FIGURE 3

mastl^kt441b homozygotes exhibit mitotic defects. (A) Phosphorylation of Histone H3 Ser10 (pHH3) in whole-mount embryos at the 6-somite stage (ss) and the 14 ss. At the 6 ss, no difference in phosphorylation was detectable among embryos containing siblings and mastl^kt441b homozygotes. At the 14 ss, the number of pHH3-positive cells was increased in mastl^kt441b homozygous embryos. For 14 ss embryos, 10 embryos of each phenotype were genotyped. (B) Time from prometaphase to the onset of anaphase of CNH cells. The duration was measured by time-lapse imaging of CNH cells with labeling of nuclei using Histone H2A-mChrery. The percentage of cells in which the duration of this process exceeded 30 min is indicated in dark gray. siblings: n = 10, mastl^kt441b homozygotes: n = 18. (C) Classification of abnormalities of mitotic cells in the tail bud of siblings and mastl^kt441b homozygous embryos. Embryos were labeled with Histone H2A-mCherry and sparsely labeled with membrane-Venus. All membrane-Venus labeled cells with condensed chromosomes within the first 30 min were tracked until the end of time-lapse imaging (2–3.5 h) or as long as we could track them (siblings: n = 50 cells, mast^kt441b homozygous: n = 66 cells, from 3 embryos each). We classified tracked cells as follows. normal: normal cell division, missegregation: chromosomes were scattered around an elongated spindle, and cytokinesis was completed to pinch off some of the chromosomes, no segregation: cytokinesis was completed without obvious chromosome segregation, prolonged metaphase: metaphase was prolonged for more than 60 min without chromosome segregation or cytokinesis; decondensation: decondensation of chromosomes without segregation. cell death: change to hypercondensed nucleus. Time lapse images of representative cells for each type are shown in (D). (D) Time lapse images of representative cells for each type shown in (C). Green indicates membrane-Venus, while magenta indicates Histone H2A-mCherry. Mitotic cell types were classified as described in (C). White arrows: mother cells with condensed chromosomes; Yellow arrows: daughter cells with condensed chromosomes; Orange arrows: hypercondensed chromosomes. Since the state of chromosome condensation was sometimes unclear, we carefully judged it referring the orthogonal views. Scale bar, 20 μm.