Figure 3(A-D) Ki67 (green; mitotically active progenitors) and Tuj1 (red; post-mitotic neurons) IF at E14.5 (A,A'), E16.5 (B,B') and E18.5 (C,C') of lateral pallia of wild-type (WT) versus mutant (KO) brains. Ki67+ proliferating cells (quantified in D) accumulate in mutant cortices. n ≥ 3 brains. Data information: Nuclei (blue) were stained with DAPI. the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone. (E-F') Tbr2 (green; IPs) and Pax6 (red; radial glia cells) IF of E13.5 (E,E') and E17.5 (F,F') lateral pallia of WT and KO brains. Data information: Nuclei (blue) were stained with DAPI. Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone. Quantification of different classes of cortical progenitors: apical radial glia cells (aRGs) as Pax6+ NPs in VZ (G), basal intermediate progenitors (IPs) as Tbr2+ cells in SVZ (H) n ≥ 3 brains. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001). Quantification of different classes of cortical progenitors: , basal RGs as Pax6+ cells in outer SVZ (I). n ≥ 3 brains. Data information: the number of positive cells was quantified in 100µm-width boxes, randomly placed across the LP. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001). (J-L) E12.5 dorsal brain views (J,J') and representative sections of posterior hemispheres (K,K'), showing a vesicle enlargement in mutant brains (K'). The extension of the ventricular surface was quantified along the A-P axis (L); red color code represents Nr2f1 gradient in WT brains. 12µm-thick sections were collected on series of 10 slides; consecutive measurement levels are 120µm apart from each other. n ≥ 3 brains. Data information: In graphs, data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001). (M) Schematic representation of different aRG cell divisions, as visualized in vivo after co-in utero electroporation (IUE) of Tis21-RFP and Sox2-GFP plasmids into E12.5-old embryos. Green: symmetric proliferative divisions; Yellow: asymmetric differentiative divisions; Red: symmetric differentiative divisions. (N-P) GFP (green; expressed under Sox2 promoter) and RFP (red; expressed under Tis21 promoter) IF of E13.5 lateral pallia, 18-hours after IUE. The proportion of single or double positive NPs is shown in pie charts in (P). n = 3 electroporated brains. Data information: Nuclei (blue) were stained with DAPI. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001). (Q-R') Ki67 (green; progenitors) and EdU (red) IF of E13.5 WT (Q,Q') and KO (R,R') embryos, injected with EdU at E12.5. Differentiating cells (EdU+Ki67-) are located in the IZ/CP; percentages shown in (Q',R') were obtained from n ≥ 4 sections of n ≥ 2 brains. Data information: Nuclei (blue) were stained with DAPI. Student t-test (Q',R'; E13.5, EdU injected at E12.5; WT/KO: *=0.03967 (*P<0.05, **P<0.01, ***P<0.001). Scale bars: 50µm. CP: cortical plate; IZ: intermediate zone; SVZ: subventricular zone; VZ: ventricular zone.