Pax6 Levels in Neural Stem Cell Self-Renewal Figure 1. Strategy to characterise the transcriptional networks regulated by Pax6 in neocortical stem cells. (A) Location analysis was performed for Pax6 binding in the genome of neocortical stem and progenitor cells at embryonic day 12.5 (E12.5) to define those promoters bound by Pax6 in vivo. (B) To define the transcriptional responses downstream of Pax6, both direct and indirect, global gene expression was compared at E12.5 between wild-type and Pax6 loss-of-function (LOF) cortices (Sey/Sey) and also between wild-type and Pax6 gain-of-function (GOF) cortices (D6- Pax6). (C) Phenotypic analyses of the Pax6 gain- and loss-of-function cortices were used to both interpret the gene expression changes observed upon altering Pax6 dosage and also to test the hypotheses generated from the combination of the Pax6 binding and regulation data (D). doi:10.1371/journal.pgen.1000511.g001 born, differentiating neurons (Sox4, Sox11) or in terminally differentiating cortical neurons (Rorb, Etv1). Together with the binding of Pax6 to sets of regulators of cell cycle progression and proliferation (for example, Ccna1, Pten, Cdkn1b, Cdk4, Fzr1 and Ctnnb1), regulation of these transcription factors confers the potential for Pax6 to control stem cell self-renewal/proliferation, neurogenesis and cell fate determination within the cortex. Transcriptome Analysis Following Changes in Pax6 Levels In Vivo Pax6 binding data defines the potential networks controlled by Pax6 in this cell type. However, binding does not necessarily equate with activity. Therefore, to characterize the transcriptional networks within which Pax6 acts, we studied gene expression in the E12.5 mouse cortex, using microarrays, following Pax6 gain and loss of function (Figure 4). Intersection of genes that are positively or negatively regulated by Pax6 defined the transcrip- tional networks dependent on Pax6, without discriminating between direct and indirect regulation. For Pax6 gain of function, we used the D6 enhancer that drives cortex-specific expression in the ventricular zone and cortical plate [35–37] to generate transgenic mice expressing the canonical Pax6 isoform, giving rise to animals with a two-fold increase in Pax6 protein in the ventricular zone, as assessed by immunohistochem- istry (Figure 4A–4C). Expression profiling of cortices from single wild-type and D6-Pax6 transgenic littermates at E12.5 found 3784 genes showing significantly altered expression (false discovery rate, FDR, of 0.1), of which 2238 (59%) were upregulated (Figure 4D; Table S2). In comparison, expression profiling of the Pax6 homozygous mutant cortex (Sey/Sey) at E12.5 identified 938 genes with significantly altered expression, of which 600 (64%) were up-regulated (Figure 4E; Table S3). Of the 938 genes showing altered expression in the Pax6 mutant cortex, 298 (32%) were also altered in expression in the D6-Pax6 cortex (Figure 4F). From the transcriptome analyses, we defined two sets of genes showing Pax6-dependency: the set of genes showing positive dependency on Pax6 was the set formed by the union of genes up-regulated in the D6-Pax6 cortex and the genes down- regulated in the Pax6 mutant cortex (Figure 4F and 4G; 2344 genes); the set of genes showing negative dependency on Pax6 was the set formed by the union of genes down-regulated in the D6- Pax6 cortex and the genes up-regulated in the Pax6 mutant cortex (Figure 4F and 4G; 1920 genes). Genes demonstrating positive dependency on Pax6 were notable for including many genes expressed in basal progenitor cells, including Eomes/Tbr2, Gadd45g, Neurod1, Tcfap2c and Hes6 (Figure 4H). Genes similarly dependent on Pax6 for expression include genes expressed in cortical stem and progenitor cells associated with self-renewal and proliferation, such as the transcrip- tional regulators Hmga2 and Hes5, and the cell cycle regulators Cdk2, Cdk4 and Ccne1. However, transcription factors associated with neurogenesis, such as Neurog2, Sox4 and Sox21, also show positive dependency on Pax6 for their expression. In addition, increasing Pax6 expression leads to an increase in expression of transcription factors that are preferentially expressed in cortical neurons of layers 5 and 6 (Tbr1, Zfp312/Fezf2, Nfia, Nfib). PLoS Genetics | www.plosgenetics.org 3 June 2009 | Volume 5 | Issue 6 | e1000511