Fat1 exencephalic cortex exhibits elongated ventricles and increased proliferation. (A) Coronal section through E14.5 Fat1^−/− exencephalic brain and control sibling stained for the dorsal cortical precursor marker Pax6 (red, top), the pan-neuronal marker βIII-tubulin (green), the intermediate neuronal progenitor marker Tbr2 (red, bottom) and with Hoechst (blue). Note that the third ventricle (yellow dotted line) is open in Fat1^−/− exencephalic brains in the thalamic region (diencephalon) and is expanded dorsally above and over the cortex (telencephalon); the lateral ventricles of the cortex are delineated with white dotted lines. (B,C) Dorsal ventricular length (B) and Pax6⁺ area (C) measured from sections as in A. ***P<0.001; n=3 Fat1^−/− exencephalic mutants and n=4 controls. (D) Coronal sections through E14.5 Fat1^−/− exencephalic cortex and a control sibling stained for the mitosis marker phospho-histone H3 (pHH3, red) and with Hoechst (blue). V, ventricle (delineated with white dotted line). (E) Percentage of pHH3⁺ cells in the cortex, calculated per unit ventricle length, using images similar to D. *P<0.05; n=3 embryos each. Data are represented as mean±s.e.m. Scale bars: 500 µm in A; 20 µm in D.

Fat1 knockdown promotes radial glial precursor proliferation. (A) Schematic of the in utero electroporation technique. (B-R) Murine cortices were electroporated at E13.5 with a nuclear EGFP expression plasmid and Fat1 shRNA (shFat1) or a scrambled shRNA (Ctrl) plasmid, and analyzed 3 days later at E16/17. (B) Fluorescence confocal micrographs of E16.5 cortex stained for EGFP (green). (C) Quantification of sections as in B for the percentage localization of EGFP⁺ cells (n=3 embryos each). (D) Confocal fluorescence micrographs of VZ/SVZ/IZ from coronal sections through E16.5 cortex immunostained for Pax6 (red) and EGFP (green); bottom panels show merge. (E,F) Quantification of sections as in D for the proportion of Pax6⁺, EGFP⁺ cells per section (E; n=3 embryos each) and relative localization in the VZ, SVZ or IZ (F; n=3 embryos each). (G) Confocal fluorescence micrographs of VZ/SVZ/IZ from coronal E16.5 cortical sections immunostained for Ki67 (red) and EGFP (green); bottom panels show merge. (H) Quantification of sections as in G for proportion of Ki67⁺, EGFP⁺ cells per section (n=3 embryos each). (I,J) Quantification of sections as in supplementary material Fig. S3B for the proportion of Pax6⁺, Ki67⁺, EGFP⁺ triple-labeled cells per section (I; n=3 embryos each) and relative localization in the VZ, SVZ or IZ (J; n=3 embryos each). (K) Fluorescence confocal micrographs of VZ/SVZ/IZ from coronal E16.5 cortical sections stained for Tbr2 (red) and EGFP (green); bottom panels show merge. (L) Quantification of sections as in K for the proportion of Tbr2⁺, EGFP⁺ cells per section (n=3 embryos each). (M) Quantification of sections as in supplementary material Fig. S3C for the proportion of Pax6⁺, Tbr2⁺, EGFP⁺ triple-labeled cells per section (n=3 embryos each). (N) Fluorescence confocal micrographs of CP from coronal E16.5 cortical sections immunostained for Satb2 (red) and EGFP (green); lower panels show merge. (O,P) Quantification of sections as in N and supplementary material Fig. S3D for the proportion of Satb2⁺, EGFP⁺ cells per section (O; n=3 embryos each) and relative localization in the VZ/SVZ/IZ versus CP (P; n=3 embryos each). (Q,R) Quantification of sections as in supplementary material Fig. S3E for the proportion of βIII-tubulin⁺, EGFP⁺ cells per section (Q; n=3 embryos each) and the relative localization in the VZ/SVZ/IZ versus CP (R; n=3 embryos each). Arrowheads indicate EGFP⁺ cells negative for the respective marker and arrows indicate double-labeled cells. *P<0.05, **P<0.01, ***P<0.001; ns, not significant; error bars denote s.e.m. Scale bars: 20 μm.

