Fig. 8

Regulation of the cellular proliferation by Lhx2 and Six3b. The number and the proportion of proliferating cells in the heads of the embryos that were rescued and not rescued at 17 hpf. Proliferating cells were detected by immunostaining with anti-phospho-Histone H3 polyclonal antibody. The nuclei in proliferating cells in the stacked confocal image of the head region (a portion from the rostral-most tip to the caudal-most edge of the optic vesicle) were counted (shown as white bars in A–G, upper panels) in the embryos that were uninjected (A), six3-AMO-injected with lhx2-uncaged (B), lhx2-AMO-injected with six3b-uncaged (C), lhx2-AMO-injected (D), six3-AMO-injected (E), lhx2-uncaged (F) and six3b-uncaged (G). The proportions of the numbers of proliferating cells among all cells in the forebrain were manifested by co-labeling with CYTOX Orange (red) in the frontal view of the cross-sections (2.5 μm) near the anterior end of the telencephalon, and quantification of the proportions (%) was carried out by counting the number of cells which were labeled with anti-phospho-Histone H3 antibody and with CYTOX Orange. (H) The quantified comparison of the proliferating cells among all cells in the cross-sections near the anterior end of the telencephalon. Measurements in each experiment are shown as percentages with the standard error. In each measurement, n = 5. (I) Uncaging of lhx2:GFP mRNA in the forebrain (upper panels) and α-Tb immunostaining of the telencephalic neurons at 24 hpf (lower panels) of the lhx2-uncaged embryos, which were not treated or treated with 75 μM aphidicolin. The arrowhead indicates impaired increase in the number of telencephalic neurons in the uncaged embryos in the presence of aphidicolin. The embryos shown in the upper and lower panels are typical examples, but they are not the same.