Figure 6(A) Real time RT-PCR analysis of Pax6 expression in neurospheres with different genotypes, as indicated. n = 3 cortices. N KO P HET: Nr2f1 KO, Pax6 HET. Data information: Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001). (B) Representative images of Nr2f1 KO Pax6 HET neurospheres obtained from E15.5 neocortices and cultured in vitro. Scale bars: 50µm. (C) Graph showing the number of neurospheres per P100 cell plate, at different steps, as indicated. While Nr2f1 KO neurospheres (orange line) proliferate for a longer time compared to WT ones (blue line), the loss of one Pax6 allele (gray line) almost restores normal proliferation rate and exhaustion time. Complete Pax6 loss is not compatible with stem cell renewal (yellow line). n ≥ 3 culture wells from n = 2 independent batches. Data information: Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001). (D-E) Paired cell analysis after neurosphere dissociation and 24-hour culture at clonal density. Couples of dividing cells were identified by Map2 (red; N, neurons) and BLBP (green; P, progenitors) IF. Pie charts in (E) show the proportion of P-P (proliferative; D), P-N (asymmetric differentiative; D') or N-N (symmetric differentiative; D'') couples in WT, Nr2f1KO and Nr2f1KO Pax6HET animals. n ≥ 3 samples from n = 2 culture batches. Data information: Nuclei (blue) were stained with DAPI. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001 Scale bars: 50µm. (F-I) Pax6 (green) and Nr2f1 (red) IF in the posterior-most region of E12.5 cortex, showing Pax6 upregulation in Nr2f1 KO animals (G,G') compared to WT (F',F''). Normal Pax6 levels can be restored by loss of one Pax6 allele (Nr2f1 KO, Pax6 HET; H,H'). n ≥ 3 brains. N KO: Nr2f1 KO; P HET: Pax6 HET. Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm. Pixel intensity quantification of Nr2f1 (blue) and Pax6 (orange) is shown in (I). n ≥ 3 brains. N KO: Nr2f1 KO; P HET: Pax6 HET. Data information: the pixel intensity or the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001). (J) Cell cycle duration at E12.5 in different mutants as indicated, quantified in posterior regions with double EdU/BrdU injection protocol n ≥ 6 sections from n = 2 brains. Data information: the pixel intensity or the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001). (K) Insets showing representative γ-Tubulin (green) and Phospho-Vimentin (red) IF of the cortical VZ surface to evaluate the orientation of the cleavage plane of dividing aRGs. Pie charts show the percentage of vertical (green), oblique (yellow) and horizontal (red) division planes of mitotic figures in the E12.5 lateral pallium of animals with different genotypes, as indicated. n ≥ 2 brains per genotype. Data information: Nuclei (blue) were stained with DAPI. Data are represented as means ± SEM. 2-way ANOVA ; *P<0.05, **P<0.01, ***P<0.001). cale bars: 50µm.