Fat1-Fat4 mutant cortices display enhanced radial precursor proliferation and delayed cell cycle exit. E13.5 Fat1-Fat4 mutant embryos and control siblings were labeled with BrdU and harvested after 24 h. (A,C,E) Coronal sections through E14.5 dorsal cortex stained for BrdU (red) and Ki67 (A), Sox2 (C) and Tbr2 (E) (green); right panel shows merge with Hoechst (blue). Arrowheads point to examples of double-labeled cells (BrdU⁺ and Ki67⁺, Sox2⁺ or Tbr2⁺) and arrows to single-labeled BrdU⁺ cells. Scale bars: 50 μm. (B,D,F) Quantification of the percentage of Ki67^–, BrdU⁺ (B), Sox2⁺, BrdU⁺ (D) and Tbr2⁺, BrdU⁺ (F) over total BrdU⁺ cells shows reduced cell cycle exit in Fat1-Fat4 mutants. *P<0.05, ***P<0.001; n=3 embryos each; data are mean±s.e.m.

Fat1 and Fat4 have additive effects on radial glial precursor proliferation. (A-J) Murine cortices were electroporated with a nuclear EGFP expression plasmid and a control shRNA (Ctrl) or combined Fat1 shRNA and Fat4 shRNA at E13.5, and analyzed 3 days later at E16/17. (A) Confocal fluorescence micrographs of E16.5 coronal cortical sections stained for EGFP (green). (B) Quantification of sections as in A for the relative localization of EGFP⁺ cells (n=3 embryos each). (C) Confocal fluorescence micrographs of the VZ/SVZ/IZ of coronal cortical sections immunostained for EGFP (green) and Pax6 (red); right panels show merge. (D,E) Quantification of sections as in C for the proportion of Pax6⁺, EGFP⁺ cells per section (D; n=3 embryos each) and the relative localization in the VZ, SVZ or IZ (E; n=3 embryos each). (F) Confocal fluorescence micrographs of the VZ/SVZ/IZ of sections stained for EGFP (green) and Ki67 (red); right panels show merge. (G) Quantification of sections as in F for the proportion of EGFP⁺ cells that are Ki67⁺ (n=3 embryos each). (H) Confocal micrographs of the CP stained for EGFP (green) and Satb2 (red); right panels show merge. (I,J) Quantification of sections as in H for the proportion of EGFP⁺ cells that are Satb2⁺ (I; n=3 embryos each) and relative localization in VZ/SVZ/IZ versus CP (J; n=3 embryos each). Dotted lines demarcate cortical regions. Arrowheads indicate EGFP⁺ cells negative for the respective marker and arrows indicate double-labeled cells. *P<0.01, **P<0.01, ***P<0.001; error bars denote s.e.m. Scale bars: 20 μm.

Fat1-Fat4 mutant radial glial precursors have apical constriction defects. (A) ZO-1 staining of thick coronal sections (10 µm). Beneath is a reconstruction of the apical surface area from z-stack confocal images. (B) Confocal fluorescence micrographs of the ventricular domain of cortical sections from E14.5 Fat1-Fat4 mutants and control sibling. Rhodamine-phalloidin was used to visualize F-actin. (C) Quantification of F-actin intensity from images as in B, showing a decrease in F-actin accumulation in Fat1-Fat4 mutants. **P<0.01; n=4 controls and n=3 Fat1-Fat4 mutants, at least 100 junctions were measured for each. (D) Reconstruction of radial precursor apical surface in Fat1^−/−; Fat4^−/− E14.5 cortex and control sibling stained for ZO-1 (green), Par3 (red) and Hoechst (blue). (E) Quantification of apical surface area. *P<0.05; n=7 controls and n=4 Fat1-Fat4 mutants, at least 100 cells were measured for each on at least two different stacks images. Scale bars: 10 µm in A,D; 20 µm in B. Data are mean±s.e.m.