Figure 7. Increased expression of Pax6 specifically in the developing cortex alters neocortical stem cell self-renewal and cell fate determination.

(A) Genes characteristic of the three main cell types in the embryonic cortex (stem/progenitor cells in the ventricular zone; basal progenitor cells and differentiating neurons) are changed in expression in the D6-Pax6 cortex: there is a striking increase in a set of genes specifically expressed in basal progenitor cells (Eomes/Tbr2, Hes6, Tcfap2c), indicating that there is an increased production of this cell type when Pax6 levels are increased. Genes specifically expressed in layer 5 and 6 neurons are upregulated when Pax6 levels are increased, as are genes associated with cortical neurogenesis (Neurog2, Sox4, Sox21). Conflicting changes in the expression of cell cycle regulators are observed, with increases in expression of 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 µm. (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 µm. (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 µm; F, 200 µm.