Pax6 Levels in Neural Stem Cell Self-Renewal genes that are positively regulated by Pax6 (both directly and indirectly), denoted by the green shading. In contrast, the union of the sets of genes downregulated in the D6-Pax6 cortex and genes up-regulated in the Sey/Sey cortex defines a set of genes that are negatively regulated by Pax6 (both directly and indirectly), denoted by the red shading. (H) Examples of the genes found in each of the four categories defined in (C). doi:10.1371/journal.pgen.1000511.g004 (Figure 5D–5F; Table S5). Ventricular zone expression was found for 188 of those genes (84%), 81 of which were solely expressed in the ventricular and subventricular zone. A noteworthy minority of Pax6 bound and regulated genes (21; 9% of detectable mRNAs) were exclusively expressed in differentiating neurons in the cortical plate. Pax6-Regulated Transcriptional and Biological Networks The combination of Pax6 binding data with the transcriptional profiling of Pax6 gain- and loss-of-function cortices enabled the delineation of the direct and indirect networks regulated by Pax6 (Figure 6) and prediction of Pax6 functions in cortical stem and progenitor cells. As shown, Pax6 positively controls sets of genes promoting neural stem cell self-renewal and/or maintenance: Pax6 positively regulates transcription of Hmga2, Tle1 and Cdk4 directly, promoting cell cycle progression and neural stem cell maintenance [39–41]. Furthermore, Pax6 controls expression of D-class cyclins, Hes5 and Notch ligands indirectly [39,42,43], underlining the importance of Pax6 in promoting neural stem cell self-renewal. However, the self-renewal functions of Pax6 are offset by its binding to, and positive regulation of, genes and transcription Figure 5. The set of genes bound and regulated by Pax6 in the embryonic cerebral cortex. (A) Identification of a set of 343 genes both bound and regulated by Pax6 by intersecting the three genomics datasets reported here: Pax6 ChIP-on-chip and the array analyses of Pax6 gain- and loss-of-function cortices. The intersection is highlighted in blue (see Table S4 for details). (B) Definition of the sets of genes bound and positively regulated by Pax6 (green shading) and negatively regulated by Pax6 (red shading). (C) Breakdown of the evidence for positive or negative regulation of genes by Pax6: 23 genes show regulation following both gain and loss of Pax6 function, 42 show regulation only upon loss of function and 278 show regulation only upon gain of function. (D–F) In situ hybridisation data for cellular expression of Pax6 bound and regulated genes in whole embryos at E14.5 was available for 279 genes (GenePaint), 56 of which were not detected at this age. Blind scoring of cellular distribution of expression patterns of the bound and regulated genes (D, E) found that the majority of genes were expressed by neocortical stem/progenitor cells (VZ), including substantial numbers of genes expressed in both the VZ and in differentiating neurons, as well as genes exclusively expressed in the VZ (E). Many Pax6 target genes were also expressed by basal progenitor cells in the SVZ. Examples of genes expressed in all three cell types (VZ, SVZ and CP) are shown in F. Scale bar, 500 mm. doi:10.1371/journal.pgen.1000511.g005 PLoS Genetics | www.plosgenetics.org 7 June 2009 | Volume 5 | Issue 6 | e1000511