Pax6 Levels in Neural Stem Cell Self-Renewal Figure 9. Pax6 protein levels are constant in cortical stem and progenitor cells, compared to the variation in Neurog2 and Hes1 levels: implications for network behaviour. (A) Confocal microscope images of immunofluorescent staining for Pax6, Neurog2 and Hes1 in the E12.5 cortex. Single channel images of staining for each antibody are shown, together with the merged image. Arrowheads highlight Pax6+ nuclei (red), Neurog2+ nuclei (blue) and Hes1+ nuclei (green). Almost all Hes1+ cells are also strongly Pax6+. Both Neurog2+/Pax6+ and Neurog2+/Pax62 cells were observed. Scale bar, 50 mm. (B) Prediction of the output of the self-renewal/neurogenesis network when Hes1 levels are high (active Notch signalling): under these conditions, Hes1 antagonises the positive regulation of Neurog2 by Pax6 to suppress neurogenesis. (C) Prediction of the output of the self-renewal/neurogenesis network when Hes1 levels are low (no active Notch signalling): under these conditions, Pax6 positively regulates Neurog2 expression, unopposed by Hes1. Pax6 and Neurog2 cooperate to promote neurogenesis and basal progenitor cell genesis. doi:10.1371/journal.pgen.1000511.g009 by repression of Neurog2, assisted by the activation of Hes5 expression by Pax6 (Figure 9B). In the absence of Notch signalling and Hes1 activity, Pax6-driven increase in Neurog2 and the resulting drive to neurogenesis are unopposed, allowing Pax6 to promote neurogenesis (Figure 9C). Therefore, the identification of the direct transcriptional targets of Pax6 in neocortical stem and progenitor cells, combined with phenotypic analyses of the Pax6 gain and loss of function cortex, have enabled the elucidation of a regulatory network controlling the balance between neurogenesis and self-renewal. The develop- ment of this model provides a framework in which to explore questions arising from the mechanism reported here. For example, only a subset of Pax6+/Hes12 stem cells in G1 phase of the cell cycle go on to generate neurons in the next round of cell division. Therefore, there are likely to be additional components of this network that promote self-renewal and suppress neurogenesis. Discussion Neural stem cell self-renewal, neurogenesis and cell fate determination are three forces that control the generation of specific classes of neurons at the correct place and time during development. Much remains to be discovered of the cellular networks operating to control these processes. We report here that the transcription factor Pax6 directly regulates genes controlling the balance between neocortical stem cell maintenance, neuro- PLoS Genetics | www.plosgenetics.org 12 June 2009 | Volume 5 | Issue 6 | e1000511