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First published online 14 September 2005
doi: 10.1242/dev.02028

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Maternal expression of the checkpoint protein BubR1 is required for synchrony of syncytial nuclear divisions and polar body arrest in Drosophila melanogaster

Daniel Pérez-Mongiovi^1,*, Nicolas Malmanche^1,*, Hassan Bousbaa^1,2 and Claudio Sunkel^1,3,

¹ Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal
² Instituto Superior de Ciências da Saúde-Norte, Grupo de Biologia Molecular e Celular, Rua Central de Gandra 1317, 4580 Gandra PRD, Portugal
³ ICBAS, Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, 4000 Porto, Portugal

View larger version (74K): [in a new window]   Fig. 1. Immunolocalisation of BubR1 during syncytial divisions. In all images, DNA is stained blue and BubR1 is stained red. (A-E) BubR1 immunolocalisation during syncytial nuclear division cycles 3-9 and (F-J) cycles 10-13. (A,F) BubR1 shows no specific localisation during interphase but (B,G) starts to accumulate at the kinetochores in prophase, (C,H) reaching a maximum in prometaphase, (D,I) decreases at metaphase and (E,J) is absent at anaphase. (K,K''') High-magnification view of a syncytial nucleus in prophase showing co-localisation of BubR1 and CID (shown in green). (K) Chromosome morphology at prophase showing initiation of condensation. (K') BubR1 co-localises with (K'') CID at kinetochores of prophase chromosomes. (K''') Merged image showing colocalisation of BubR1 and CID. (L,L''') High-magnification view of a syncytial nucleus at metaphase showing co-localisation of BubR1 over spindle microtubules (green). (L) Chromosome morphology at metaphase showing alignment along the metaphase plate. (L') At metaphase, BubR1 is sometimes detected along spindle microtubules (see also inset in D). (L'') Spindle microtubules were detected with anti--tubulin antibodies. (L''') Merged image showing co-localisation of BubR1 along spindle microtubules. Scale bar: 10 µm.

View larger version (46K): [in a new window]   Fig. 2. Molecular and phenotypic characterisation of bubR1Rev1. (A) Genomic organisation of bubR11, bubR1Rev1 and bubR1Rev74 alleles. bubR1Rev1 and bubR1Rev74 were isolated from a genetic screen designed to identify imprecise excisions of the P element inserted in bubR11 (l(2)K03110). Arrows indicate pairs of primers used for PCR to determine whether the ends of the P elements were intact. (B) Southern blot showing the difference between wild-type and bubR1 alleles. Although bubR1Rev74 and wild-type strains are identical, indicating that a perfect P element excision had taken place, bubR1Rev1 genomic organisation shows a smaller band at 4.3 kb (asterisk), indicating a 2 kb deletion within the P element. The genomic DNA was digested with HindIII and EcoRI. (C) Western blot analysis of BubR1 protein levels in wild-type and bubR1 alleles. Tubulin was used as a loading control. Total proteins from wild-type third instar larval brains and female ovaries shows high levels of BubR1. BubR1 could only be detected from a minimum of two wild-type embryo extract at 30-90 minutes AEL. Homozygous bubR11 and bubR1Rev1 third instar larval brains show a significant reduction in BubR1 protein levels. BubR1 protein level is significantly decreased in homozygous bubR1Rev1 ovaries and undetectable from protein extract of two bubR1Rev1 embryos. (D) Quantification of the mitotic phenotype in third instar larval brains from wild-type and bubR1 mutant allele combinations. In mutant genetic background, we observed a decrease in prometaphase-metaphase mitotic figures and an increase in the level of Sister Chromatid Separation (SCS). (E,F) Third instar larval neuroblasts incubated in 10 µM colchicine for 30 minutes induces a prometaphase-like arrest in wild-type cells (E), whereas most bubR1Rev1 mutant cells (F) undergo SCS. Scale bar: 5 µm.

