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Fig. 3. Mitotic phenotypes in syncytial bubR1^Rev1 embryos. (A) bubR1^Rev1 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) bubR1^Rev1 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 bubR1^Rev1 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 bubR1^Rev1 embryo showing micronuclei (arrowhead) and pyknotic nuclei (arrow). Nuclei from wild-type (I) or bubR1^Rev1 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 bubR1^Rev1 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 bubR1^Rev1 embryos (n=131) was performed on fixed material. Wild-type embryos show synchronous nuclei, while approximately half of the bubR1^Rev1 embryos show desynchronised nuclear cycles. There is also a relative increase in prophase figures and we failed to detect any proper metaphases in bubR1^Rev1 embryos. Scale bars: 100 µm in A,C; 25 µm in B,D-G; 50 µm in H; 10 µm in I,J.

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