Figure 6. Phenotypes of Cre-mediated Rapgef2 knockout and compensatory effects of artificial Rap1 expression. A, Efficient knockout of Rapgef2 by introduction of Cre recombinase. Rapgef2-f/f (2-f/f) embryos at E14.5 were subjected to IUE-mediated transduction of pCAG-NLS-Cre and pCAG-FloxP-EGFP-N1 (Cre/cEGFP). As controls, Rapgef2-cKO (2-cKO), and Rapgef2-f/f embryos at E14.5 were subjected to IUE-mediated transduction of CAG=EGFP (EGFP). Coronal sections of the brains of Cre/cEGFP and EGFP/Rapgef2-f/f embryos at E16.5 were subjected to immunostaining for GFP (green) and Rapgef2 (red), as indicated. GFP+ cells apparently lacking Rapgef2 expression are indicated by arrowheads. The images are representative of four biological replicates of each group. Scale bars, 20 µm. B, C, Zonal distribution of GFP-labeled cells and their neural progenitor markers. The sections prepared as in A were subjected to immunostaining for GFP (green), Pax6 (red), and Tbr2 (purple; B). The images are representative of four biological replicates of each group. Scale bars, 50 µm. The percentages of GFP+ cells located in the VZ, SVZ, and IZ/CP (left), and those displaying Pax6−/Tbr2− (D.N.), Pax6+, and Tbr2+ markers (right) are shown as mean ± SD values derived from four each of EGFP/Rapgef2-ff (white bars) and Cre/cEGFP (purple bars) embryos (C). Student’s t test: *p < 0.05, **p < 0.01. D, Disruption of the apical surface structures by Cre-mediated Rapgef2 knockout. The sections prepared as in A were subjected to immunostaining for GFP (green), afadin (red), and Pax6 (blue), as indicated. The images are representative of four biological replicates of each group. Arrowheads indicate the end feet of the apical fibers of GFP-labeled RGCs on the apical surface. Scale bars, 50 µm. E, F, Effects of the overexpression of constitutively active Rap1 on zonal distribution of GFP-labeled cells and their neural progenitor markers. pCAG-Myc-Rap1WT (Rap1WT), pCAG-Myc-Rap1G12V (Rap1G12V), or pCAG-Myc (Vector) were cotransduced by IUE with pCAG-NLS-Cre and pCAG-FloxP-EGFP-N1 into Rapgef2-f/f (2-f/f) embryos at E14.5. Coronal sections of the brains at E16.5 were subjected to immunostaining for GFP (green), Pax6 (red), and Tbr2 (purple), as indicated (E). The images are representative of three biological replicates of each group. Scale bars, 50 µm. The percentages of GFP+ cells located in the VZ, SVZ, and IZ/CP (left) and those displaying Pax6−/Tbr2− (D.N.), Pax6+, and Tbr2+ markers (right) are shown as mean ± SD values derived from three each of mice cotransduced with pCAG-Myc (purple bars), pCAG-Myc-Rap1WT (yellow bars), and pCAG-Myc-Rap1G12V (red bars; F). Student’s t test: **p < 0.01. G, Effects of Rap1 overexpression on the apical surface structures of Cre-mediated Rapgef2 knockout. The sections prepared as in E were subjected to immunostaining for GFP (green), ZO-1 (red), and Myc (blue), as indicated. The images are representative of three biological replicates of each group. Arrowheads indicate the end feet of the apical fibers of GFP-labeled cells on the apical surface. Scale bars, 50 µm.