluorescent signal: the signal along the dorsal-ventral axis was quantified in the ventricular zone by histogram (ImageJ, NIH), using the mean intensity. For this quantification the Developmental Studies Hybridoma Bank anti-Pax6 antibody was used at 1:1000. TUNEL analysis was performed on 20 mm cryostat sections using the Apoptag Kit following the manufacturer’s recommendations (Milli- pore, CA, USA). Similar results were observed with activated Caspase 3 staining (data not shown). Immunohistochemistry for Pax6, Neurog2, and Hes1 Triple immunohistochemistry for Pax6, Hes1 and Neurog2 was carried out on sections of E12.5 cortex by standard techniques (rabbit anti-Pax6 antibody, Covance PRB-278P; guinea pig anti- Hes1 antibody, gift from Ryoichiro Kageyama; goat anti- Neurog2, Santa Cruz Biotechnology). Quantification of the fluorescent intensity of nuclear staining for each antibody was carried out on confocal microscrope images (Radiance, Biorad) of a minimum of three sections using Volocity software (Improvision). mRNA Expression Profiling Cortices were dissected from individual embryos in two litters of E12.5 Pax6Sey (Edinburgh Small-eye, [6]) mutant embryos and from one litter of E12.5 D6-Pax6 transgenic mice. Total RNA was extracted and cDNA synthesised using the SMART system, as described [64]. Gene expression in neocortices from three single Pax6Sey/Sey embryos was compared to that in four single wildtype littermates on six dye-swapped oligonucleotide microarrays; while gene expression in neocortices from three single D6-Pax6 embryos was compared to that in three single wildtype littermates at E12.5 in a set of 6 paired dye-swapped hybridizations. Arrays of the MEEBO oligonucleotide set (Invitrogen), produced by the Pathology Department, University of Cambridge were used for all studies. cDNA labeling, array hybridization, slide scanning (Axon GenePix microarray scanner, Molecular Devices) and data extraction were performed as described [64]. Expression data were archived and lowess normalized using the Acuity system (Molecular Devices). Log ratios of all expression measurements for each array were median-centered and expression ratios variance normalized across all of the arrays (without adjusting the average fold-change). Genes were filtered on the basis of the number of arrays on which they were detected, being required to be present in 4 of the 6 microarrays. For the D6-Pax6 analysis, dye-swapped replicate pairs of arrays were averaged, and in the case of one missing value, the single value used. Significant differences in gene expression were identified using the signifi- cance analysis of microarrays (SAM) algorithm (Version 2; one class, at least 200 permutations), using a false discovery rate (FDR) of 0.1 [65]. Data Integration and Network Modelling Dataset intersections between array datasets were performed using gene symbols. Gene symbols were retrieved using the SOURCE (http://smd-www.stanford.edu/cgi-bin/source/sourceSearch) data- base and the gene accession numbers provided by the array/ oligonucleotide manufacturers. Network modeling was carried out using BioTapestry (www.biotapestry.org) [66]. Supporting Information Figure S1 Analysis of cell death in the Pax6-overexpressing cortex by TUNEL staining. PLoS Genetics | www.plosgenetics.org 14 June 2009 | Volume 5 | Issue 6 | e1000511