Figure 3. ARHGAP11B expression in developing ferret neocortex results in an extended neurogenic period.

Ferret E33 neocortex was electroporated in utero with a plasmid encoding FP, together with either a plasmid encoding ARHGAP11B or empty vector (Control), followed by analysis at E40/P0 (A, B left), P10 (B center) and P16 (B right, (C–E). (A) Double immunofluorescence for FP (green) and Satb2 (magenta), combined with DAPI staining (white), of the E40/P0 ferret neocortex. The immunofluorescence of the same cryosection for Tbr1 is shown in Figure 3—figure supplement 1A. Images are single optical sections. Scale bars, 50 μm. (B) Distribution of Satb2+ FP+ neurons at E40/P0 (left), P10 (center) and P16 (right), between the cortical plate (CP, green) and germinal zones plus intermediate zone (GZ + IZ, yellow), upon control (Con, left) and ARHGAP11B (11B, right) electroporations. Data are the mean of 3 (P0 and P10) or 4 (P16) experiments. Error bars indicate SD; **, p <0.01; n.s., not statistically significant; two-way ANOVA with Bonferroni post-hoc tests (P10, Control CP vs. ARHGAP11B CP, p =0.0015). (C) Triple (immuno)fluorescence for FP (green), Satb2 (magenta) and EdU (yellow), combined with DAPI staining (white), of the P16 ferret neocortex, upon EdU injection at P5. Images are single optical sections. Scale bars, 1 mm. (C') Higher magnification of a FP+ Satb2+ EdU+ neuron upon electroporation of the plasmid encoding FP together with the plasmid encoding ARHGAP11B. Dashed lines, cell body. Images (single optical sections) are oriented with the apical side facing down and are 50 μm wide. (D) Percentage of FP+ cells that are EdU+ upon control (white) and ARHGAP11B (black) electroporations. (E) Percentage of EdU+ FP+ cells that are Satb2+ upon control (white) and ARHGAP11B (black) electroporations. (D, E) Data are the mean of 3 experiments. Error bars indicate SD; ***, p <0.001; *, p <0.05; Student's t-test.

Figure 3—figure supplement 1. Almost all neurons generated from ARHGAP11B-expressing progenitors are Satb2+.

Ferret E33 neocortex was electroporated in utero with a plasmid encoding FP together with either a plasmid encoding ARHGAP11B or empty vector (Control), followed by triple immunofluorescence for FP, Tbr1 and Satb2, combined with DAPI staining, at E40/P0. (A) Overview of the electroporated areas showing the immunofluorescence for Tbr1 (yellow) and FP (green) and the DAPI staining (white) (single optical sections). Note that the DAPI staining and FP immunofluorescence images are the same as in Figure 3A; for Satb2 staining, see Figure 3A. Scale bars, 50 μm. (B) Percentages of FP+ cells that are Satb2+ Tbr1– (blue), Satb2+ Tbr1+ (purple) and Satb2– Tbr1+ (yellow), upon control (left) and ARHGAP11B (right) electroporations. Data are the mean of 3 experiments. Error bars indicate SD.

Figure 3—figure supplement 2. FP and Satb2 immunostaining patterns of control and ARHGAP11B-expressing developing ferret neocortex at P10.

Ferret E33 neocortex was electroporated in utero with a plasmid encoding FP together with either a plasmid encoding ARHGAP11B or empty vector (Control), followed by double immunofluorescence for FP (green) and Satb2 (magenta), combined with DAPI staining (white), at P10. Images are maximum intensity projections of 5 optical sections. Scale bars, 1 mm.