Fig. 4: Four-cell replication stress as a source of chromosome errors.

a, Live imaging of mEGFP–PCNA (S-phase marker, green) and H2B–mCherry (chromosomes, magenta) in mouse embryos. Magnified z-projection snapshots of 4-cell sister blastomeres are shown, along with 3D-reconstructed images of 2-to-4-cell and 4-to-8-cell divisions (h:min, time after 2-cell anaphase onset). The image frame colours reflect the cell-cycle phases in b. The asterisks show PCNA foci (late-S phase marker). b–d, Extended S phase often precedes chromosome bridge formation during 4-to-8-cell division (error). In b, each line represents a single cell, and the average cell-cycle durations are shown in c, based on durations plotted in d. The number (n) of cells from 24 embryos is shown. e, Nucleoside-supplementation experiment. Embryos cultured for 6 h in +nucleoside medium from 4-cell G1 were sequentially labelled with IdU and CldU for 30 min each, then analysed using the DNA fibre spreading assay (f and g). Control embryos were sampled simultaneously at mid/late-S phase. f, DNA fibre classification of +nucleoside 4-cell embryos based on fork categories (Fig. 2h). Control 4-cell embryos differ slightly from Fig. 2i, possibly reflecting differences between mid/late-S phase (f) and early-S phase (Fig. 2i). g, Mobile fork speed. h, Nucleosides suppressed p-CHK1 at the 4-cell late-S phase. The numbers of nuclei from 30 (control) and 12 (+nucleosides) embryos are shown. i, Nucleosides suppressed γH2AX at the 4-cell late-S phase. Histone H3, magenta. The numbers (n) of nuclei from 23 (control) and 15 (+nucleosides) embryos are shown. j, Nucleosides suppressed chromosome segregation errors. The chromosome-aberration frequency at anaphase during 4-to-8-cell division was monitored using live-cell imaging. The numbers (n) of cell divisions analysed live are shown. Scale bars, 10 μm (a (blue and grey boxes)) and 5 μm (a (magenta boxes), h and i). Error bars represent the mean ± s.d. (d, g–i).