FIGURE 6.

ATF4 degradation is required for proper cell positioning in the developing embryonic brain. A, in situ hybridization of wild type and ATF4 knock-out mouse brains from E12.5 and E16.5. B, ATF4 was immunoprecipitated from RIPA brain lysates from the indicated ages and subjected to immunoprecipitation and immunoblot analysis with ATF4 antibody. Input lysates were probed with antibodies against p35, CDK5, and actin. C, in utero electroporation scheme. D, RT-PCR analysis of ATF4 and actin brain mRNA from the indicated ages. E, brains were electroporated with pCAG, pCAG-WT, pCAG-S218A, pCAG-5A, or pCAG-9A (all proline-directed sites mutated) at E11.5 and fixed 2 days later at E13.5. Brain sections (12 μm) were prepared and stained with an anti-GFP antibody (green). Nuclei were stained with Hoechst 33258 (blue). F, quantification of cell positioning from D. Positioning of GFP-positive cells were scored as the percentage of cells in the VZ, IZ, or CP. Data are presented as the mean ± S.E. (n = 3). Ventricular zone: a, GFP to WT, S218A, 5A, and 9A p < 0.001; b, WT to S218A, 5A, and 9A, p < 0.001. Intermediate zone: c, GFP to S218A and 5A, p < 0.01, GFP to 9A, p < 0.001; d, WT to 5A, p < 0.01, WT to S218A, 9A, p < 0.001. Ventricular zone: e, GFP to WT, S218A, 5A, and 9A, p < 0.001; f, WT to S218A, 5A, and 9A, p < 0.001. Intermediate zone + cortical plate: g, GFP to WT, S218A, 5A, and 9A, p < 0.001; h, WT to S218A, 5A, and 9A, p < 0.001. p values were obtained by one-way analysis of variance.