Author Summary Neural stem cells make all of the neurons in the brain. A key feature of these cells is the ability to regulate the balance between making more neural stem cells, the process of self-renewal, and making nerve cells, the process of neurogenesis. Too much self-renewal would result in a brain with too few neurons and abnormal circuitry; too much neurogenesis would deplete all of the neural stem cells too quickly, resulting in a small brain and neurological abnormalities. Little is currently known of the how neural stem cells control this fundamental choice. We used one transcription factor, Pax6, which is important for this decision, as an entry point to define the cellular networks controlling neural stem cell self-renewal and neurogenesis in the developing mouse brain. We found that the relative amount of Pax6 controls the balance between self-renewal and neurogenesis in neural stem cells. Increasing Pax6 levels drives the system towards neurogenesis, at the expense of self-renewal, by turning on a genetic programme for making neurons, whereas decreasing Pax6 turns off the genetic programme for neural stem cell self-renewal. In both cases, altering the levels of Pax6 ultimately leads to a small brain, but through very different mechanisms. Given the functions of Pax6 in stem cell self-renewal/ proliferation and neurogenesis, a potentially fruitful approach to uncovering cellular pathways controlling these processes is to identify the downstream targets of Pax6 in neocortical stem cells. Therefore, in this study we used Pax6 as an entry point to define the cellular networks regulating neural stem cell self-renewal and neurogenesis by combining chromatin immunoprecipitation (ChIP) to identify Pax6-bound promoters with the anatomical phenotypes and gene expression changes observed in Pax6 mutant cortices. To do so, we identified where in the genome Pax6 binds in mouse neocortical stem cells during normal development, defining the potential cellular networks regulated by Pax6. As binding does not necessarily imply regulation, we also studied the transcrip- tional consequences of altering Pax6 levels in the neocortex in vivo. Developing tissues are sensitive to Pax6 dosage: heterozygous human mutations in Pax6 result in aniridia and forebrain abnormalities [24,25], as do a number of mouse mutations (for reviews, see [7,26]), humans and mice homozygous for Pax6 mutations typically lack eyes and have marked microcephaly and absent olfactory bulbs [5,6,19], whereas increasing Pax6 levels in transgenic mice results in microphthalmia and forebrain abnor- malities [27–29]. Therefore, we examined the transcriptional consequences of both increasing and decreasing Pax6 levels in the developing cortex to identify those promoters actively regulated by Pax6 in neocortical stem and progenitor cells. We find that Pax6 controls the balance between self-renewal and differentiation in neural stem cells in a dose dependent manner, positively and directly regulating cohorts of genes that promote self-renewal, basal progenitor cell genesis and neurogen- esis. In addition, we found that Pax6 interacts with three other regulators of cortical stem cell neurogenesis, Neurog2, Ascl1 and Hes1. The four transcription factors regulate one another and many of the same target genes in an antagonistic manner, defining a core self-renewal/neurogenesis network that is dependent on a critical level of Pax6. In support of this, we found in phenotypic analyses of Pax6 gain and loss of function cortices that an increase in Pax6 levels drives the system towards neurogenesis and basal Pax6 Levels in Neural Stem Cell Self-Renewal progenitor cell genesis at the expense of self-renewal, whereas removing Pax6 reduces cortical stem cell self-renewal. In both cases, altering the levels of Pax6 ultimately leads to microcephaly, but through different cellular and biological pathways. Results The goal of this research was to define the genetic networks directly and indirectly regulated by Pax6 in neocortical stem and progenitor cells and to characterise the biological networks of which Pax6 is a component. To do so, we combined location analysis of Pax6 bound genomic binding sites in neocortical stem and progenitor cells in vivo with transcriptome analysis of the changes in gene expression resulting from altered levels of Pax6, together with phenotypic data from these and previous studies of Pax6 gain- and loss-of-function in the developing mouse cerebral cortex (Figure 1). Finally, we integrated these data with expression data from previous studies of other transcription factors that have roles in controlling neocortical neurogenesis, most notably Neurogenin2 (Neurog2), Hes1 and Ascl1/Mash1 [15,30,31], in order to extend our knowledge of the biological networks in which Pax6 operates. Identification of Pax6 Target Genes in the Developing Neocortex by Chromatin Immunoprecipitation To identify promoters targeted by Pax6, we applied in vivo location analysis to identify where in the genome Pax6 is bound in neocortical stem and progenitor cells. We carried out chromatin immunoprecipitation (ChIP) for Pax6-bound DNA in the developing mouse neocortex at embryonic day 12.5 (E12.5), at which stage Pax6 is expressed by all neocortical ventricular zone stem and progenitor cells, but not by post-mitotic neurons [32]. A set of three biologically independent Pax6 ChIP experiments was carried out using oligonucleotide microarrays spanning ,8.5 kb around the transcriptional start sites of over 17,000 mouse genes (see Materials and Methods for details). As the arrays used sample the proximal promoters of these genes, this analysis will not include intronic or distally located enhancers bound by Pax6, such as the Pax6-bound cortical enhancer of Neurog2 expression, found ,9 kb upstream of the Neurog2 start site [33,34]. Data from the three experiments were analysed to identify regions of promoters bound by Pax6 in each ChIP and the individual analyses combined to define a set of 1560 genes with Pax6-bound promoters (Figure 2; Table S1), of which 1172 are genes with known or predicted functions (Figure 2H). A set of five genes defined as Pax6-bound by the array study were confirmed as bound by qPCR analysis of additional Pax6 ChIP from the E12.5 cortex using two different Pax6 polyclonal antibodies (Figure 2G). Pax6 Directly Binds to Genes that Control Self-Renewal and Neurogenesis Functional annotation of the set of Pax6-bound genes by Gene Ontology analysis found a significant enrichment for develop- mental processes among Pax6 target genes, including nervous system and eye development (Figure 3). Broad functional categories enriched in the Pax6-bound set included cell cycle regulation, regulation of proliferation, cell-cell signalling, neuro- genesis and neuron differentiation. At the functional level, Pax6- bound genes are enriched for genes with transcription factor activity and chromatin binding (Figure 3A and 3D). Pax6 binds transcription factors that are expressed in stem and progenitor cells (Pax6 itself, Hmga2, Cutl1, Nr2f2, Emx2, Sox9, Neurog3, Tle1) and basal progenitor cells in the cortex (Eomes/Tbr2, Neurod1), as well as to transcription factors expressed in newly PLoS Genetics | www.plosgenetics.org 2 June 2009 | Volume 5 | Issue 6 | e1000511