Figure 5(A-B') Pax6 (green; aRGs) and Nr2f1 (red) IF of E12.5 WT (A,A') and KO (B,B') lateral pallia. Data information: nuclei (blue) were stained with DAPI. Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone. (C) Pax6 and Nr2f1 pixel intensity quantification at E12.5 and E14.5 indicating increased Pax6 levels upon Nr2f1 removal. n ≥ 3 brains. Data information: the pixel intensity was quantified in 100µm-width boxes, randomly placed across the lateral pallium. Data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001). (D) Real time RT-PCR quantification of Nr2f1 and Pax6 expression in E12.5 cortices. n ≥ 3 cortices. Data information: Data are represented as means ± SEM. 2-way ANOVA (*P<0.05, **P<0.01, ***P<0.001). (E-G'') GFP (green; electroporated cells), Pax6 (red) and Nr2f1 (blue) IF of pCIG2-Nr2f1-IRES-GFP electroporated brains at E13.5 (24-hours after IUE). VZ (F-F'') and CP (G-G'') regions are shown at high magnification. Arrowheads point to GFP+ electroporated cells over-expressing Nr2f1 (blue in F',G'), down-regulating Pax6 (F'') and rapidly migrating out of the VZ (G-G''). Scale bars: 50µm. SVZ: subventricular zone; VZ: ventricular zone. (H) Scatter plot showing Pax6 and Nr2f1 pixel intensity of VZ electroporated progenitors, comparing Nr2f1 overexpressing cells (orange dots) with control pCIG2-GFP electroporated cells (blue dots). Average Nr2f1 and Pax6 pixel intensities of the 2 populations were compared by 2-way ANOVA and resulted significantly different (***= <0.0001). n = 2 electroporated brains. Data information Data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001). (I) Real time RT-PCR of cell cycle genes comparing WT (blue line) and KO (orange line) cortices. CyclinD1 and P21 transcripts are down-regulated whereas Dct is up-regulated in mutants. n = 3 cortices. Data information: Data are represented as means ± SEM. 2-way ANOVA *P<0.05, **P<0.01, ***P<0.001). (J-L) GFP (green; electroporated cells) and P21 (red) IF of E14.5 cortex electroporated 48 hours earlier with control PX458 plasmid (J) or CRISPR/Cas9-expressing plasmid directed against Nr2f1 sequence (PX458-αNr2f1; K). Percentage of P21/GFP double positive cells (L). Arrowheads in (J,K) point to P21+ cells. n = 2 electroporated brains. Data information , nuclei (blue) were stained with DAPI. Student t-test (L; *P<0.05 (M) Schematic model of Nr2f1 action on NP cell cycle and neural differentiation. Nr2f1 promotes neurogenesis by repressing Pax6 and Dct expression and thus cell cycle progression, and by activating P21-mediated cell cycle exit. Both actions modulate the G1-phase length. (N) Graph showing the number of differentially expressed genes (DEG up-regulated (UP) or down-regulated (DOWN) in KO compared to WT, as detected by RNA-Seq of E15.5 neocortices. Data information: For statistical analysis of RNA-Seq data , see Methods. (O) Gene ontology (GO) categories significantly enriched among DEG genes (DAVID Gene Ontology software (P) Hierarchical clustering and heatmap of the expression level of DEGs belonging to the "neuron differentiation" Heat map color scale indicates normalized gene expression from high (red) to low (white) level. Data information: For statistical analysis of RNA-Seq data see Methods.