FIGURE 5

FIGURE 5

Cell detachment is related to aberrant mitosis in mastl^kt441b homozygous embryos. Time-lapse imaging was carried out focusing on the tailbud region of mastl^kt441b homozygous embryos carrying the Bac(foxa2:Kaede) transgene at the 8 to 16-somite stage. Kaede-positive cells that detached from the CNH were reverse tracked. (A) Summary of the tracking of detached cells in mastl^kt441b homozygous embryos. Among 21 cells, 12 cells were successfully tracked back to the CNH, and all of them detached during aberrant mitosis. A schematic summary of tracked cells is shown in Supplementary Figure S3(B) Time-lapse images of one detached cell in an embryo. Only one detached cell was detached among 5 sibling embryos. It was detached from the CNH at time 0 (white arrows) and died (orange arrows) soon thereafter. Chromosomes were not condensed. Scale bar, 20 μm. (C) A representative image of cells detaching from the CNH in mastl^kt441b homozygous embryos. A mother cell in the CNH (white arrows) caused aberrant chromosome segregation at time 0. Both daughter cells detached from the CNH and maintained condensed chromosomes after segregation for around 2 h (yellow arrows). Their chromosomes appeared to decondense later (blue arrows). One of the daughter cells (upper panel) returned to the CNH. In this experiment, mastl^kt441b homozygous embryos harboring the Bac(foxa2:Kaede) transgene were used. Nuclei were labeled by injection of Histone H2A-mCherry mRNA into 1 blastomere of the 8-cell stage. Green: CNH cells, magenta: Histone H2A-mCherry. (D) A representative cell that was detached and then divided near the CNH in mastl^kt441b homozygous embryos. White arrows: the mother cell; Yellow arrows: condensed chromosomes; Gray arrows: chromosomes that could not be judged condensed or decondensed. (E) A representative image of normal cell division of CNH cells in sibling embryos. White arrows: metaphase; Yellow arrows: anaphase; Blue arrows: decondensed chromosomes in telophase. (F) Duration of the onset of chromosome segregation (anaphase) to chromosome decondensation. Detached cells were analyzed in mastl^kt4441b homozygous embryos, comparing with CNH cells in siblings. In many cases, daughter cells in mastl^kt4441b homozygotes could not be tracked until chromosome decondensation, because some daughter cells framed out, displayed reduced Kaede signals, or maintained condensed chromosomes until the time end of imaging (shown by open circles). On the other hand, examples in which time from chromosome segregation to chromosome decondensation was measured in the period of time-lapse imaging is shown by closed circles. Box plots of duration in each genotype show the first and third quartile, a line represents the median, and whiskers indicate the minimum and maximum inside 1.5 IQR (inter-quartile range). The time difference was statistically evaluated using the exact Wilcoxon-Mann-Whitney test. ***p = 0.00016 (G) The number of detached cells in 4 mastl^kt441b homozygous embryos at the 12-somite stage and the 16-somite stage were classified by pHH3 staining and chromosome condensation. Note that all cells with condensed chromosomes were pHH3-positive.