Fig. 6. Ngn2 is expressed in a subset of cycling cells. (A,B) Ngn2 and phosphorylated vimentin (p-vim) double staining at E13 to compare percentage Ngn2⁺ between S- and NS-dividing cells. Two NS mitoses (arrows in B, merged image photographed with a 100x lens is shown in C) were weakly positive for Ngn2, whereas S mitoses in this field were all Ngn2-negative (some are magnified in D). See Table 3 for quantification. Pial surface is indicated with asterisk. (E,F) Ngn2 and Ki67 double staining to examine Ngn2 expression in relation to cell cycling. Our grading of Ngn2 and Ki67 intensity is exemplified using a picture taken at the ventricular zone (VZ)/subventricular zone (SV) border of an E14 section. The presented 7 cells included a Ngn2⁺⁺⁺Ki67^– cell, a Ngn2⁺⁺Ki67⁺ cell, a Ngn2⁺Ki67⁺⁺⁺ cell, a Ngn2⁺Ki67⁺⁺ cell, and three Ngn2^–Ki67⁺ cells. In separate staining of equivalent sections, Ki67⁺⁺⁺ cells were nearly always pH3⁺ (not shown). (G) There was an observed relationship between the intensity of Ki67 immunoreactivity and that of Ngn2 immunoreactivity. Graph shows the proportion of cells in each of the 3x3 fractions (043%) out of the total cells that were simultaneously positive (++++) for Ki67 and Ngn2 (100%); indicated values are obtained from 458 cells examined using a confocal microscope (see also Table 4). (H-K) Milder treatment with anti-Ki67 visualizes cells during or just prior to division. By using diluted anti-Ki67 antibody (5-10 times lower in concentration compared with one used for Fig. 1B,C, Fig. 6F,G), Ki67⁺⁺⁺ or Ki67⁺⁺ cells were more clearly discriminated from Ki67⁺ cells which were only faintly visualized. Under these conditions, the proportion of Ngn2-positive cells out of the Ki67⁺⁺⁺ cells was obtained for either of the S-dividing and NS-dividing populations (Table 3). In this field, one NS-dividing cell was Ngn2⁺, but S-dividing cells were all Ngn2^–. Of the two Ki67⁺⁺ cells (outlined), the upper cell was Ngn2⁺, whereas the lower, triangular-shaped cell was Ngn2^–. (L-Q) Anti-Ngn2 staining on time-lapse monitored cells to examine cell cycle-dependent and lineage-restricted expression of Ngn2. Strongest immunoreactivity was observed in some (but not all) of the cells 4-8 hours after generation [L,M: the indicated daughter cell was Ngn2⁺⁺⁺, whereas the other was Ngn2^– (not shown); cerebral wall thickness at 0 hour was approximately 180 µm]. (N,O) Pin-like cells losing the ventricular process (arrowheads in N) showed a moderate level of Ngn2 expression (O, photographed at two different planes). (P,Q) We could not detect Ngn2 expression in cells quickly moving towards the ventricular surface. Scale bars: 10 µm in A-F,H-K,M,O,Q.