, 1999; Di Stefano et al., 2003; Lee et al., 2000; Polager and Ginsberg, 2008; Goto et al., 2006; Malumbres and Barbacid, 2005). We found evidence that this pathway is directly regulated by Pax6. At the onset of corticogenesis, around embryonic day 12.5 (E12.5), Pax6 is expressed in a gradient across the embryonic cortex with high levels rostrolaterally and low levels caudomedially (Figures 1A–1C;
Bishop et al., 2000; Manuel et al., 2007). We used iododeoxyuridine (IdU) and bromodeoxyuridine (BrdU) double labeling as summarized in Figure 1D to calculate the cell-cycle and S phase times (Tc and Ts, respectively; Martynoga et al., 2005; Figures selleck chemical 1E and 1F) in regions of E12.5 Pax6+/+ and Pax6−/− cortex expressing high, medium, or low levels of Pax6 ( Figures 1C and 1G–1I). In Pax6+/+ embryos, the mean Tc was longest in areas expressing high or medium levels of Pax6 ( Figures 1G–1I). In Pax6−/− embryos, the mean Tc was significantly shorter by ∼25% in these two areas. Neither the mean Tc in the area of lowest Pax6 expression nor the mean Ts in any area was affected in
Pax6−/− embryos. We also tested the effect of an acute (conditional) loss of Pax6, since the rapid onset of a defect would strengthen the possibility that Pax6 influences the cell cycle directly. We analyzed mice carrying a tamoxifen-induced, cortex-specific deletion of Pax6. Their Volasertib genotypes were as follows: (1) Pax6loxP/loxP; Emx1-CreERT2; R26R-YFP (inducible knockout [iKO] embryos); and (2) Pax6loxP/+; Emx1-CreERT2; R26R-YFP embryos (controls; deletion of only one copy of Pax6 has no detectable effect on cortical progenitor proliferation; Figure S1 available online). Tamoxifen administered at E9.5 resulted in loss of Pax6 from the cortex (sparing the ventral pallium, which does not express Emx1) by E12.5 ( Figures 1J and 1K). The results obtained from iKOs were similar to those from
Pax6−/− embryos, with significant effects of both genotype and cortical region on mean Tc (two-way ANOVA). In rostral and central-lateral areas (i.e., [Pax6]high in controls), the mean Tc was longer than in central-medial and caudal areas in controls (p < 0.0001, Sidak’s multiple-comparisons Oxymatrine test) and was significantly reduced in iKOs ( Figures 1L–1O). These results show an association between the spatial distribution of Pax6 across the cortex and both progenitor Tc in wild-types (WTs) and the effect of Pax6 absence on Tc in mutants at E12.5. The expression of Pax6 across the cortex becomes increasingly uniform with embryonic age, with similar levels being attained in all areas by E15.5 (Figures 2A–2C), suggesting that the regional effects of Pax6 loss on progenitor proliferation might be different at these ages. In a first set of experiments, iKOs were generated by tamoxifen administration at either E10.5 or E13.5 (Figures 2D–2Q). Activation of YFP from the R26R-YFP allele occurred within 48 hr ( Figure S2A).
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