Figure 4.
Figure 4. Evidence for chromosome breakage in two transgenic lines overexpressing Plk4. (A) Time-lapse images of chromosomes labeled with histone-RFP followed in Plk4 OE (line A) during meiosis I. Black arrows point at the smallest chromosome fragment moving back and forth on the metaphase plate. Chromosomes appear in gray levels. Timing (expressed in hours and minutes) is relative to NEBD. Scale bar is 10 µm. The right panel shows the smallest DNA fragment (gray) at a higher magnification harboring a Mad2-YFP signal (yellow) at NEBD + 6h10. Scale bar is 1 µm. (B and D) Immunofluorescent costaining of DNA (white), CREST (pink), and Tubulin (green) in Plk4 OE line A (B) and line B (D) oocytes observed at NEBD + 6h00. White arrows point at the chromosome pieces. Higher magnification of DNA pieces is shown in the right panels: the upper right panel corresponds to the largest one and the lower right panel to the smallest one. Scale bar is 20 µm in the left panels, 5 µm in the middle panels, and 1 µm in the right panels. (C and E) Examples of fixed oocytes with broken bivalents in line A (C) and line B (E) observed at NEBD + 6h00. Broken bivalents have been artificially colored in blue for line A (C) and in green for line B (E). Scale bar is 5 µm. (F) Quantitative analysis of the volume of the smallest chromosome fragment in oocytes from Plk4 OE lines A and B observed at NEBD + 6h00; ****, P < 0.0001 (two-tailed t test with Welch correction); n is the number of oocytes. (G) Quantification of the percentage of oocytes with one broken bivalent in Plk4 OE line A and B observed at NEBD + 6h00; *, P = 0.0104 (Fisher’s exact test); N is the number of mice, n is the number of oocytes. Error bars correspond to SD. (H) Scheme representing the positions of the two mCherry-Plk4 transgenes: the insertion (in blue) is closer to the centromeric region (in pink) of chromosome 9 for line A and the insertion (in green) is closer to the telomeric region of chromosome 12 for line B. (I) Two examples of FISH labeling of chromosome 9 (blue) in line A observed at NEBD + 6h00. 36 oocytes were collected from three mice. Among them, 16.67% had a broken bivalent. These broken bivalents were always labeled by the FISH probe for chromosome 9. DNA is labeled in gray. The FISH signal appears in blue. Scale bar is 10 µm for the left upper panel. Higher magnifications are shown in the middle and right upper panels; scale bar is 2 µm. Chromosome fragments labeled by the FISH probe appear in black on the right panels. (J) Proposed model of bivalent breakage occurring in the two Plk4 OE lines. MTs appear in dark green. Kinetochores are in pink. The mCherry-Plk4 transgene insertion sites appear in blue (line A) and green (line B).

Evidence for chromosome breakage in two transgenic lines overexpressing Plk4. (A) Time-lapse images of chromosomes labeled with histone-RFP followed in Plk4 OE (line A) during meiosis I. Black arrows point at the smallest chromosome fragment moving back and forth on the metaphase plate. Chromosomes appear in gray levels. Timing (expressed in hours and minutes) is relative to NEBD. Scale bar is 10 µm. The right panel shows the smallest DNA fragment (gray) at a higher magnification harboring a Mad2-YFP signal (yellow) at NEBD + 6h10. Scale bar is 1 µm. (B and D) Immunofluorescent costaining of DNA (white), CREST (pink), and Tubulin (green) in Plk4 OE line A (B) and line B (D) oocytes observed at NEBD + 6h00. White arrows point at the chromosome pieces. Higher magnification of DNA pieces is shown in the right panels: the upper right panel corresponds to the largest one and the lower right panel to the smallest one. Scale bar is 20 µm in the left panels, 5 µm in the middle panels, and 1 µm in the right panels. (C and E) Examples of fixed oocytes with broken bivalents in line A (C) and line B (E) observed at NEBD + 6h00. Broken bivalents have been artificially colored in blue for line A (C) and in green for line B (E). Scale bar is 5 µm. (F) Quantitative analysis of the volume of the smallest chromosome fragment in oocytes from Plk4 OE lines A and B observed at NEBD + 6h00; ****, P < 0.0001 (two-tailed t test with Welch correction); n is the number of oocytes. (G) Quantification of the percentage of oocytes with one broken bivalent in Plk4 OE line A and B observed at NEBD + 6h00; *, P = 0.0104 (Fisher’s exact test); N is the number of mice, n is the number of oocytes. Error bars correspond to SD. (H) Scheme representing the positions of the two mCherry-Plk4 transgenes: the insertion (in blue) is closer to the centromeric region (in pink) of chromosome 9 for line A and the insertion (in green) is closer to the telomeric region of chromosome 12 for line B. (I) Two examples of FISH labeling of chromosome 9 (blue) in line A observed at NEBD + 6h00. 36 oocytes were collected from three mice. Among them, 16.67% had a broken bivalent. These broken bivalents were always labeled by the FISH probe for chromosome 9. DNA is labeled in gray. The FISH signal appears in blue. Scale bar is 10 µm for the left upper panel. Higher magnifications are shown in the middle and right upper panels; scale bar is 2 µm. Chromosome fragments labeled by the FISH probe appear in black on the right panels. (J) Proposed model of bivalent breakage occurring in the two Plk4 OE lines. MTs appear in dark green. Kinetochores are in pink. The mCherry-Plk4 transgene insertion sites appear in blue (line A) and green (line B).

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