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FIGURE 1:. Individual condensed chromosome size is not constant during early embryogenesis. (A–I) Chromosomes in squashed embryos at each cell stage; bar = 5 μm. (J) The lengths and (K) widths of chromosomes at each cell stage are shown in the box plot. The middle line of each box is the median. The top and bottom lines are the third and first quartiles, respectively, and the whiskers indicate the 90th and 10th percentiles. The distributions of chromosome length and width in each cell stage are shown in histograms (Supplemental Figures S1 and S2).
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FIGURE 2:. Dynamics of chromosome condensation in various cell stages. (A) Representative nuclear images at various cell stages of a control embryo and one-cell-stage smc-4 (RNAi) embryo. Images are at 50-s intervals. Time corresponds to NEBD of 0. Bar, 5 μm. (B) Average of condensation parameter versus time. Colors indicate data from different cell stages of wild-type embryos (blue, 1-cell stage, male pronucleus, n = 6; light blue, 2-cell stage, n = 5; green, 4-cell stage, n = 7; brown, 8-cell stage, n = 8; yellow, 16-cell stage, n = 2; pink, 28-cell stage, n = 8) and one-cell-stage smc-4 (RNAi) embryos (gray, n = 5). The average condensation parameter was calculated after aligning the sequences to NEBD. Error bars are SE. The sudden increase in the condensation parameter in smc-4 (RNAi) around NEBD likely reflects the diffusion of free histones throughout the cytoplasm and might not reflect a change in chromosome condensation.
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FIGURE 3:. Reduced or increased DNA content leads to increased or reduced condensed chromosome size, respectively. The lengths of condensed chromosomes at each cell stage are shown in the box plot. Data for wild-type (blue), klp-18 (RNAi) haploid (yellow), klp-18 (RNAi) polyploid (pink), and mei-1 (RNAi) haploid (green) embryos. Chromosome lengths at almost all stages, except for the one-cell stage (haploid per pronucleus only at the one-cell stage) in klp-18 (RNAi) haploid or mei-1 (RNAi) haploid embryos, were significantly larger than those of wild-type embryos (Student's t test). *p < 0.005; **0.005 < p < 0.05. The chromosome lengths in klp-18 (RNAi) polyploid embryos were significantly smaller than those of wild-type embryos at each cell stage. Note that the pronuclei contained a haploid genome in wild-type and klp-18 or mei-1 (RNAi) embryos at the one-cell stage. The middle line of each box is the median. The top and bottom lines are the third and first quartiles, respectively, and the whiskers indicate 90th and 10th percentiles. N.D., not determined. Mean length, SD, and sample size are shown in Table 1.
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FIGURE 4:. Nucleus size affects condensed chromosome size. (A) Relationship between measured nuclear diameter and cell length at various cell stages of wild-type embryos. Different shapes and colors represent each cell stage: blue circle, 1-cell stage; light blue diamond, 2- and 4-cell stages; green triangle, 8- and 16-cell stages; orange cross, 28- and 50-cell stages. (B) Mean chromosome length at each cell stage was plotted as function of the calculated relative DNA density. Data for wild-type (blue diamond), klp-18 (RNAi) haploid (yellow rectangle), mei-1 (RNAi) haploid (green rectangle), ran-3 (gray triangle), ima-3 (brown circle), and C27D9.1 (RNAi) embryos (pink cross). We do not include data from klp-18 (RNAi) polyploid embryo because the exact ploidy of the embryos could not be determined. The regression line for the wild-type data is shown. Error bars are SE. (C) Individual nuclear diameters for wild-type (blue), ran-3 (gray), ima-3 (brown), and C27D9.1 (RNAi) embryos (pink) at each cell stage are shown in the box plot. The nuclear diameters in each RNAi embryo in all cell stages were significantly different from those of wild-type embryos (Student's t test). (D) Chromosome length at most cell stages in ran-3, ima-3, C27D9.1 (RNAi) embryos significantly differed from those of wild-type embryos (Student's t test). *p < 0.005; **0.005 < p < 0.05. The middle line of each box is the median. The top and bottom lines are the third and first quartiles, respectively, and the whiskers indicate the 90th and 10th percentiles. Mean chromosome length, nuclear diameter, SD, and sample size are shown in Tables 1 and 2.
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FIGURE 5:. Chromosome sizes reconstructed in vitro using X. laevis egg extracts. (A) Examples of nuclei reconstructed from control extracts, with WGA, or with anti–lamin LIII antibody. (B) Quantified nuclear diameters in the extracts for control, WGA, and anti-lamin LIII antibody. (C) Representative chromosomes reconstituted with the extracts after nuclear formation. (D, E) Length (D) and width (E) of the reconstituted chromosomes. The middle line of each box is the median. The top and bottom lines are the third and first quartiles, respectively, and the whiskers indicate the 90th and 10th percentiles. Mean chromosome length, chromosome width, nuclear diameter, SD, and sample size are shown in Table 3. *p < 0.005.
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FIGURE 6:. Curve fit of the estimated packing ratio vs. intranuclear DNA density. Linear packing ratio was plotted vs. intranuclear DNA density, which was calculated by dividing DNA content (Mbp) by the nuclear radius (μm). Data are summarized in Supplemental Table S3. The plot was fitted to the exponential curve with the least-squares method. Black circle, C. elegans wild type (diploid); black diamond, C. elegans klp-18 (RNAi) haploid; gray diamond, C. elegans mei-1 (RNAi) haploid; gray triangle, C. elegans ran-3 (RNAi); black triangle, C. elegans ima-3 (RNAi); black square, C. elegans C27D9.1 (RNAi); white circle, X. laevis.
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