Fig. 2. Olomoucine induces lengthening of the cell cycle in WEC. (A) Size (crown to rump) of E9.5 embryos developed for 24 hours in WEC in the absence (control) or presence of 80 µM iso-olomoucine (iso-olo) or olomoucine (olo) followed by fixation. Arrows indicate the reported (Kaufman, 1992) size of fixed embryos with 22-26 pairs of somites (E9.5) and 30-35 pairs of somites (E10.25-10.5). Bars represent the s.d.; control, n=5; iso-olo, n=8 and olo, n=7; olo vs. iso-olo P<0.0001. (B) Analyses of NE cells in the rostral telencephalon of E9.5 littermate embryos developed in WEC in the presence of 80 µM iso-olomoucine (iso-olo) or olomoucine (olo). (a,b) TUNEL staining after 24 hours WEC. (c,d) BrdU immunoreactivity after 19 hours WEC, with 50 µM BrdU added at 3 hours. (a-d) Scale bar: 50 µm. (e) Cumulative BrdU labeling, with 50 µM BrdU added at 3 hours (time=0) and WEC being continued for the indicated times. The BrdU labeling index indicates the proportion of DAPI-stained cell nuclei in the neuroepithelium that were stained for BrdU (all nuclei stained for BrdU=1.0). For each time point, one littermate embryo each was analyzed for iso-olo (filled circles) and olo (open squares); data are the mean of two independent litters (except for the 1 hour time point); bars indicate the variation of the duplicate values from the mean; correlation coefficients are r²=0.995 and r²=0.998 for iso-olo and olo, respectively. The dotted line indicates the BrdU labeling index observed after 16 hours of BrdU labeling, which was the same for iso-olo and olo (for clarity, the filled circle and open square symbols at the 16 hours time point are placed next to each other). The extrapolated intercept of the iso-olo and olo best-fit lines with the dotted line is indicated by the solid and dashed arrow on the abscissa, respectively, and provides an estimate of the sum of the lengths of the G2, M, plus G1 phases (plus the fraction of S phase required to detect BrdU incorporation).