The distribution of polycomb-group proteins during cell division and development in Drosophila embryos: impact on models for silencing.

The subcellular three-dimensional distribution of three polycomb-group (PcG) proteins-polycomb, polyhomeotic and posterior sex combs-in fixed whole-mount Drosophila embryos was analyzed by multicolor confocal fluorescence microscopy. All three proteins are localized in complex patterns of 100 or more loci throughout most of the interphase nuclear volume. The rather narrow distribution of the protein intensities in the vast majority of loci argues against a PcG-mediated sequestration of repressed target genes by aggregation into subnuclear domains. In contrast to the case for PEV repression (Csink, A.K., and S. Henikoff. 1996. Nature. 381:529-531), there is a lack of correlation between the occurrence of PcG proteins and high concentrations of DNA, demonstrating that the silenced genes are not targeted to heterochromatic regions within the nucleus. There is a clear distinction between sites of transcription in the nucleus and sites of PcG binding, supporting the assumption that most PcG binding loci are sites of repressive complexes. Although the PcG proteins maintain tissue-specific repression for up to 14 cell generations, the proteins studied here visibly dissociate from the chromatin during mitosis, and disperse into the cytoplasm in a differential manner. Quantitation of the fluorescence intensities in the whole mount embryos demonstrate that the dissociated proteins are present in the cytoplasm. We determined that <2% of PH remains attached to late metaphase and anaphase chromosomes. Each of the three proteins that were studied has a different rate and extent of dissociation at prophase and reassociation at telophase. These observations have important implications for models of the mechanism and maintenance of PcG- mediated gene repression.

The dynamic nuclear redistribution of an hnRNP K-homologous protein during Drosophila embryo development and heat shock. Flexibility of transcription sites in vivo.

The Drosophila protein Hrb57A has sequence homology to mammalian heterogenous nuclear ribonucleoprotein (hnRNP) K proteins. Its in vivo distribution has been studied at high resolution by confocal laser scanning microscopy (CLSM) in embryos injected with fluorescently labeled monoclonal antibody. Injection of antibody into living embryos had no apparent deleterious effects on further development. Furthermore, the antibody-protein complex could be observed for more than 7 cell cycles in vivo, revealing a dynamic redistribution from the nucleus to cytoplasm at each mitosis from blastoderm until hatching. The evaluation of two- and three-dimensional CLSM data sets demonstrated important differences in the localization of the protein in the nuclei of living compared to fixed embryos. The Hrb57A protein was recruited to the 93D locus upon heat shock and thus serves as an in vivo probe for the activity of the gene in diploid cells of the embryo. Observations during heat shock revealed considerable mobility within interphase nuclei of this transcription site. Furthermore, the reinitiation as well as the down regulation of transcriptional loci in vivo during the recovery from heat shock could be followed by the rapid redistribution of the hnRNP K during stress recovery. These data are incompatible with a model of the interphase nucleus in which transcription complexes are associated with a rigid nuclear matrix.

In vivo observation of the puff-specific protein no-on transient A (NONA) in nuclei of Drosophila embryos.

The spatial distribution of no-on transient A (NONA), a protein associated with specific puffs on polytene chromosomes, was followed in nuclei of living Drosophila embryos by microinjection of fluorescently labeled monoclonal antibody to NONA. The injected antibodies remained active until the larval stage, revealing the distribution of the NONA protein throughout embryogenesis. Most injected animals completed embryonic development and hatched as normal larvae. NONA was restricted to the cytoplasm until the end of cycle 11. We document an active uptake of the NONA-antibody complex into early interphase nuclei from nuclear cycle 14 onwards, following each mitosis. Significant differences in the distribution of the protein between fixed and living embryos were apparent, particularly at high resolution. The NONA protein was localized in the nuclei of living embryos at discrete sites, most of which lay at the periphery and some of which were tightly clustered. The constellation of sites changed with time; in some nuclei these changes were fast whereas in other nuclei the pattern was quite stable. These data suggest that specific protein complexes associated with active interphase chromatin, and possibly chromatin in general, are mobile in the living organism.

Consequences of topoisomerase II inhibition in early embryogenesis of Drosophila revealed by in vivo confocal laser scanning microscopy.

The regulation of DNA topology by topoisomerase II from Drosophila melanogaster has been studied extensively by biochemical methods but little is known about its roles in vivo. We have performed experiments on the inhibition of topoisomerase II in living Drosophila blastoderm embryos. We show that the enzymatic activity can be specifically disrupted by microinjection of antitopoisomerase II antibodies as well as the epipodophyllotoxin VM26, a known inhibitor of topoisomerase II in vitro. By labeling the chromatin of live embryos with tetramethylrhodamine-coupled histones, the effects of inhibition on nuclear morphology and behaviour was followed in vivo using confocal laser scanning microscopy. Both the antibodies and the drug prevented or hindered the segregation of chromatin daughter sets at the anaphase stage of mitosis. In addition, high concentrations of inhibitor interfered with the condensation of chromatin and its proper arrangement into the metaphase plate. The observed effects yielded non-functional nuclei, which were drawn into the inner yolk mass of the embryo. Concurrently, undamaged nuclei surrounding the affected region underwent compensatory division, leading to the restoration of the nuclear population, and thereby demonstrating the regulative capacity of Drosophila blastoderm embryos.