Pax6 Levels in Neural Stem Cell Self-Renewal Figure 4. Expression analysis of Pax6 gain and loss-of-function cortices identifies sets of genes dependent on Pax6 for expression. (A) Generation of transgenic mice over-expressing Pax6 specifically in the developing cerebral cortex, using the D6 promoter [35]. (B) Fluorescent immunohistochemistry for Pax6 in control and D6-Pax6 E12.5 cortices demonstrates the marked increase in Pax6 protein in the transgenic cortex. Scale bar, 100 mm. (C) Quantification of the intensity of anti-Pax6 immunofluorescence in WT and D6-Pax6 cortical ventricular zones, relative to signal in the basal ganglia in which the D6 transgene is not expressed. Error bars, s.e.m. (D) Microarray analysis of gene expression changes in the cortex of D6-Pax6 mice at E12.5. Gene expression in cortices from single E12.5 embryos was compared between mice carrying the D6-Pax6 transgene and control littermates in a set of 6 paired, dye-swapped array hybridisations. Paired hybridisations were averaged and genes showing reproducible, statistically significant (FDR,0.1) changes in expression were clustered to identify 1567 up-regulated and 2238 down-regulated transcripts (see Table S2 for details). (E) Microarray analysis of gene expression changes in the cortex of Pax6 mutant (Sey/Sey) mice at E12.5. Gene expression in cortices from single E12.5 embryos was compared between Sey/Sey mice and control littermates in a set of 6 dye-swapped array hybridisations. A set of 938 genes showing reproducible, statistically significant (FDR,0.1) changes in expression were clustered to identify 338 down-regulated and 600 up- regulated transcripts (see Table S3 for details). (F) A set of 298 genes was found to have altered expression in both the Sey/Sey and the D6-Pax6 E12.5 cortex to a statistically significant level. (G) From the total set of 4404 genes showing altered expression in one of more of the Sey/Sey and D6-Pax6 E12.5 cortices, the union of the sets of genes upregulated in the D6-Pax6 cortex and genes down-regulated in the Sey/Sey cortex defines a set of PLoS Genetics | www.plosgenetics.org 6 June 2009 | Volume 5 | Issue 6 | e1000511