Figure 6.

Smaug2 and nanos1 mRNA are associated with 4E-T in a P-Body-like granule in Pax6-positive apical precursors. A, Western blot analysis for Smaug2 (Smg2) and 4E-T in lysates of E12.5 cortical precursors cultured for 3 d and immunoprecipitated with anti-Smaug2 or with control, nonspecific rabbit IgG. As a positive control, 10% of the input homogenate was loaded. B, Western blot analysis for Smaug2 and 4E-T in lysates of E12.5 cortical precursors cultured for 3 d and immunoprecipitated with anti-4E-T or with control, nonspecific mouse IgG. As a positive control, 10% of the input homogenate was loaded. C, Confocal images of E12.5 cortical precursors cultured for 3 d and immunostained for Smaug2 (green) and 4E-T (magenta). Cultures were also counterstained with Hoechst (blue). Top, Boxed areas are shown at higher magnification in the bottom panels. Arrows indicate granules that are positive for both Smaug2 and 4E-T. Scale bar, 5 μm. D, Confocal images of E12.5 3 d cortical precursor cultures after the PLA with Smaug2 and 4E-T antibodies. Cultures were also counterstained with Hoechst (blue). Left, Boxed areas are shown at higher magnification to the right. Scale bar, 10 μm. E, Confocal images of E12.5 cortical precursors cultured for 3 d and immunostained for Smaug2 (red) and Dcp1 (green). Cultures were also counterstained with Hoechst (blue). Top, Boxed areas are shown at higher magnification in the bottom panels. Arrows indicate granules that are double labeled for Smaug2 and Dcp1. Scale bar, 10 μm. F, Confocal images of cortical precursor cultures after PLA with Smaug2 and Dcp1 antibodies. Cultures were also counterstained with Hoechst (blue). Left, Boxed areas are shown at higher magnification on the right. Scale bar, 10 μm. G, RT-PCR analysis for nanos1 mRNA in 4E-T immunoprecipitates (4E-T IP) from the E12.5 cortex. As a control, similar lysates were immunoprecipitated with a control, nonspecific mouse IgG (IgG). H, qRT-PCR analysis for nanos1 mRNA enrichment in multiple independent 4E-T immunoprecipitates from the E12.5 cortex, in comparison with control IgG immunoprecipitates. I, Confocal images of E12.5 cortical precursors cultured for 3 d and analyzed by FISH for nanos1 mRNA (red or magenta) and immunostaining for 4E-T or Smaug2 (both green). Cultures were also counterstained with Hoechst (blue). Arrows and arrowheads indicate nanos1 mRNA-positive foci that are or are not positive for the relevant protein, respectively. Scale bar, 5 μm. J, Quantification of cultures as in I for the percentage of total nanos1 mRNA-positive foci that also colocalized with Smaug2 (Smg2) or 4E-T alone, or with both together. *p < 0.05. **p < 0.01. ***p < 0.01. n = 3. K, Confocal images of the E12.5 cortical VZ immunostained with 4E-T (green) and subjected to FISH (magenta) with a nanos1 mRNA probe shown at low magnification (left) and high magnification (right). Cell nuclei were counterstained with Hoechst (blue). Bottom, Merge. Left, Boxed regions are shown at high magnification to the right. Arrows indicate foci positive for both nanos1 mRNA and 4E-T. Arrowheads indicate nanos1 mRNA foci that are negative for 4E-T. v, Ventricle. Scale bar, 10 μm. L, Quantification of sections similar to that shown in K for the relative proportion of nanos1 mRNA-positive foci that colocalized with 4E-T in each bin of the VZ/SVZ, as defined in Figure 4H. **p < 0.01. n = 3. M, N, Quantification of sections similar to those shown in K for the proportion of nanos1, nanos2, or nanos3 mRNA foci that colocalized with Smaug2 across the entire E12.5 VZ/SVZ (M) or only in Bin1 (N), the apical-most region of the VZ. *p < 0.05. n = 3. Statistics were performed with ANOVA and Tukey's post hoc multiple comparisons test. Error bars indicate SEM.