Figure 4. ARHGAP11B expression results in a greater abundance of upper-layer neurons and expansion of the developing ferret neocortex.

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 P16. (A) Double immunofluorescence for FP (green) and Satb2 (magenta), combined with DAPI (white) and Nissl (yellow) staining, of the CP (single optical sections). Neuronal layers are marked on the left. Arrowheads, increased thickness of layer II upon ARHGAP11B expression. Scale bars, 200 μm. (B) Distribution of Satb2+ FP+ neurons between the neuronal layers upon control (Con, left) and ARHGAP11B (11B, right) electroporations. Data are the mean of 6 experiments. Error bars indicate SD; ***, p <0.001; two-way ANOVA with Bonferroni post-hoc tests (Layer V, Control vs. ARHGAP11B, p <0.0001; Layer III, Control vs. ARHGAP11B, p =0.0073) (C) Percentage of FP+ cells in the CP that are Satb2+, upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 6 experiments. Error bars indicate SD; ***, p <0.001; Student's t-test. (D) Percentage of FP+ cells in layers II + III that are Brn2+, upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 5 experiments. Error bars indicate SD; *, p <0.05; Student's t-test. (E) Quantification of the gyrus thickness of control (Con) and ARHGAP11B-expressing (11B) ferret neocortex. Measurements were performed as described in Figure 4—figure supplement 3. All data are expressed as ratio between electroporated hemisphere (IUE) and non-electroporated contralateral hemisphere (non-IUE). Data are the mean (red lines) of 20 gyri per condition from six neocortices per condition. Error bars indicate SD; *, p <0.05; Student's t-test. (F) Quantification of layers II-IV thickness, upon control (Con, white) and ARHGAP11B (11B, black) electroporations. Data are the mean of 6 experiments. Error bars indicate SD; *, p <0.05; Student's t-test. (G) Quantification of the lateral length of the entire areas harbouring FP+ cells, measured as depicted in Figure 4—figure supplement 5A top, upon control (Con, white) and ARHGAP11B (11B, black) electroporations. Data are the mean of 6 experiments. Error bars indicate SD; **, p <0.01; Student's t-test. (H) Quantification of the lateral length of the dorsal neocortex, measured as depicted in Figure 4—figure supplement 5A bottom, upon control (Con) and ARHGAP11B (11B) electroporations. All data are expressed as ratio between electroporated hemisphere (IUE) and non-electroporated contralateral hemisphere (non-IUE). Data are the mean of 6 experiments. Error bars indicate SD; *, p <0.05; Student's t-test.

Figure 4—figure supplement 1. ARHGAP11B expression results in a greater abundance of FP+ cells and FP+ Satb2+ neurons in the CP.

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 P16. (A, C) Quantification of FP+ cells (A) and Satb2+ FP+ cells (C) in a 200 µm-wide field in the CP, upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 6 experiments. Error bars indicate SD; **, p <0.01; *, p <0.05; Student's t-test. (B) Distribution of FP+ cells between the neuronal layers upon control (Con, left) and ARHGAP11B (11B, right) electroporations. Data are the mean of 6 experiments. Error bars indicate SD; **, p <0.01; two-way ANOVA with Bonferroni post-hoc tests (Layer V, Control vs. ARHGAP11B, p =0.014).

Figure 4—figure supplement 2. ARHGAP11B expression results in a greater abundance of FP+ Brn2+ neurons in cortical layers II and III.

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 and Brn2, combined with DAPI staining, at P16. (A) Overview of the CP of the electroporated area showing the immunofluorescence for FP (green) and Brn2 (magenta) and the DAPI staining (white) (single optical sections). Layers II and III are indicated on the left. Scale bars, 100 μm. Boxes (50 × 50 μm) indicate a Brn2+ FP+ neuron in layer II, shown at higher magnification on the right. Dashed lines, cell body. (B) Quantification of Brn2+ FP+ cells in a 200 µm-wide field in layers II + III (left), in layer II only (center) and in layer III only (right), upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 5 experiments. Error bars indicate SD; **, p <0.01; *, p <0.05; Student's t-test.

Figure 4—figure supplement 3. ARHGAP11B expression in developing ferret neocortex does not increase neocortical folding.

