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Mutations in Drosophila myb lead to centrosome amplification and genomic instability

Siau-Min Fung, Gary Ramsay and Alisa L. Katzen*

Department of Molecular Genetics, University of Illinois at Chicago, College of Medicine, Chicago, IL 60607-7170, USA



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  		Fig. 1. Dm myb mutants have missing and improperly oriented abdominal bristles, and patches of undifferentiated tissue. (A-H) Comparison of wild-type and mutant abdomens. Dorsal (A-D,H) and ventral (E-G) cuticles from female adult abdomens: control w/Df(1)sd72a (A,E) flies raised at 24°C; myb2 (B,F) flies raised at 24°C; (C) myb1 raised at 18°C; (H) esg mutant (esgk0060/Df(2L)TE35D-2) raised at 24°C; pharate adults of myb2/Df(1)sd72a raised at 24°C (D); and pharate adults of myb2 raised at 28°C (G). (I-L) Comparison of wild-type and mutant abdominal histoblast nuclei in third instar larvae raised at 24°C. All nuclei within the field were visualized by DAPI staining. (I'-L') Abdominal histoblast nest cells were identified by using ß-galactosidase antibodies and the enhancer trap insert esgP3. (I) Control w; esgP3/+; (J) myb2; esgP3/+; (K) myb2/Df(1)sd72a; esgP3/+; (L) esgVS8/esgP3. Scale bars: in A, 0.5 mm for A-H; in I', 0.05 mm in I-L'.

 


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  		Fig. 2. Abdominal development is delayed in Dm myb mutants. At specified timepoints (hours after puparium formation), the abdominal epidermis was dissected from females that had been raised at 18°C until puparium formation and then shifted to 24°C for continued development. Samples were stained with DAPI to visualize nuclei. Large nuclei represent larval polyploid cells; small nuclei correspond to the diploid nuclei of abdominal histoblast cells. (A-C) w/Df(1)sd72a controls at 13 (A), 30 (B), and 42 (C) hours APF; (D-F) myb2 at 18 (D), 42 (E) and 51 (F) hours APF; (G) myb2/Df(1)sd72a at 42 hours APF; (H) myb2/Df(1)sd72a; P(w+, myb+) at 30 hours APF; and (I) myb1 at 36 hours APF. Arrowheads indicate small histoblast nests. Scale bar: 0.05 mm.

 


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  		Fig. 3. Chromosome condensation is delayed in abdominal histoblasts that are mutant for Dm myb. Abdominal epidermal samples from females were doubly stained with DAPI to visualize nuclei (blue in merged panels) and with the PH3 antibody to identify mitotic cells (red in merged panels, but appearing as magenta because of superimposition on blue). Scale bars: in A, 0.01 mm for A-C; in E, 0.01 mm for D,E. (A,D) w/Df(1)sd72a controls at 36 hours APF; (B) myb2 at 36-42 hours APF; (C,E) myb2/Df(1)sd72a at 42-47 hours APF. Asterisks in (B,C) indicate examples of ‘pre-prophase’ cells. For comparison, examples of prophase (D) and ‘pre-prophase’ (E) cells are shown at higher magnification.

 


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  		Fig. 4. Most defective mitoses in abdominal histoblasts that are mutant for Dm myb display an aberrant number of centrosomes with abnormal spindles and nuclear morphology. Abdominal epidermal samples from females were treated with DAPI to visualize nuclei (blue in merged panels), and immunostained with antibodies against ß-tubulin (green in merged panels) and against two centrosomal proteins, CP60 and CP190 (red in merged panels). Interphase nuclei appear magenta in merges due to combined signal from DAPI and the nuclear CP60/190 proteins. Scale bar in A: 0.01mm. One centrosome was visible at each pole in control cells during anaphase (A) w/Df(1)sd72a, 40 hours APF; whereas two centrosomes were frequently seen at each pole in Dm myb mutant homozygotes (B) myb2, 44 hours APF. (C-F) Additional representatives of mutant cells with abnormal numbers of centrosomes. (C) myb2/Df(1)sd72a, 48 hours APF, five centrosomes; relatively normal anaphase with evidence of straggling DNA (arrow). (D) myb2/Df(1)sd72a, 48 hours APF, three centrosomes; disrupted organization of spindles and chromosomes during metaphase. (E) myb1/Df(1)sd72a, 67 hours APF at 18°C, four centrosomes; two metaphase plates. (F) myb2, 42 hours APF, one centrosome organizing a monopolar spindle.