View larger version (54K): [in a new window]   Fig. 3. Mitotic phenotypes in syncytial bubR1Rev1 embryos. (A) bubR1Rev1 embryos at nuclear cycle 5 in anaphase configuration. (B) Higher magnification view from a selected nuclei in A showing abnormal anaphase with asymmetric distribution of chromatin and lagging chromosomes. (C) bubR1Rev1 embryo at late syncytial stage showing irregular distribution and size of nuclei (see higher magnification in D) at the cortex (arrow) or at the interior of the embryo (arrowhead). (E-G) Abnormal mitotic progression in bubR1Rev1 embryos. (E) Prometaphase nuclei showing precocious sister chromatid separation, (F) anaphase nuclei showing lagging chromosomes and (G) telophase nuclei showing DNA bridges. (H) Mitotic domain of a gastrulated bubR1Rev1 embryo showing micronuclei (arrowhead) and pyknotic nuclei (arrow). Nuclei from wild-type (I) or bubR1Rev1 embryos (J) were imaged by time-lapse confocal microscopy of the Histone-GFP transgene during mitosis. Recording was performed from early stages of chromosome condensation until late telophase; the time is indicated in minutes in every frame (see Movies 1 and 2 in the supplementary material). Wild-type nuclei show normal chromosome congression and segregation. In bubR1Rev1 embryos, chromosomes never reach a stable metaphase configuration (arrowhead) and adjacent nuclei show highly asynchronous mitotic figures from prophase (asterisk) to anaphase (arrow). Aberrant chromosome segregation, such as lagging chromosomes (arrowhead at 11 minutes) and chromatin bridges (arrowhead at 14 minutes) are visible during anaphase and telophase. (K) Quantification of syncytial nuclear cycle stage in wild-type (n=259) and in bubR1Rev1 embryos (n=131) was performed on fixed material. Wild-type embryos show synchronous nuclei, while approximately half of the bubR1Rev1 embryos show desynchronised nuclear cycles. There is also a relative increase in prophase figures and we failed to detect any proper metaphases in bubR1Rev1 embryos. Scale bars: 100 µm in A,C; 25 µm in B,D-G; 50 µm in H; 10 µm in I,J.

View larger version (96K): [in a new window]   Fig. 4. Centrosome replication in bubR1Rev1 embryos. In all panels, DNA is in red, centrosomes are in (A-C) green or (E-H) blue with tubulin in green. (A) Cortical section of a 3 hour AEL wild-type embryo showing a homogeneous distribution of nuclei, each associated with a pair of centrosomes. (B) bubR1Rev1 embryo at 1 hour AEL showing well-separated centrosomes at the cortex and almost complete absence of nuclear DNA. (C) Cortical section of bubR1Rev1 embryo at 3 hours AEL showing regions with no nuclei and areas with large number of centrosomes. (D) Quantification of the number of centrosomes per area in either wild-type (black squares) or mutant embryos (white circles) at different times AEL (minutes). Asterisk refers to embryos in A,C. (E-H) Mitotic spindles at nuclear cycles 10-11 in bubR1Rev1 embryos. (E) Abnormal distribution of barrel-shaped spindles (arrow) showing centrosomes (arrowhead) abnormally close to a centrosome from an adjacent spindle. (F) Fused spindles without proper separation of centrosomes at the spindle poles (arrow); other spindles assemble normally but show lagging chromosomes during anaphase (arrowheads). (G) Monopolar spindles resulting from loss of centrosomes (arrowhead), spindles that share centrosomes (asterisk) and free centrosomes (arrow) are able to nucleate microtubules. (H) Excess centrosomes perturb spindle assembly and chromosomes segregation. Scale bars: 20 µm in A,C; 50 µm in B; 10 µm in E-H.

