Nuclear division and migration during early embryogenesis of Bradysia tritici coquillet (syn. Sciara ocellaris) (diptera : Sciaridae)

Nuclear division and migration of cleavage nuclei in the embryos of Bradysia tritici (Diptera : Sciaridae) have been studied by light microscopy and nuclear staining. There are 8 cleavage cycles up to the syncytial blastoderm stage (4.5 hr), and during the 11th cycle cellularization begins (6.5 hr). The first 3 divisions take about 30 min each. During the 5th and 6th cycles, the maximum rate of division is reached (12 min/cycle at 22°C). After pole cell formation, the duration of the following mitotic cycles increases progressively. During nuclear migration, the presumptive germ line nuclei reach the egg cortex first, followed by anterior somatic nuclei and finally, posterior somatic nuclei reach the egg cortex. Possibly as a result of this region-specific nuclear migration, nuclear divisions become parasynchronous after 3 hr of embryogenesis (4th cycle). Several mitotic cycles later, between the 8th and 10th cycle in different embryos, X-chromosome elimination in somatic nuclei begins at the anterior egg pole and progresses in anteroposterior direction. Our observations suggest that the observed region-specific differences may be due to the activity of localized factors in the egg that control migration and nuclear cycle of the somatic nuclei.     

Aster migration determines the length scale of nuclear separation in the Drosophila syncytial embryo

In the early embryo of many species, comparatively small spindles are positioned near the cell center for subsequent cytokinesis. In most insects, however, rapid nuclear divisions occur in the absence of cytokinesis, and nuclei distribute rapidly throughout the large syncytial embryo. Even distribution and anchoring of nuclei at the embryo cortex are crucial for cellularization of the blastoderm embryo. The principles underlying nuclear dispersal in a syncytium are unclear. We established a cell-free system from individual Drosophila melanogaster embryos that supports successive nuclear division cycles with native characteristics. This allowed us to investigate nuclear separation in predefined volumes. Encapsulating nuclei in microchambers revealed that the early cytoplasm is programmed to separate nuclei a distinct distance. Laser microsurgery revealed an important role of microtubule aster migration through cytoplasmic space, which depended on F-actin and cooperated with anaphase spindle elongation. These activities define a characteristic separation length scale that appears to be a conserved property of developing insect embryos.     

Hidden polyteny in giant embryonic nuclei of Drosophila melanogaster gnu mutants

Drosophila melanogaster embryos, whose mothers are homozygous for the gnu (a recessive lethal mutation with maternal effect) undergo DNA synthesis but are defective in nuclear division. This leads to formation of giant nuclei in the syncytial blastoderm. The interior spatial chromatin organization and possibility of obtaining polytene chromosomes in these nuclei was analysed. Partial conjugation of homologous chromatids, which is an evidence for cryptic polyteny in the gnu embryos nuclei, was shown.     

Drop out: a third chromosome maternal-effect locus required for formation of the Drosophila cellular blastoderm.

drop out (dop) is a recessive maternal-effect locus identified in a screen for female-sterile mutations in Drosophila polytene region 71C-F. Phenotypic analyses of the dop mutation indicate that the gene is required for proper formation of the cellular blastoderm. In embryos derived from either homozygous or hemizygous dop mothers, cytoplasmic clearing, nuclear migration and division, and pole cell formation appear normal. However, developmental defects are observed prior to and during cellularization of the blastoderm. At the beginning of nuclear cycle 14, the distinct separation of the internal yolk mass and the cortical cytoplasm breaks down. Subsequently, a population of somatic nuclei located at the periphery of the syncytial blastoderm becomes irregularly spaced and nonuniform in their distribution. Despite a somewhat regular formation of the cortical actin network, cellularization in mutant embryos is extremely variable. Such embryos fail to gastrulate normally and produce variable amounts of defective cuticle. Overall, our analyses suggest that the dop gene functions in maintaining the separation of yolk and cortical cytoplasm and in stabilizing the distribution of somatic nuclei in the Drosophila syncytial blastoderm.     

A role for cyclin J in the rapid nuclear division cycles of early Drosophila embryogenesis

The nuclear division cycles of early Drosophila embryogenesis have a number of unique features that distinguish them from later cell cycles. These features include the lack of some checkpoints that operate in later cell cycles, the absence of gap phases, and very rapid DNA synthesis phases. The molecular mechanisms that control these rapid nuclear division cycles are poorly understood. Here we describe analysis of cyclin J, a previously uncharacterized cyclin which has an RNA expression pattern that suggests a possible role in early embryogenesis. We show that the cyclin J protein is present in early embryos where it forms active kinase complexes with cyclin-dependent kinase (Cdk) 2. To determine whether cyclin J plays a role in controlling the early nuclear cycles we isolated peptide aptamers that specifically bind to cyclin J and inhibit its ability to activate Cdks. We injected the inhibitory aptamers into syncytial Drosophila embryos and demonstrated that they caused defects in chromosome segregation and progression through mitosis. We obtained similar results by injecting cyclin J antibodies into embryos. Our results suggest that a cyclin J-associated kinase activity is required for the early embryonic division cycles.     

