Abstract.
The chromatin protein Polycomb (PC) is necessary for keeping homeotic genes repressed in a permanent and heritable manner. PC is part of a large multimeric complex (PcG proteins) involved in generating silenced chromatin domains at target genes, thus preventing their inappropriate expression. In order to assess the intranuclear distribution of PC during mitosis in different developmental stages as well as in the germ line we generated transgenic fly lines expressing a PC-GFP (Green Fluorescent Protein) fusion protein. Rapidly dividing nuclei were found to display a rather homogeneous PC-GFP distribution. However, with increasing differentiation a pronounced subnuclear pattern was observed. In all investigated diploid somatic tissues the bulk of PC-GFP fusion protein is depleted from the chromosomes during mitosis: however, a detectable fraction remains associated. In the male germ line in early spermatogenesis, PC-GFP was closely associated with the chromosomal bivalents and gradually lost at later stages. Interestingly, we found that PC is associated with the nucleolus in spermatocytes, unlike somatic nuclei. In contrast to mature sperm showing no PC-GFP signal the female germ line retains PC in the germinal vesicle.
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Received: 10 November 1998; in revised form: 30 January 1999 / Accepted: 30 January 1999
Electronic Supplementary Material
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Movie 1: Embryonic Development
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Movie 2: Intracellular PC-GFP distribution during blastoderm nuclear cycles
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Movie 3: Spermatocyte nuclei
All movies were generated from confocal data sets with NIH Image and saved in the QuickTime format on a Macintosh. To reduce file size the original image size was halved. Since virtually every computer system is made up of different components including graphics card and monitor, it might be necessary to adjust monitor brightness and/or contrast to optimize the display of the 256 graylevels used. We recommend using the “double size” feature of the player software to allow easier visualization.
Movie 1: Embryonic Development
An embryo is shown from the blastula stage until hatching of the larvae which can be observed at the end of this movie. The hatching of the larva demonstrates that the observation conditions, e.g. phototoxicity, were not lethal. The embryo is viewed laterally, with the ventral side left and anterior towards the top of the image. Image size is 0.5 mm from top to bottom.
The stack of four confocal sections shown in each frame was recorded at one time point (that is one section after the other, starting with the one at the bottom. The waiting time between the recording of two stacks was 15 minutes (excluding scanning time). This movie contains 126 frames; the total duration of development is about 31 hours. During the later stages of embryonic development the embryo starts to move within the egg. Since it took about 14 seconds to record one image (frame averaged), the images are somewhat blurred due to that motion. The movement of the larvae also caused the rotation of the egg towards the end of movie. The left section is relatively close to the center of the embryo, the right section is grazing through the outer edge of the embryo.
Movie 2: Intracellular PC-GFP distribution during blastoderm nuclear cycles
Complete time sequence of the images shown in Figure 3A. Left: GFP fluorescence, right: simultaneous differential interference contrast (DIC) recording. Image size is 40 µm from top to bottom. Confocal 3D stacks were recorded subsequently with a 5 second break in between. From each stack the same section was extracted for display in this movie. The recording of one stack took 30 seconds; the images are thus 35 seconds apart. The 40 frames span about 23 minutes.
The sequence starts in mitosis between cycles 10 and 11 and ends in interphase of cycle 13. At the onset of every mitosis the majority of the PC-GFP signal leaks into the cytoplasm. However a very weak but clearly detectable signal remains associated with the chromosomal area. This area is defined by the GFP-signal itself but can also be seen in the DIC image because of its higher optical density.
Movie 3: Spermatocyte nuclei
Two spermatocyte nuclei from a testis prepared as those shown in Figure 6. A 360 degree rotation in 10 degree steps was made from a 3D confocal data set from fixed tissue. Parts of neighboring nuclei which would have obscured the view were cut away electronically. Left: DNA staining (DAPI), right: PC-GFP fluorescence. The images in each frame are about 20 µm wide. Some areas are overexposed to allow a better visualization of weaker signals.
Note that the two PC-GFP signals in the corners of each nucleus colocalize much better with their respective DNA signals than the PC-GFP signals that surround the nucleoli.
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Dietzel, S., Niemann, H., Brückner, B. et al. The nuclear distribution of Polycomb during Drosophila melanogaster development shown with a GFP fusion protein. Chromosoma 108, 83–94 (1999). https://doi.org/10.1007/s004120050355
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DOI: https://doi.org/10.1007/s004120050355