View larger version (35K): [in a new window]   Fig. 5. Development of bubR1Rev1 embryos following spindle damage. Wild-type and bubR1Rev1 embryos at 0-3 hours AEL were incubated with colchicine during 30 minutes, fixed and immunostained with anti Phospho-Histone H3 antibodies. Merged images show DNA (blue), anti-PH3 (green) and BubR1 (red). Following colchicine treatment, (A,A'') Wild-type nuclei arrest in a prometaphase-like stage and are Phospho-Histone H3 positive, while a significant proportion of bubR1Rev1 embryos are Phospho-Histone H3 negative (B,B''). Following colchicine treatment, BubR1 accumulates at kinetochores of (C) wild-type prometaphase-like nuclei but is not detectable in (D) nuclei from bubR1Rev1 embryos. (E) Quantification of Phospho-Histone H3-positive embryos treated with colchicine during cycle 3-6, 7-9 and 10-13 (wild-type embryos: n=32 for cycle 3-6, n=21 for cycle 7-9 and n=37 for cycle 10-13. bubR1Rev1 embryos: n=54 for cycle 3-6, n=18 for cycle 7-9 and n=47 for cycle 10-13. We considered embryos positive when more than 80% of nuclei were Phospho-Histone H3 positive. Scale bar: 10 µm.

View larger version (70K): [in a new window]   Fig. 6. Organisation of the polar body in wild-type and bubR1Rev1 embryos. (A-C) In merged images, BubR1 is in red, DNA in blue and CID in green. (A-A''') BubR1 localises at kinetochores of polar body chromosomes in wild-type embryos. (B,B''') In unfertilised bubR1Rev1 embryos, polar body chromosomes fail to maintain a prometaphase arrest and appear decondensed. (C,C''') In fertilised bubR1Rev1 embryos, polar body chromosomes are mostly decondensed and show an increased number of CID-positive signals. (D-G) DNA is in blue and Phospho-Histone H3 in red in merged images. (D,D'') In wild-type embryos, polar bodies have condensed Phospho-Histone H3 positive chromosomes. (E,E'') In unfertilised bubR1Rev1 embryos, polar bodies show DNA condensation with a low Phospho-Histone H3 signal and alteration of chromosome configuration. (F,F'',G,G'') In fertilised bubR1Rev1 embryos, the polar bodies follow cycles of condensation-decondensation. (F') The polar body is Phospho-Histone H3 negative and shows a decondensed interphase-like state or (G-G'') shows a low Phospho-Histone H3 positive labelling and partial DNA condensation. (H) Quantification of Phospho-Histone H3-positive polar bodies in wild-type and bubR1Rev1 embryos: fertilised wild-type embryos (Ft Wt, n=30), unfertilised bubR1Rev1 embryos (Unf bubR1Rev1, n=30), fertilised bubR1Rev1 embryos with syncytial nuclei in interphase (Int bubR1Rev1, n=11) and fertilised bubR1Rev1 embryos with syncytial nuclei in mitosis (Mit bubR1Rev1, n=30). Scale bar: 10 µm.

View larger version (64K): [in a new window]   Fig. 7. Live imaging and BrdU incorporation of the polar body of wild-type and bubR1Rev1 embryos. (A,B) Time lapse imaging of polar bodies from fertilised (A) wild-type or (B) bubR1Rev1 embryos. In wild-type embryos, polar bodies do not change in structure and Histone-GFP signal level remains constant. In bubR1Rev1 embryos, polar bodies show cyclical changes in Histone-GFP signal intensity (see Movies 3 and 4 in the supplementary material). (C) Graphical representation of the relative Histone-GFP signal intensity/pixel in polar bodies of different embryos (wt, wild type; S1-4, bubR1Rev1 embryos). Oscillations in Histone-GFP signal intensity represent differences in DNA condensation and decondensation. (D) Graphical representation of the dynamic range of the raw data from C. (E,F) BrdU incorporation into fertilised (E,E'') wild type or (F,F'') bubR1Rev1 embryos. Arrows indicate polar bodies and arrowheads syncytial nuclei. BrdU incorporation occurs in the DNA of the bubR1Rev1 polar body and in all the nuclei in the interior of the embryo, but not in the polar body of wild-type embryos. In all merged images, DNA is stained blue and BrdU is stained red. Insets in merged images show higher magnifications of the polar body. Scale bars: 10 µm in A,B; 100 µm E,F.