5% (±7.9 SEM; n = 3 independent experiments) of control values within 48 hr of E10.5 tamoxifen administration, and to 9.8% (±7.0; n = 3) and 1.6% (±0.6; n = 3) by 72 hr and 96 hr, respectively. Immunohistochemistry showed no obvious

loss of Pax6 protein from the cortex 48 hr after tamoxifen administration ( Figures S2B and S2F), presumably due to residual protein perdurance. Within 72 hr of tamoxifen administration, however, Pax6 protein was removed from most cells in Emx1’s cortical expression domain ( Figures S2C, S2G, S2E, and S2I). We compared the numbers of YFP-positive cells in S phase in rostral, central, and caudal areas of the cortex (high, medium, and low Pax6-expressing, respectively; Figures 2D–2E″, 2J, 2K, selleck 2N, and 2O) in iKO and control embryos. Most cortical cells were YFP labeled in these embryos (Figures 2D’ and 2E’), and the proportions that were not ranged from 5% to 15% in both iKO and control cortices. Cells in S phase were identified by a 1 hr pulse of BrdU. The average numbers of YFP-positive cells that were in S phase at different times after tamoxifen administration at E10.5 (iKOE10.5tamox) or E13.5 (iKOE13.5tamox) are shown in Figures 2J, 2K, 2N, and 2O. In E13.5 iKOE10.5tamox embryos, i.e., shortly after loss of almost all Pax6 protein, increases in the numbers of cells in S phase occurred specifically in the rostral cortex (Figure 2J), indicating rapid onset of

overproliferation confined to this region. Two days later, see more however, in E15.5 iKOE10.5tamox embryos, significant increases in the number of cells in S phase were found in all parts of the cortex (Figure 2K). Similarly, between E15.5 and E16.5 in iKOE13.5tamox embryos, significant increases in the number

of cells in S phase occurred in all cortical areas (Figures 2N and 2O). In a second set of experiments, we estimated (as in Figures 1D–1F) values for mean Tc and Ts in iKOE9.5tamox embryos at E14.5 (Figures CYTH4 2R–2U; Figures S2D and S2H). The mean Tc varied significantly with genotype and cortical area (two-way ANOVA). It was reduced significantly in all cortical areas in iKOs (Figures 2R–2U). In controls, the mean Tc was slightly lower in the caudal cortex than in the rostral and central-lateral cortex (p < 0.0003 and < 0.015, respectively; Sidak’s multiple-comparisons test). The mean Ts did not show differences with genotype or cortical area. These results indicate that loss of Pax6 causes shortening of the cell cycle across all cortical areas by E14.5. Given that Tcs are shortened after loss of Pax6, either in specific cortical regions or across the entire cortex, depending on age, we predicted that these changes should correlate with an increased incidence of cells in M phase (identified by their expression of phosphorylated histone 3 [PH3]). This proved to be true (Figures 2F–2I, 2L, 2M, 2P, and 2Q; Figure S3). Interestingly, the positions of these additional M phase cells were abnormal.