Pax6 Levels in Neural Stem Cell Self-Renewal positive regulators of G1 progression (Cdk2, Cdk4, Hmga2) and negative regulators of proliferation (Pten, Fzr1), accompanied by decreased expression of genes specifically transcribed in S- and M-phase (Foxm1, Mcm3, 5 & 7). (B) Over-production of basal progenitor cells (Eomes/Tbr2) and layer 6 neurons (Tbr1) observed at E12.5, resulting from increased Pax6 expression that also upregulates Neurog2 expression. Arrowheads indicate the region of Pax6 over-expression within the cortex, which does not extend throughout the entire lateral dimension of the VZ. Scale bars, 100 mm. (C) The increased expression of Pax6 in the D6-Pax6 cortex results in microcephaly at E14.5 that is more pronounced caudally (where the D6 transgene drives the highest increase in Pax6 expression). The cortical plate is reduced in thickness compared to wildtype, with an overall reduction in the number of neurons (as assessed by Tbr1 staining). Increased Neurog2 expression is maintained at this stage, as is increased Eomes/Tbr2 expression. Scale bar, 200 mm. (D,E) Increased Pax6 expression results in a reduction in total cortical size at E12.5. At both E12.5 and E14.5 the microcephaly is not associated with a marked change in mitotic index, as assayed by phopho-histone H3 staining for cells in M-phase at the ventricular surface. Green, Tuj1; red, phospho-histone H3; blue, DAPI. Scale bars, D, 100 mm; F, 200 mm. doi:10.1371/journal.pgen.1000511.g007 determination in the Pax6 gain-of-function cortex (D6-Pax6 transgenic mouse; Figure 7). As predicted, increased Pax6 resulted in a marked increase in the expression of Eomes/Tbr2 at E12.5, demonstrating the increase in the basal progenitor cell population. Furthermore, there was with an increase in early born, layer 6 neurons (Tbr1-expressing cells), without an increase in the total number of neurons produced by this stage of development, compared to controls (as evidenced by the number of cells expressing neuron-specific tubulin, Tuj1, Figure 7). However, by E14.5 the D6-Pax6 transgenic cortex was significantly smaller than that of controls, with a reduction in total neuron number (Figure 7). The reduction in cortical neuron number at this stage is consistent with the early increase in Pax6 expression driving cortical stem and progenitor cells towards an inappropriately early basal progenitor and neuronal fate. The reduction in cortical size is not due to an increase in cell death in the Pax6 overexpressing cortex, as no significant increase in apoptosis could be detected by TUNEL staining (Figure S1). The early depletion of the stem cell pool reduces the number of stem cells available for neurogenesis at subsequent stages, resulting in an overall reduction in cortical size and total cortical neurogenesis by E14.5 (Figure 7). A Cortical Neurogenesis/Self-Renewal Regulatory Circuit Controlled by Pax6 Neurogenin2 (Neurog2) and Ascl1/Mash1 are two proneural bHLH transcription factors with well-established roles in neocor- tical neurogenesis and cell fate [15,31]. Both interact with Pax6: Pax6 positively regulates Neurog2 via an enhancer [34], whereas Ascl1 expression is upregulated in Pax6-null cortices [52]. Gene expression profiling of the Neurog2 mutant cortex and the Ascl1- ventral telencephalon has been analysed to define genes downstream of both factors [53]. As with Hes1, there are striking overlaps in the sets of genes downstream of Ascl1, Neurog2 and Pax6 in the developing telencephalon. Placing Pax6 in the context of these three transcription factors controlling cortical neurogenesis, Hes1, Ascl1 and Neurog2, enables the definition of a core transcriptional circuit controlling cortical neural stem cell self-renewal and neurogenesis (Figure 8A). Pax6 both positively regulates the expression of Neurog2 and also synergises with Neurog2 to promote basal progenitor cell genesis and thus production of cortical excitatory projection neurons, whereas Hes1 opposes this process by repressing many of the same genes. Ascl1 has complex functions in cortical neurogenesis: while it also promotes basal progenitor cell genesis, it also drives expression of transcription factors to promote inhibitory interneu- ron genesis (Lhx8 and Isl1, for example). Pax6 and Hes1 both repress Ascl1, and Pax6 represses Lhx8 and Isl1, to inhibit the interneuron-producing functions of Ascl1. This network also leads to clear predictions of the normal functions of Pax6 in regulating neurogenesis and the consequences of altered Pax6 expression in the early cortex (Figure 8). The network indicates that Pax6 is essential for the cortical identity of the basal progenitor cells produced in the cortex, as it is an essential driver of Eomes/Tbr2 expression, a key determinant of cortical basal progenitor cell identity [47,48]. Given the repression of Ascl1 by Pax6 in cortical stem cells and the interneuron- promoting function of Ascl1, loss of Pax6 function should result in a basal progenitor cell population lacking cortical identity (due to loss of Eomes/Tbr2 expression), a decrease in the genesis of cortical pyramidal neurons and an increase in the production of inhibitory interneurons (Figure 8B). These are all phenotypes observed in the Pax6 cortex [8,9,14,52]. In contrast, increasing Pax6 expression would be predicted to increase basal progenitor genesis early in cortical development and increase pyramidal cell genesis both directly from the ventricular zone and indirectly from basal progenitor cells (Figure 8C). In principle, Pax6 could increase cortical stem cell self-renewal/ proliferation, but this would be in competition with all of the functions of Pax6 that promote neurogenesis: reduced prolifera- tion/cell cycle exit (via Pten and Fzr1/Cdh1 expression), basal progenitor cell genesis (via Eomes/Tbr2 and Neurod1) and neurogenesis (via Neurog2 and Sox4). In the D6-Pax6 cortex, as described above, an increase in basal progenitor genesis is observed early in development, accompanied by an increase in the production of early-born cortical pyramidal cells. Levels of Pax6 Regulate the Balance between Neurogenesis and Self-Renewal Our in vivo analysis supports the finding that Pax6 operates in and regulates a core transcriptional network that controls the balance between neurogenesis and stem cell self-renewal in a highly dosage-sensitive manner. Altering the amount of Pax6 in neural stem cells has profound effects on the output of neural stem cells, ultimately compromising the ability of neural stem cells to generate all of the required neurons for normal assembly of the cerebral cortex. This raises the question as to whether Pax6 levels do vary or oscillate in vivo, as has been reported for several other genes in developing systems [54]. The levels of Hes1 and Neurog2 proteins vary with cell cycle stage and have also been shown to oscillate in neocortical stem and progenitor cells with a 2–3 hour frequency [51], in a Notch- dependent fashion in the case of Hes1. Immunohistochemistry for Pax6, Neurog2 and Hes1 shows that Pax6 does not show obvious cell cycle-dependent changes in levels, unlike Hes1 and Neurog2, as assessed by nuclear location in the ventricular zone, as it is expressed in all neocortical stem and progenitor cells at relatively high levels (Figure 9A; Figure S2). This approach cannot resolve oscillations with a short periodicity, as observed for Hes1 and Neurog2 by live imaging [51]. However, Hes1-expressing cortical stem and progenitor cells also express high levels of Pax6, so it is unlikely that Hes1 represses Pax6 expression as it does Neurog2 [51]. In the presence of Notch signalling, increased Hes1 and Hes5 activity suppresses neurogenesis and promotes self-renewal, in part PLoS Genetics | www.plosgenetics.org 10 June 2009 | Volume 5 | Issue 6 | e1000511