Drosophila Wee1 kinase regulates Cdk1 and mitotic entry during embryogenesis

Cyclin-dependent kinases (Cdks) are the central regulators of the cell division cycle. Inhibitors of Cdks ensure proper coordination of cell cycle events and help regulate cell proliferation in the context of tissues and organs. Wee1 homologs phosphorylate a conserved tyrosine to inhibit the mitotic cyclin-dependent kinase Cdk1. Loss of Wee1 function in fission or budding yeast causes premature entry into mitosis. The importance of metazoan Wee1 homologs for timing mitosis, however, has been demonstrated only in Xenopus egg extracts and via ectopic Cdk1 activation . Here, we report that Drosophila Wee1 (dWee1) regulates Cdk1 via phosphorylation of tyrosine 15 and times mitotic entry during the cortical nuclear cycles of syncytial blastoderm embryos, which lack gap phases. Loss of maternal dwee1 leads to premature entry into mitosis, mitotic spindle defects, chromosome condensation problems, and a Chk2-dependent block of subsequent development, and then embryonic lethality. These findings modify previous models about cell cycle regulation in syncytial embryos and demonstrate that Wee1 kinases can regulate mitotic entry in vivo during metazoan development even in cycles that lack a G2 phase.     

Maternal effect mutations of the sponge locus affect actin cytoskeletal rearrangements in Drosophila melanogaster embryos

In the syncytial blastoderm stage of Drosophila embryogenesis, dome-shaped actin "caps" are observed above the interphase nuclei. During mitosis, this actin rearranges to participate in the formation of pseudocleavage furrows, transient membranous invaginations between dividing nuclei. Embryos laid by homozygous sponge mothers lack these characteristic actin structures, but retain other actin associated structures and processes. Our results indicate that the sponge product is specifically required for the formation of actin caps and metaphase furrows. The specificity of the sponge phenotype permits dissection of both the process of actin cap formation and the functions of actin caps and metaphase furrows. Our data demonstrate that the distribution of actin binding protein 13D2 is unaffected in sponge embryos and suggest that 13D2 is upstream of actin in cortical cap assembly. Although actin caps and metaphase furrows have been implicated in maintaining the fidelity of nuclear division and the positions of nuclei within the cortex, our observations indicate that these structures are dispensible during the early syncytial blastoderm cell cycles. A later requirement for actin metaphase furrows in preventing the nucleation of mitotic spindles between inappropriate centrosomes is observed. Furthermore, the formation of actin caps and metaphase furrows is not a prerequisite for the formation of the hexagonal array of actin instrumental in the conversion of the syncytial embryo into a cellular blastoderm.     

The dissociation of nuclear and centrosomal division in gnu, a mutation causing giant nuclei in Drosophila

We describe a recessive, maternal-effect lethal mutation of Drosophila, gnu. gnu uncouples nuclear division from many cytoplasmic events of mitosis in the Drosophila embryo. Embryos from homozygous females are defective in nuclear division, but not in DNA replication, and therefore develop a small number of giant nuclei. Centrosomes divide independently of nuclear division and migrate to the surface of the syncytial blastoderm. There they nucleate microtubules into asters, which appear normal at first but become very large. Only later, when the giant nuclei begin to break down, are spindles sometimes formed. The cortical actin of these embryos develops into a characteristic network.     

A morphological and developmental study of Drosophila embryos ligated during nuclear multiplication

In the Drosophila embryo, determination is established at the cellular blastoderm and a mosaic type development is observed after this time. Before the blastoderm stage, however, development is not of the mosaic type, as ligation during the nuclear multiplication stage causes a change in the spatial organization of the larval pattern. An aberration in determination leads to an increase in segment size, an increase in the number of cells per segment, and a decrease in segment number. This abnormal determination of blastoderm cells has also been demonstrated experimentally by marking corresponding regions of the blastoderm in ligated (posterior fragments only) and nonligated embryos. When the blastoderms of nonligated and ligated embryos are punctured at the same site, ligated embryos produce larvae with damage in segments posterior to the segments damaged in larvae from nonligated embryos. Ultrastructurally, no abnormalities were observed in the plasma membrane at the time of ligation or later in blastoderm cells which formed in the ligation area of these embryos. Evidence from this study, as well as other sources, indicates that determination of segmentation is under maternal control.     

