Figure 1. ARHGAP11B increases the abundance of BPs in the developing ferret neocortex.

Ferret E33 neocortex was electroporated in utero with a plasmid encoding a fluorescent protein (FP) together with either a plasmid encoding ARHGAP11B or empty vector (Control), followed by analysis at E40/P0. (A) Double immunofluorescence for FP (green) and PCNA (magenta) (for the images of the single channels and DAPI staining, see Figure 1—figure supplement 2A). Images are single optical sections. Scale bars, 100 μm. Boxes (50 × 50 μm) indicate FP+ BPs in the OSVZ (1, top), ISVZ (2, middle) and VZ (3, bottom), shown at higher magnification in (A). (A) Dashed lines indicate a cell body contour. (B) Percentage of FP+ cells in the germinal zones (GZ total) and in the VZ, ISVZ and OSVZ that are PCNA+ upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 3 experiments. Error bars indicate SD; *, p <0.05; n.s., not statistically significant; Student's t-test. (C) Double immunofluorescence for FP (green) and phospho-vimentin (PhVim, magenta), combined with DAPI staining (white). Images are single optical sections. Scale bars, 50 μm. Vertical arrowheads, apical mitosis; horizontal arrowheads, basal mitosis. (D) Quantification of FP+ mitotic cells, as revealed by PhVim immunofluorescence, in a 200 µm-wide field of the cortical wall, upon control (white) and ARHGAP11B (black) electroporations. Apical, mitoses lining the ventricular surface; basal, mitoses away from the ventricle (Abv.VZ, abventricular VZ; ISVZ; OSVZ). Data are the mean of 4 experiments. Error bars indicate SD; **, p <0.01; *, p <0.05; n.s., not statistically significant; Student's t-test. (E) Mitotic bRG (single optical sections). Double immunofluorescence for FP (green) and phospho-vimentin (PhVim, magenta), combined with DAPI staining (white), upon electroporation of the plasmid encoding FP together with the plasmid encoding ARHGAP11B. Arrowheads, PhVim+ basal process of the mitotic bRG. Images are oriented with the apical side facing down and are 25 μm wide. (F) Quantification of mitotic bRG (FP+ PhVim+ cell bodies in the SVZ that contain a PhVim+ process), in a 200 µm-wide field of total SVZ (left), ISVZ (middle) and OSVZ (right), upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 4 experiments. Error bars indicate SD; **, p <0.01; *, p <0.05; Student's t-test.

Figure 1—figure supplement 1. Forced expression of ARHGAP11B in the developing ferret neocortex.

(A) Scheme of ferret neocortex development and experimental approach. ARHGAP11B was expressed in developing ferret neocortex by in utero electroporation at E33, when the OSVZ begins to form. Analyses were performed at E37, E40/P0, P10 and P16. Analysis of cortical progenitors was performed at E40/P0, analysis of post-mitotic cells at E40/P0, P10 and P16, and analysis of brain size and neocortex morphology at P16. (B–D) mRNA expression analysis by RT-qPCR at E37, P0, P10 and P16. RNA was isolated from cryosections of paraformaldehyde-fixed brain tissue following ferret in utero electroporation at E33. Expression of the housekeeping gene Hprt1 (B, note the lack of signal in the absence of reverse transcriptase (–RT)) was used for normalization of ARHGAP11B expression detected with two different primer pairs (C, ARHGAP11B_1; D, ARHGAP11B_2). Two control (Con_1, Con_2; white) and two ARHGAP11B-electroporated (11B_1, 11B_2; black) embryos were analyzed at each stage, except for P0 when one control and one ARHGAP11B-electroporated embryo were analyzed. Error bars represent SD of three PCR amplifications. (E, F) ARHGAP11B protein expression analysis by immunofluorescence. 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 E37. (E) Triple immunofluorescence for FP (green), ARHGAB11B (11B, magenta) and Arhgap11a (11a, yellow), combined with DAPI staining (white). Arrows, an ARHGAP11B+ FP+ cell that is Arhgap11a–; arrowheads, an Arhgap11a+ cell that is FP– ARHGAP11B–. Note that the exposure of images of ARHGAP11B staining in the control neocortex was longer than in the ARHGAP11B-electroporated neocortex, in order to show the lack of a specific signal in the control, where only unspecific signal at blood vessels was detected (asterisks). Images are single optical sections. Scale bars, 50 μm. (F) Triple immunofluorescence for FP (green), ARHGAB11B (11B, magenta) and Ki67 (yellow), combined with DAPI staining (white), upon electroporation of a plasmid encoding FP and a plasmid encoding ARHGAP11B. Images are single optical sections. Scale bar, 20 μm. Boxes (25 μm wide), indicating an ARHGAP11B-expressing BP (upper box) and an ARHGAP11B-expressing AP (lower box), are shown at higher magnification on the right. Dashed lines, cell bodies.

Figure 1—figure supplement 2. ARHGAP11B increases the abundance of BPs in the developing ferret neocortex.

Ferret E33 neocortex was electroporated in utero with a plasmid encoding a fluorescent protein (FP) together with either a plasmid encoding ARHGAP11B or empty vector (Control), followed by analysis at E40/P0. (A) Double immunofluorescence for FP (green) and PCNA (magenta), combined with DAPI staining (white). Images are single optical sections. Scale bars, 100 μm. See also Figure 1A. (B) Quantification of FP+ neural progenitors, as revealed by PCNA immunofluorescence, in a 200 µm-wide field in the total SVZ (left), ISVZ (middle) and OSVZ (right), upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 4 experiments. Error bars indicate SD; **, p <0.01; *, p <0.05; Student's t-test. (C) Quantification of FP– neural progenitors, as revealed by PCNA+ immunofluorescence, in a 200 µm-wide field in the total SVZ (left), ISVZ (middle) and OSVZ (right), of embryos subjected to control (white) and ARHGAP11B (black) electroporations. Data are the mean of 4 experiments. Error bars indicate SD; n.s., not statistically significant; Student's t-test. (D) Quantitative analysis of BP morphology at mitosis (PhVim+ cells in the SVZ), as determined by the presence (radial, green) or absence (nonpolar, yellow) of PhVim+ radial processes, in non-electroporated (FP–), control-electroporated (Con) and ARHGAP11B-electroporated (11B) embryos. Data are the mean of 4 experiments. Error bars indicate SD; n.s., not statistically significant; two-way ANOVA with Bonferroni post-hoc tests (Radial; Con vs. 11B, p = 0.14; FP– vs. 11B, p >0.99; Con vs. FP–, p =0.059). (E) Quantification of FP– mitotic cells, as revealed by PhVim immunofluorescence, in a 200 µm-wide field of the cortical wall of embryos subjected to control (white) and ARHGAP11B (black) electroporations. Apical, mitoses lining the ventricular surface; basal, mitoses away from the ventricle (Abv.VZ, abventricular VZ; ISVZ; OSVZ). Data are the mean of 4 experiments. Error bars indicate SD; n.s., not statistically significant; Student's t-test. (F) Quantification of the thickness of VZ (left), ISVZ (middle) and OSVZ (right), upon control (white circles) and ARHGAP11B (black circles) electroporations. Data are the mean of 4 experiments. Error bars indicate SD; n.s., not statistically significant; Student's t-test.