Figure 3.

Rb deletion causes ectopic proliferation and abnormal radial migration of neuroblasts in the developing OE. (A–D′) Double immunostaining with Hoechst (blue), anti-Tuj1 (green), and anti-BrdU (red) performed on sagittal sections in Rb+/− (A–D) vs. Rb−/− (A′–D′) embryos at E15.5. (D,D′) are higher magnification images of the regions shown in white boxes in (B,B′), respectively. Loss of Rb leads to increased progenitor proliferation and abnormal radial migration in the OE. Many (Brdu+Tuj1+) cells are found scattered in the intermediate zone (IZ) in Rb−/− OE (arrowheads in D′) with randomly orientated neurites (D′; green and red insets) compared with controls where they are primarily located in the apical and basal layers with parallel neurite orientation (insets in D). (E,E′) Immunostaining with PH3 (phospho-histone H3, M phase marker) performed at E18.5 and showing increase in the number of post-mitotic cells in Rb−/− vs. Rb+/− embryos (arrowheads in E′), thus suggesting the absence of major defects in cell cycle exit. (F) Quantification revealed a significant increase in the average surface area of OE in Rb−/− embryos vs. Rb+/− control littermates at P0, consistent with the increased proliferation described above. (G) Quantifications of BrdU+, BrdU+Tuj1+ and BrdU+Tuj1+ (IZ, intermediate zone) cells in the OE at E15.5 and E18.5 showing 2–3-fold increase in the numbers of these cells in the absence of Rb (see text for detail). (H) Graph showing no change in the ratios of double positive cells to BrdU+ cells between genotypes at the ages examined (H). Error bars represent SD of measurements from n = 3 per genotype and asterisks indicate a statistical significant difference between genotypes using 2-way ANOVA tests, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Scale bars = 100 μm.