Figure 5.
Figure 5. Chromosome segregation defects in SSX2IP2 MO–treated eggs. (A) Low concentrations of SSX2IP antibodies (100 ng/µl, injected at one-cell stage eggs of medaka) were used to localize SSX2IP in embryos developed to stage 10–11. Colors of labels indicate colors of respective proteins. Magnified panels (magn.) show enlarged views of the boxed regions. (B) Medaka eggs were coinjected with either control MO (Cont. MO) or SSX2IP2-MO, and Histone H2B-GFP (H2B-GFP) mRNA. Developing embryos were analyzed around stage 8 with DSLM. (top) Representative global images of MO-treated embryos. Red arrowheads in SSX2IP-MO show abnormal cell divisions. (bottom) Still images of dividing blastomeres in Cont.MO and SSX2IP-MO injected eggs. Yellow arrowheads indicate dividing cells; red arrows indicate lagging chromosomes. Each image was taken with an interval of 1 min. (C) Quantification of abnormal chromosomes/lagging chromosomes in control and MO-injected eggs. The significance was calculated by a Student’s t test (two-tailed) and scored ***, P < 0.001. Error bars indicate SD. (D–F) Rescue of SSX2IP-knockdown eggs by EGFP-OlSSX2IP2 (EGFP-SSX2IP) expression. Eggs were coinjected with SSX2IP2-MO and EGFP-SSX2IP. (D) Quantification of γ-tubulin foci in MO-treated and rescued eggs. Numbers of γ-tubulin foci were categorized as shown in representative images of γ-tubulin staining (top panels). The significance was calculated using six or seven independent experiments, in which at least 100 structures were counted. Significance was scored by a Student’s t test (two-tailed) as follows: ***, P < 0.001; ****, P < 0.0001. Error bars indicate SD. (E) Quantification of abnormal spindles in MO-treated and rescued eggs. The significance was calculated by a Student’s t test (two-tailed) and scored as follows: *, P < 0.05; **, P < 0.01. Error bars indicate SD. (F) γ-tubulin and α-tubulin, in MO-treated and rescued eggs at stage 10, was visualized as indicated by the colored legends. Dashed lines crossed by a bar indicate telophase spindles. Magnified panels (magn.) show enlarged views of the boxed regions. Arrowheads indicate abnormal numbers of centrosomes. Arrows indicate localization of EGFP-SSX2IP at the centrosome. Bars: (A) 10 µm; (B, top) 40 µm; (B, bottom) 20 µm; (D) 20 µm; (F) 10 µm.

Chromosome segregation defects in SSX2IP2 MO–treated eggs. (A) Low concentrations of SSX2IP antibodies (100 ng/µl, injected at one-cell stage eggs of medaka) were used to localize SSX2IP in embryos developed to stage 10–11. Colors of labels indicate colors of respective proteins. Magnified panels (magn.) show enlarged views of the boxed regions. (B) Medaka eggs were coinjected with either control MO (Cont. MO) or SSX2IP2-MO, and Histone H2B-GFP (H2B-GFP) mRNA. Developing embryos were analyzed around stage 8 with DSLM. (top) Representative global images of MO-treated embryos. Red arrowheads in SSX2IP-MO show abnormal cell divisions. (bottom) Still images of dividing blastomeres in Cont.MO and SSX2IP-MO injected eggs. Yellow arrowheads indicate dividing cells; red arrows indicate lagging chromosomes. Each image was taken with an interval of 1 min. (C) Quantification of abnormal chromosomes/lagging chromosomes in control and MO-injected eggs. The significance was calculated by a Student’s t test (two-tailed) and scored ***, P < 0.001. Error bars indicate SD. (D–F) Rescue of SSX2IP-knockdown eggs by EGFP-OlSSX2IP2 (EGFP-SSX2IP) expression. Eggs were coinjected with SSX2IP2-MO and EGFP-SSX2IP. (D) Quantification of γ-tubulin foci in MO-treated and rescued eggs. Numbers of γ-tubulin foci were categorized as shown in representative images of γ-tubulin staining (top panels). The significance was calculated using six or seven independent experiments, in which at least 100 structures were counted. Significance was scored by a Student’s t test (two-tailed) as follows: ***, P < 0.001; ****, P < 0.0001. Error bars indicate SD. (E) Quantification of abnormal spindles in MO-treated and rescued eggs. The significance was calculated by a Student’s t test (two-tailed) and scored as follows: *, P < 0.05; **, P < 0.01. Error bars indicate SD. (F) γ-tubulin and α-tubulin, in MO-treated and rescued eggs at stage 10, was visualized as indicated by the colored legends. Dashed lines crossed by a bar indicate telophase spindles. Magnified panels (magn.) show enlarged views of the boxed regions. Arrowheads indicate abnormal numbers of centrosomes. Arrows indicate localization of EGFP-SSX2IP at the centrosome. Bars: (A) 10 µm; (B, top) 40 µm; (B, bottom) 20 µm; (D) 20 µm; (F) 10 µm.

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