 


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  		Fig. 5. Ectopic expression of RBF delayed proliferation of abdominal histoblasts, but did not result in abnormal numbers of centrosomes during mitosis. White prepupae from either UAS-RBF alone as a control (A) or hsp70-GAL4/UAS-RBF (B-D) were picked and subjected to a 40 minute heat shock at 37°C. Heat shock treatment was repeated every 12 hours until pupae were dissected at 40-43 hours APF. Samples were treated with DAPI to visualize nuclei (A,B, and blue in merged panels), and immunostained with antibodies against ß-tubulin (green in merged panels) and two centrosomal proteins, CP60 and CP190 (red in merged panels). Low magnification views show normal development of the heat shocked UAS-RBF control (A) and the developmental delay caused by ectopic expression of RBF (B). Arrows indicate areas where larval polyploid cells have not been replaced by histoblasts. Representative metaphase (C) and anaphase (D) nuclei from the delayed mitoses have appropriate numbers of centrosomes. Scale bar: in A, 0.1 mm for A,B; in C, 0.01 mm for C,D

 


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  		Fig. 6. Chromosomal dynamics are disturbed during mitosis in mutant Dm myb abdominal histoblasts. Abdominal epidermal samples from females were doubly stained with DAPI to visualize nuclei (blue in merged panels) and with antibodies against the kinetochore associated protein, Bub1 (red in merged panels). (A-C) Mutations in Dm myb disturbed kinetochore arrangement during metaphase. (A) w/Df(1)sd72a control, 24 hours APF. (B) myb2, 42 hours APF. (C) The presence of more than 16 Bub1 signals in this myb2/Df(1)sd72a cell (42 hours APF) is indicative of polyploidy and demonstrates that such cells continue to progress into mitosis. Scale bar: 0.01 mm.

 


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  		Fig. 7. Aneuploidy, polyploidy and variable nuclear morphology in mutant Dm myb abdominal epidermal cells. (A,B) Abdominal epidermal samples from female pupae hybridized with an X-chromosome probe (red), and stained with DAPI (blue) and PH3 antibodies (green). (A) Two signals were seen in each of two separating nuclei in w/Df(1)sd72a controls during anaphase. (B) By contrast, a different number of signals (1, 4 and 6) were evident in each of three separating nuclei in a mutant myb2 cell. (C-K) Abdominal epidermal samples from females at 30 hours APF for controls and at 44-46 hours APF for Dm myb mutants were stained with DAPI for DNA quantitation (blue in D-K) and rhodamine-labeled phalloidin (red in D-K), and optically sectioned by confocal microscopy under identical conditions. For each of three experiments, nuclear fluorescent intensities were measured. The average value of the G1 control nuclei was determined and used as the base value of 1x. Values of other nuclei were adjusted accordingly to calculate relative fluorescent intensities. (C) Results of analyzing 121 control (w/Df(1)sd72a); 124 myb2, and 83 myb2/Df(1)sd72a nuclei are graphically represented. The range of values included in each category are indicated in parentheses as follows: <= 0.5x (<= 0.6); 1x (0.7-1.5x); 2x (1.6-2.5x) and >= 3x (>= 2.6). (D-K) Examples of control and mutant cells with relative DNA quantitation values as indicated. (D) Three control w/Df(1)sd72a G1 nuclei; (E) a control G2 nucleus is centered; (F) enlarged myb2 nucleus; (G) binucleate myb2 cell; (H) mis-shapen and enlarged myb2/Df(1)sd72a nucleus; (I) multilobed myb2/Df(1)sd72a nucleus; (J) a myb2/Df(1)sd72a nucleus with subdiploid DNA content (arrow); (K) a myb2/Df(1)sd72a cell containing a large nucleus and a micronucleus indicated by arrow. Scale bars: in A, 0.01 mm in A,B; in K, 0.01 mm in D-K.

 

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© The Company of Biologists Ltd 2002