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 P16. (A) Image of an electroporated brain, with the electroporated area in orange. R, rostral; C, caudal. (B) Schematic representation of the P16 ferret brain (dorsal view). Lines 1 and 2 indicate the positions at which all the morphological measurements were performed. Position one is exemplified in (C) and position two in (D). Gyri and sulci used for the morphological measurements are indicated in green and blue, respectively. For morphological parameters of gyri, the posterior sigmoid gyrus (psg, position 1), lateral gyrus (lg, position 2) and the coronal gyrus (cg, positions 1 and 2) were analyzed; for morphological parameters of sulci, the cruciate sulcus (cs, position 1), lateral sulcus (ls, position 2) and the suprasylvian sulcus (ss, positions 1 and 2) were analyzed. Please refer to Sawada and Watanabe (2012) for the ferret gyri and sulci nomenclature. (C) Immunofluorescence for FP (green), combined with DAPI staining (white), of electroporated brains at position 1 (see B). Images are single optical sections. Scale bar, 1 mm. Two gyri and two sulci used for the morphological measurements at position one are indicated in green and blue, respectively (see (B) for abbreviations). (D) Graphical definition of the measured morphological parameters. Double immunofluorescence for FP (green) and GFAP (magenta), combined with DAPI staining (white), of electroporated brain at position 2 (see B). Image (shown twice) is a single optical section. Yellow lines indicate measured morphological parameters. Two gyri and two sulci used for the morphological measurements at position two are indicated in green and blue, respectively, in the left image (see (B) for abbreviations). See also Materials and methods section for details of morphological measurements. (E) Quantification of the weight of the control (Con) and ARHGAP11B-expressing (11B) ferret brains. Data are the mean (red lines) of 7 (control) or 9 (ARHGAP11B) brains. (F–I) Quantification of the indicated morphological parameters (see (D)), that is the gyrification index of the electroporated area (referred to as local GI) (F), gyrus size (G), sulcus depth (H) and sulcus thickness (I), of control (Con) and ARHGAP11B-expressing (11B) ferret neocortex. Measurements were performed at positions 1 and 2 using the gyri and sulci indicated in (B–D), yielding up to four data points for gyrus morphology and four data points for sulcus morphology per single brain. All data are expressed as ratio between electroporated hemisphere (IUE) and non-electroporated contra-lateral hemisphere (non-IUE). (F–H) Data are the mean (red lines) of 6 neocortices per condition. (I) Data are the mean (red lines) of 18 (control) and 19 (ARHGAP11B) sulci from six neocortices per condition. (E–I) Error bars indicate SD; n.s., not statistically significant; Student's t-test.

Figure 4—figure supplement 4. ARHGAP11B expression in developing ferret neocortex does not lead to increased cell death.

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, G), P10 (C, D, G) and P16 (A, E–G). (A) Quantification of the number of FP+ cells in a 200 µm-wide field of cortical wall at E40/P0 (left) and P16 (right), upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 6 experiments. Error bars indicate SD; ***, p <0.001; **, p <0.01; *, p <0.05; Student's t-test. (B) Immunofluorescence for FP (green), combined with DAPI staining (white) and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining (magenta), of the E40/P0 ferret neocortex. Image width, 200 μm. (C) Double immunofluorescence for FP (green) and Caspase-3 (magenta), combined with DAPI staining (white), of the P10 ferret neocortex. Scale bar, 500 μm. (D) Immunofluorescence for FP (green), combined with staining using fluorescently labeled annexin V (yellow) and DAPI staining (white), of the P10 ferret neocortex. TUNEL staining (magenta) is shown for the ARHGAP11B-expressing neocortex. Image width, 200 μm. (E) Immunofluorescence for FP (green), combined with DAPI staining (white) and TUNEL staining (magenta), of the P16 ferret neocortex. Scale bar, 500 μm. (F) Immunofluorescence for FP (green), combined with staining using fluorescently labeled annexin V (yellow) and DAPI staining (white), of the cortical plate of the P16 ferret neocortex. Scale bar, 300 μm. (B–F) All images are single optical sections. (G) Percentage of FP+ cells that are apoptotic. Note that at all stages analyzed and by all detection methods, ARHGAP11B-expressing neocortex does not show an increase in apoptosis. Data are the mean of at least two neocortices per condition.

Figure 4—figure supplement 5. ARHGAP11B expression in developing ferret neocortex leads to its tangential expansion and an increase in cell density.

