EdU pulse labeling and PH3 expression. (A-D) Confocal images of E11.5 frontal sections showing double staining of PH3 and EdU pulsed for different durations. Midline is to the left. (A) With a 0.5-hour EdU pulse, PH3 shows no co-localization with EdU in both apically and basally dividing cells. (B) With a 2-hour EdU pulse, some cells show co-localization in both apical and basal locations (arrowheads). Compared with the 0.5-hour pulse, more EdU-labeled cells are found close to the ventricle (arrow). (C) With a 4-hour EdU pulse, many cells express both PH3 and EdU both apically and basally (arrowheads). (D) With an 8-hour EdU pulse, no co-localization of EdU and PH3 is detected. Dashed lines indicate the large, central cluster of EdU-labeled cells. (E) Cell counts showing the proportion of PH3+/EdU+ cells in total PH3+ cells at E11.5 and E12.5. The greatest proportion of co-localization is found with a 4-hour pulse, suggesting that most cells enter M phase within 4 hours after S phase. Cell counts are shown as mean ± standard error of the mean; one-way ANOVA test F = 27.70, P < 0.0001. Post-hoc Tukey test shows significant difference between EdU 0.5 hours versus 2 hours (P < 0.01), EdU 0.5 hours versus 4 hours (P < 0.001), EdU 2 hours versus 4 hours (P < 0.05), EdU 2 hours versus 8 hours (P < 0.01), and EdU 4 hours versus 8 hours (P < 0.001) at E11.5 and a significant difference between EdU 0.5 hours versus 4 hours (P < 0.01), EdU 2 hours versus 4 hours (P < 0.01), and EdU 4 hours versus 8 hours (P < 0.01) at E12.5. No significance is detected for ratios between apically and basally dividing cells at the same pulse time. Scale bar: 50 μm. (F,G) Distribution of EdU-positive cells for each pulse time at E11.5 (F) and E12.5 (G). Each bin is 20-μm wide and bin 1 is at the surface of the third ventricle. Three sections for each pulse time at each stage were averaged.