Cells labeled with BrdU at E13.5 or E15.5 were analyzed for their neuronal identity at E18.5 using Cux1 (A–D) and Ctip2 (E–H) as markers for upper and deeper layer cortical neurons, respectively. White boxed area is shown at higher magnification in separate channels to show the cells co-expressing BrdU and indicated marker (arrowheads) or only BrdU (dashed outline). Bar = 50µm. (I,J) Quantification of BrdU+ cells show that there is persistent production of Ctip2+ neurons in Gli3^cko at E13.5 (I) and E15.5 (J).

A–D. Tbr2-expressing IPCs (green) are greatly reduced in Gli3^cko at E18.5 (D). N = 3 at E14.5 and N = 4 at E18.5 per genotype. E–H. Mitotic cells are labeled with antibody against phospho-histone H3 (pHH3, green) and the VZ is identified with Pax6 expression (red). Note the pHH3+ cells in the basal VZ and SVZ (white arrowheads) at E14.5 Gli3^cko (F). I. Quantification of Tbr2+ IPCs shows a slight increase at E14.5 followed by a great reduction at E18.5 in the mutants compared to the controls. J. Quantification of pHH3+ cells based on their location (I) shows that more IPCs are dividing at E14.5 in Gli3^cko (Basal pHH3+) and the number of mitotic RGCs in the mutants is less than control at E18.5 (Apical pHH3+; white bracket). Bars: A–D, 50µm; E–H, 20µm.

A–H. E13.5 Gli3^F/F;ROSA26^R/R embryos were electroporated in utero with indicated DNA constructs and analyzed at E18.5. Control cells electroporated with pRFP (A,C) or pGFP (E,G) are mostly found in the upper cortical layers and express Cux1 (A,E) and rarely in deeper layers expressing Ctip2 (C,G). In contrast, cells co-electroporated with Cre plasmid show scattered distribution throughout the cortex (B,D) and more likely to express Ctip2 (D). Introduction of Gli3R construct in Cre-expressing cells reverse this phenotype by becoming more similar to control electroporated cells in their location to upper cortical layers (F,H) and expression of Cux1 (F) and not Ctip2 (H). Higher magnification images of boxed areas of both upper and deeper cortical layers are shown underneath each experimental condition. Dotted circles highlight the single positive cells and the white arrowheads, the double-positive cells. N = 5 for −Cre, = 4 for + Cre, and = 7 for +Cre + Gli3R. Bar = 50µm. I,J. Quantification of the electroporated cells (identified by RFP or GFP expression) shows that Cre-expressing Gli3 mutant cells express less Cux1 (p = 0.01) and more Ctip2 (p = 0.005) compared to control (−Cre). However, Gli3R can rescue the phenotype of Cre-expressing mutant cells by converting to Cux1+ cells (p < 0.05) and rarely becoming Ctip2+ cells (p < 0.001) as in control cells. In addition, over-expression of Gli3R reversed the mutant phenotype by generating more Cux1+ cells (p < 0.05) and suppressing Ctip2+ cells (p < 0.001) compared to the controls (−Cre).

BrdU was injected to label cells born at the indicated embryonic stages and analyzed at E18.5. Representative immunofluorescent images for BrdU (green) and distribution of BrdU+ cells in 10 bins spanning the cortical thickness are shown as histograms. While E11.5-born cells show similar distribution in both control (A) and Gli3^cko (B), greater fractions of cells born at E13.5 (D) and E15.5 (F) are found in deeper cortical layers compared to respective controls (C,E). Percentage of cells in upper layers (Bins 2–4) and deeper layers (6–8) are indicated in the histograms. N = 3 per genotype per experimental condition, except N = 5 for BrdU injection at E13.5 in Gli3^cko. *, p < 0.001. control = Nestin-Cre;Gli3^F/+ and Gli3^cko = Nestin-Cre;Gli3^F/xt.

A–D. TUNEL assay on E18.5 (A,B) and P3 (C,D) brains show more apoptotic cells (green) and especially in upper cortical layers of Gli3^cko brain (D, bracket). E. The number of apoptotic cells per unit area (Apoptotic index) shows that increase in cell death does not occur in Gli3^cko cortex until E18.5 and is greatly increased at P3. N = 3 per genotype. F,G. Lower magnification images of Cux1 immunostaining show very weak Cux1 immunoreactivity in most of the upper cortical layers in P21 Gli3^cko brain (G) in contrast to the strong uniform Cux1 expression (F). Within the mutant cortex, patches of Cux1+ cells with bigger nuclei are found (g’) adjacent to the areas with low Cux1 immunoreactivity and smaller nuclei (g”). Scale bars = 1mm (F,G); = 20µm (g’,g”). (H,I) Nissl staining of P21 Gli3^cko brain shows reduced cortical thickness (line with arrows), reduced corpus callosum (arrows) and larger lateral ventricle compared to the control. Axonal projections in the presumptive corpus callosum is also greatly reduced in Gli3^cko brain as indicated by myelin basic protein (MBP) staining (h,i).

A–C. Cell cycle exit was studied by labeling cells in the S-phase of cell cycle with EdU and BrdU at E14.5 and E15.5, respectively, and analyzed for co-expression of EdU (green), BrdU (blue) and Ki67 (red) at E16.5. Boxed areas of cortical plate (CP) and SVZ were shown at higher magnification in separate channels to indicate cells remaining in cell cycle from E14.5 to E16.5 (EdU+ BrdU+ Ki67+, white dashed outline with white arrowheads), cells that exited cell cycle by E15.5 (EdU+ BrdU− Ki67−, green dashed outline) and cells that exited cell cycle by E16.5 (BrdU+ Ki67−, yellow dashed outline). Note that more EdU+ cells are found in the CP in Gli3^cko than in control. D. Cells that were dividing at E14.5 (EdU+) were quantitatively analyzed at E16.5 for their cell cycle status. A higher percentage of EdU+ became post-mitotic by E15.5 (EdU+ BrdU− Ki67−, green shaded area) in Gli3^cko than in control. Similarly, more mutant cells exited cell cycle by E16.5 (EdU+ BrdU+ Ki67−, blue shaded area) compared to control. In contrast, there was decrease in number of EdU+ cells remaining in cell cycle by E16.5 (EdU+ BrdU− Ki67+, yellow shaded area and EdU+ BrdU+ Ki67+, white area). E. Cells dividing at E15.5 (BrdU+) also increased the cell cycle exit by E16.5 as evidenced by the ratio of BrdU+ Ki67− cells to the total number of BrdU+ cells. F. More EdU+ cells were found to be post-mitotic in the CP of Gli3^cko compared to the control. N = 3 per genotype for E14.5-dividing cell analysis. N = 8 per genotype for E15.5-dividing cell analysis.

Schematic diagram of cortical development shows that the loss of Gli3 in RGC results in decreased production of IPCs, which further deplete themselves via increased cell cycle exit. In addition, the cortical neurons generated from mutant IPCs adopt Ctip2+ deeper cortical neuron identity at the expense of Cux1+ upper cortical neurons, which also exhibit abnormalities in their survival and maturation.