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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00606

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The maternal-effect gene futile cycle is essential for pronuclear congression and mitotic spindle assembly in the zebrafish zygote

Marcus P. S. Dekens, Francisco J. Pelegri^*, Hans-Martin Maischein and Christiane Nüsslein-Volhard

Max-Planck Institut für Entwicklungsbiologie, Abteilung Genetik, Spemannstrasse 35, 72076 Tübingen, Germany

View larger version (72K): [in a new window]   Fig. 1. fue embryos undergo several rounds of anucleate cleavage. Fixed wild-type (A) and fue (B) embryos at the eight-cell stage, labelled with DAPI to reveal nuclei (red) and fluorescein phalloidin to visualise the actin cytoskeleton (green). (C) A wild-type embryo at the sphere stage and (D) a living fue embryo shortly after it has arrested cleavage. In both cases development took place for the same period of time. In most of the fue embryos, the characteristic cleavage orientation pattern is not perturbed, and the cells progress through several cleavage cycles, indicating that the core cell cycle engine is not affected. (E,F) Unfertilized wild-type and fue eggs, showing that the sperm is required to initiate cleavage of the fue embryo as in the wild type.

View larger version (177K): [in a new window]   Fig. 2. fue zygotes resume and complete meiosis normally. DAPI was applied in order to label chromosomes (blue) and fluorescein phalloidin to visualise the actin cytoskeleton (green). (A,B) Chromosomes during meiotic arrest at metaphase II in wild-type (A) and fue (B) unfertilized, non-activated oocytes. Directly after fertilization meiosis is resumed and completed with the extrusion of one set of chromosomes into a vesicle on the outside of the cell membrane. Extrusion of this second polar body (arrowhead) takes place around 7 minutes post-fertilization in wild-type (C) and fue zygotes (D). This demonstrates that the terminal event of meiosis is completed in fue zygotes as in the wild type. Scale bars: 10 µm.

View larger version (55K): [in a new window]   Fig. 3. The pronuclei fail to congress in fue zygotes. By labelling the nuclei of zygotes, fixed at consecutive time points after in vitro fertilization, we have documented the following sequence of pronuclear events. (A,D) Congression of pronuclei around 12 minutes post-fertilization, in wild-type (A) and fue (D) zygotes. (B,E) Around 14-20 minutes post-fertilization the pronuclei fuse in wild type (B) but are separate in fue zygotes (E). (C,F) Around 22 minutes post-fertilization one zygotic nucleus is present in wild type (C) and two pronuclei are observed in fue (F) zygotes. Eggs fertilized with in vivo BrdU-labelled sperm, were fixed, and nuclear BrdU was immunodetected at the two-cell stage. In wild-type embryos (G), two BrdU-labelled nuclei (arrowheads) are detected, in contrast to fue embryos (H) where only one BrdU-labelled nucleus (arrowhead) is detected, although two nuclei are always present in both fue (J) and wild-type embryos (I). This confirms that fue zygotes have a defect in pronuclear congression, and therefore fusion of the pronuclei does not occur as opposed to wild-type zygotes. Scale bars: 10 µm.

View larger version (59K): [in a new window]   Fig. 4. DNA replication immunodetected after BrdU incorporation. (A,C,E) Wild type, (B,D,F) fue embryos. (A,B) Embryos injected with BrdU directly after fertilization show nuclear BrdU labelling at the beginning of the two-cell stage. (C,D) Embryos injected with BrdU at the onset of the second cleavage show nuclear BrdU labelling at the eight-cell stage. (E,F) When BrdU is injected at the 32-cell stage, we observed labelled nuclei at the 64-cell stage. These images illustrate that, fue zygotes undergo S phase and go through consecutive S phases during the subsequent cleavage cycles.

View larger version (50K): [in a new window]   Fig. 5. fue mutants exhibit a defect in mitotic spindle assembly. Mitotic spindles were visualised using an antibody directed against -tubulin (green) and chromosomes were labelled with DAPI (blue). (A) The first mitotic spindle is assembled in wild-type zygotes at 25 minutes post-fertilization. (B,C) In fue zygotes mitotic spindle assembly is perturbed because of a defect in chromosome-dependent microtubule nucleation (B), however, in about 30% of fue zygotes microtubule nucleation on one or both chromosome clusters (arrowhead, C) does occur. In all cases the formation of asters does not seem to be affected (B,C). (E) A mitotic spindle as observed in wild-type eight-cell stage embryos. (F,G) In the majority of fue embryos at post-zygotic stages, microtubules are associated with the chromosome clusters during mitosis (F), except in a minority of embryos (about 5%), where aberrant spindles are observed, which can randomly segregate DNA (G). (D,H) In all fue cells at post-zygotic stages a pair of asters is formed during mitosis (H), which is similar to that observed in the anucleate cells of wild-type embryos that were injected with the DNA replication inhibitor, aphidicolin (D). Therefore, fue zygotes demonstrate a defect in spindle assembly, arising from a defect in the chromatin-dependent generation of microtubules (Karsenti and Vernos, 2001). Scale bars: 20 µm.

View larger version (40K): [in a new window]   Fig. 6. Centrosome duplication is not affected in fue mutants. Centrosomes at the eight-cell stage during interphase, visualised with an antibody directed against -tubulin (green) and nuclei labelled with DAPI (blue) from (A) a wild-type embryo, (B) fue embryo, (C) an embryo treated with the DNA replication inhibitor aphidicolin. (D-F) Enlargements of single centrosome pairs of A-C, respectively. Centrosomes are present and are able to duplicate in all fue cells as well as the (anucleate) cells of aphidicolin-treated embryos, demonstrating that the centrosomes can duplicate independently from nuclei. Scale bars: 20 µm.

View larger version (118K): [in a new window]   Fig. 7. Transcription is initiated earlier in fue embryos. Initiation of mRNA transcription coincides with phosphorylation of the H5 epitope of RNA polymerase II. Labelling with an antibody directed against the phosphorylated form of this epitope is in red, nuclei are labelled with DAPI (blue) while the outline of the cell is visualised with an antibody directed against ß-catenin (green). (A,C) Wild-type and (B,D) fue cells from (A,B) 16-cell stage and (C,D) 32-cell stage embryos. (E,F) Wild-type embryos at the 256-cell stage (E) and 512-cell stage (F). Phosphorylation of the H5 epitope occurs first at the 512-cell stage in wild-type embryos and at the 32-cell stage in fue embryos, showing that the onset of mRNA transcription occurs at an earlier stage in fue embryos. Scale bars: 20 µm.

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