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 of tangential expansion (A–F) and cell density (G–I) at E40/P0 (D, E) and P16 (all other panels). (A) Graphical definition of the lateral length of the FP +area (top) and the lateral length of dorsal neocortex (bottom). Double immunofluorescence for FP (green) and GFAP (magenta), combined with DAPI staining (white), of electroporated brain at position 2 (same image as in Figure 4—figure supplement 3D). Image (shown twice) is a single optical section. Yellow lines indicate the two morphological parameters measured. Lateral length of the FP+ area is defined by the position of FP+ cells. Lateral length of the dorsal neocortex is defined as the distance between the cingulate gyrus (cig) and the ectosylvian gyrus (eg). See also Materials and methods section for details. (B) Immunofluorescence for FP (green), combined with DAPI staining (white), of the areas harbouring FP+ cells (single optical sections). Dashed lines, basal contour of the FP+ area. Lateral length of the respective contour is indicated in the bottom right corner. Note that the images and the countours depict only the areas with a high abundance of FP+ cells, which is distinct from the quantification shown in (C). Scale bars, 1 mm. (C) Quantification of the lateral length of the entire areas harbouring FP+ cells, measured as depicted in (A top), at three different positions along the rostro-caudal axis (positions 1 and 2, and an intermediate position referred to as 1.5) as depicted at the bottom of the panel, upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 6 experiments. Error bars indicate SD; *, p <0.05; Student's t-test. (D) Immunofluorescence for FP (green), combined with DAPI staining (blue), of an area harbouring FP+ cells at E40/P0 (single optical sections). Thick dashed line, basal contour of the FP+ area; thin dashed line, apical contour of the electroporated area. Numbers indicate basal and apical lateral length of the FP+ area and the electroporated area, respectively. Scale bar, 500 µm. (E) Quantification of the apical (left) and basal (right) lateral lengths of the areas harbouring FP+ cells at E40/P0, measured as depicted in (D), upon 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. (F) Quantification of the lateral length of the dorsal neocortex, measured as depicted in (A bottom), at positions 1, 1.5 and 2 along the rostro-caudal axis, upon control (Con) and ARHGAP11B (11B) electroporations. Data are expressed as ratio between electroporated hemisphere (IUE) and non-electroporated contra-lateral hemisphere (non-IUE). Data are the mean of 6 experiments. Error bars indicate SD; *, p <0.05; Student's t-test. (G) Immunofluorescence for FP (green) and Satb2 (cyan), combined with DAPI staining (white), of the indicated upper layers of the CP at position 2. Images are single optical sections. Scale bars, 20 μm. (H, I) Cell density quantification. Cell density was measured in 50 μm x 50 μm fields of layer II (left), layer III (center) and layer IV (right) of the CP, upon control (white) and ARHGAP11B (black) electroporations. (H) Quantification of the total cell density, as revealed by DAPI staining of nuclei. (I) Quantification of the neuronal density, as revealed by Satb2 staining. Data are the mean (red lines) of 4 (control) and 5 (ARHGAP11B) experiments, with three fields per layer per experiment. Error bars indicate SD; **, p <0.01; *, p <0.05; n.s., not statistically significant, Student's t-test.

Figure 4—figure supplement 6. ARHGAP11B induces the appearance of astrocytes from the targeted progenitors in the developing ferret neocortex.

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 P16. (A, B) Double immunofluorescence for FP (green) and GFAP (magenta), combined with DAPI staining (white), of the CP (A, single optical sections). Scale bars, 100 μm. (B) High magnification of an FP+ GFAP+ ARHGAP11B-expressing cell in the CP from a different experiment than (A). Arrows, cell body; arrowheads, cell processes. Image width, 36.7 μm. (C) Percentage of FP+ cells in the CP that are GFAP+, upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 4 experiments. Error bars indicate SD; *, p <0.05, Student's t-test. (D) Double immunofluorescence for FP (green) and S100ß (magenta), combined with DAPI staining (white), of the CP (single optical sections). Scale bars, 100 μm. Boxes (75.5 μm wide) indicate an FP+ S100ß+ cell in the CP, shown at higher magnification in the bottom images. Arrows, cell body; arrowheads, cell process. (E) Double immunofluorescence for FP (green) and Olig2 (magenta), combined with DAPI staining (white), of the CP (single optical sections). Scale bars, 100 μm. Boxes (52 × 52 μm) indicate an FP+ Olig2– cell in the CP, shown at higher magnification on the right. Note that no cells were found to be FP+ Olig2+.