Pax6 Levels in Neural Stem Cell Self-Renewal Figure 6. Pax6-regulated transcriptional networks in neocortical stem cells. Pax6 directly regulates sets of genes that control four major processes in cortical neurogenesis: stem cell maintenance/self-renewal, neurogenesis, production of basal progenitor cells and cortical identity (black lines). These direct programmes are underpinned by indirect control of larger numbers of genes involved in each of these processes, as shown by the grey lines. Hes1 suppresses neurogenesis when continuously over-expressed in cortical stem/progenitor cells [51] and that is associated with repression (blue lines) of many of the same genes positively regulated by Pax6, including those genes required for genesis of basal progenitor cells. doi:10.1371/journal.pgen.1000511.g006 factors that promote neurogenesis, including the tumour suppres- sor genes Pten and Fzr1/Cdh1 and the transcription factors Neurog2 and Sox4 [31,44–46]. In addition, Pax6 directly and positively controls genes that promote basal progenitor cell genesis, including one of the major determinants of the basal progenitor fate, Eomes/Tbr2, as well as Neurod1 and Gadd45g [47–49]. Together, these functions of Pax6 indicate that one biological role for Pax6 is to promote neurogenesis by increasing the number of neocortical stem and progenitor cells either exiting the cell cycle or becoming basal progenitor cells, and thus neurons at their next division. In addition to roles in neurogenesis and self-renewal, Pax6 controls genes that regulate the neocortical identity of the neurons produced in the dorsal pallium. It does so by primarily negatively regulating genes associated with neuronal cell fates, and subpallially-derived inhibitory neuron and interneuron identity in particular, through repression of key transcription factors that confer interneuron fates (Isl1, Foxd1, Ascl1, Dlx1, Lhx8; Figure 6; [50]). Many of the findings reported here on the functions of Pax6 in neurogenesis and self-renewal are in contrast with the recently described functions of the bHLH transcription factor Hes1, sustained overexpression of which represses many of the genes upregulated by Pax6 [51]. Comparison of the expression data from Hes1 overexpression with the Pax6 data presented here shows that Pax6 and Hes1 have opposing functions on neurogenesis and basal progenitor genesis through the same sets of effector genes (Figure 6): Pax6 positively regulates Neurog2 and Eomes/Tbr2, for example, whereas both of these key transcription factors are repressed by Hes1. As discussed in more detail below, this overlap suggests that Pax6 and Hes1 are both regulating a core mechanism for controlling neural stem cell self-renewal and neurogenesis. Pax6 Gain-of-Function Results in an Increase in Neurogenesis and Production of Basal Progenitor Cells To test the prediction of the functions of the Pax6-regulated network described above, we studied neurogenesis and cell fate PLoS Genetics | www.plosgenetics.org 8 June 2009 | Volume 5 | Issue 6 | e1000511