Wolbachia-mediated male killing is associated with defective chromatin remodeling

Male killing, induced by different bacterial taxa of maternally inherited microorganisms, resulting in highly distorted female-biased sex-ratios, is a common phenomenon among arthropods. Some strains of the endosymbiont bacteria Wolbachia have been shown to induce this phenotype in particular insect hosts. High altitude populations of Drosophila bifasciata infected with Wolbachia show selective male killing during embryonic development. However, since this was first reported, circa 60 years ago, the interaction between Wolbachia and its host has remained unclear. Herein we show that D. bifasciata male embryos display defective chromatin remodeling, improper chromatid segregation and chromosome bridging, as well as abnormal mitotic spindles and gradual loss of their centrosomes. These defects occur at different times in the early development of male embryos leading to death during early nuclear division cycles or large defective areas of the cellular blastoderm, culminating in abnormal embryos that die before eclosion. We propose that Wolbachia affects the development of male embryos by specifically targeting male chromatin remodeling and thus disturbing mitotic spindle assembly and chromosome behavior. These are the first observations that demonstrate fundamental aspects of the cytological mechanism of male killing and represent a solid base for further molecular studies of this phenomenon.     

daughterless-abo-like, a Drosophila maternal-effect mutation that exhibits abnormal centrosome separation during the late blastoderm divisions

daughterless-abo-like (dal) is a maternal-effect semilethal mutation in Drosophila. The nuclear divisions of embryos derived from homozygous dal females are normal through nuclear cycle 10. However, during nuclear cycles 11, 12 and 13, a total of about half of the nuclei in each embryo either fail to divide or fuse with a neighboring nucleus during telophase. These abnormal nuclei eventually sink into the interior of the embryo, leaving their centrosomes behind on the surface. The loss of about one-half of the peripheral nuclei into the interior of the embryo results in these embryos cellularizing during nuclear cycle 14 with about one-half the normal number of cells. Surprisingly, many of these embryos develop a nearly normal larval cuticle and 8% develop to adulthood. Observations of live embryos doubly injected with tubulin and histones that have been fluorescently labeled allows nuclear and centrosomal behavior to be directly followed as the embryo develops. We find that the abnormal nuclei arise from nuclei whose centrosomes have failed to separate normally in the previous interphase. These incompletely separated centrosomes can cause a non-functional spindle to form, leading to a nuclear division failure. Alternatively, they can form an abnormal spindle with a centrosome from a neighboring nucleus, causing two nuclei to share a common spindle pole. Such nuclei with a shared centrosome will undergo telophase fusions, unequal divisions, or division failures later in mitosis. These findings have helped us to understand the function of the centrosome in the Drosophila embryo.     

蟾蜍种间核移植胚胎发育早期LDH同工酶的表现

利用聚丙烯酰胺凝胶电泳,对中华大蟾蜍(Bufo bufo比gargarizans)与花背蟾蜍(Bufo raddei)种间核移植胚胎发育早期六个不同阶段中全胚胎的乳酸脱氢酶(LDH)同工酶进行了分析。酶谱比较的结果表明:在正、反种间移核胚胎中,供体核LDH基因的活动开始表现于尾芽胚期;此前,杂种胚胎中LDH同工酶谱类型与受体一致。     

The secretory membrane system in the Drosophila syncytial blastoderm embryo exists as functionally compartmentalized units around individual nuclei

Drosophila melanogaster embryogenesis begins with 13 nuclear division cycles within a syncytium. This produces >6,000 nuclei that, during the next division cycle, become encased in plasma membrane in the process known as cellularization. In this study, we investigate how the secretory membrane system becomes equally apportioned among the thousands of syncytial nuclei in preparation for cellularization. Upon nuclear arrival at the cortex, the endoplasmic reticulum (ER) and Golgi were found to segregate among nuclei, with each nucleus becoming surrounded by a single ER/Golgi membrane system separate from adjacent ones. The nuclear-associated units of ER and Golgi across the syncytial blastoderm produced secretory products that were delivered to the plasma membrane in a spatially restricted fashion across the embryo. This occurred in the absence of plasma membrane boundaries between nuclei and was dependent on centrosome-derived microtubules. The emergence of secretory membranes that compartmentalized around individual nuclei in the syncytial blastoderm is likely to ensure that secretory organelles are equivalently partitioned among nuclei at cellularization and could play an important role in the establishment of localized gene and protein expression patterns within the early embryo.     

Observations by a novel method of surface changes during the syncytial blastoderm stage of the Drosophila embryo

Up to now the cleavage process during the syncytial blastoderm stage has proved hard to follow in living Drosophila embryos. However, this can be achieved by microinjection of TMRITC-BSA into the fluid between the vitelline membrane and the embryo surface. The superficial bulges are then visible with epifluorescence optics. Once formed the bulges go through cycles of flattening, expansion, division, and rounding up. A tendency was noted for more divisions to occur at angles closer to 90° than at more acute angles relative to the previous cleavage. However, the cleavage angles were frequently determined by the distribution of surface space around the bulges. As caps became more numerous they squeezed and pushed around each other while expanding. As a result of these movements the surface of the blastoderm becomes uniformly covered with nuclei by the time of cellularization.