Cytochalasin induces spindle fusion in the syncytial blastoderm of the early Drosophila embryo.

Microfilament integrity is needed to maintain the regular arrangement of the spindle microtubules and to guarantee the normal progression of the last syncytial mitoses in Drosophila embryo. To investigate when and how microfilaments participate in this process, we incubated permeabilized embryos with the inhibitor of actin polymerization, cytochalasin B, at different times during the nuclear cycle. Our results suggest that the correct microfilament distribution is only required for the appropriate segregation of nuclei during the 11th, 12th and 13th syncytial mitoses rather than during the 10th mitosis when the spindles are too far apart to interact. When cytochalasin B treatment was performed during the last syncytial mitoses many spindles fuse among them and the regular mitotic progression is perturbed.     

Distribution of F-actin during cleavage of the Drosophila syncytial blastoderm

The process of cleavage during the syncytial blastoderm stage of the Drosophila embryo was studied in fixed whole-mounts using a triple- staining technique. Plasmalemma was stained with Concanavalin A conjugated to tetramethylrhodamine isothiocyanate, the underlying cortical F-actin with a fluorescein derivative of phalloidin, and nuclei with 4',-6 diamidine-2-phenylindole dihydrochloride. The surface caps, which overlie the superficial nuclei at this stage, were found to be rich in F-actin as compared with the rest of the cortex. After the caps formed, they extended over the surface and flattened. Whilst this was occurring the F-actin network within the caps became more diffuse. By the end of the expansion process F-actin had become concentrated at both poles of the caps. The caps then split in two. The cleavage was not accompanied by the formation of any apparent contractile ring of microfilaments across the cap, rather the break region was depleted in F-actin. The cortical actin associated with each half of the old cap then became reorganized around a nucleus to form a new daughter cap, and the cycle began again.     

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.     

Microtubule distribution reveals superficial metameric patterns in the early Drosophila embryo

Microtubule distribution was examined in whole mounts of Drosophila embryos from the cellularization of the syncytial blastoderm (stage 6) to the completion of the gastrulation (stage 7) by fluorescence microscopy. During ventral furrow formation, the fluorescence of tubulin network was not uniform, but disposed in zebra stripes. Antibodies against alpha-tubulin showed 14 alternating pairs of darker and brighter transverse areas. The possible significance of this pattern is discussed.     

Involvement of microtubules and microfilaments in centrosome dynamics during the syncytial mitoses of the early Drosophila embryo.

To examine the role of microfilaments and microtubules in centrosome dynamics we exposed Drosophila embryos to culture medium containing cytochalasin B and to low temperature. The results show that the splitting of the centrosomal material does not occur when the embryos are treated with cytochalasin before centrosome duplication at late telophase. The fragmentation of the centrosomal material, caused by cold exposure, is also prevented by cytochalasin incubation. These results indicate that both microtubules and microfilaments may be involved in determining centrosome shape during the syncytial mitoses which lead to the formation of the blastoderm in early Drosophila embryos.     

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.     

DRhoGEF2 regulates actin organization and contractility in the Drosophila blastoderm embryo

Morphogenesis of the Drosophila melanogaster embryo is associated with a dynamic reorganization of the actin cytoskeleton that is mediated by small GTPases of the Rho family. Often, Rho1 controls different aspects of cytoskeletal function in parallel, requiring a complex level of regulation. We show that the guanine triphosphate (GTP) exchange factor DRhoGEF2 is apically localized in epithelial cells throughout embryogenesis. We demonstrate that DRhoGEF2, which has previously been shown to regulate cell shape changes during gastrulation, recruits Rho1 to actin rings and regulates actin distribution and actomyosin contractility during nuclear divisions, pole cell formation, and cellularization of syncytial blastoderm embryos. We propose that DRhoGEF2 activity coordinates contractile actomyosin forces throughout morphogenesis in Drosophila by regulating the association of myosin with actin to form contractile cables. Our results support the hypothesis that specific aspects of Rho1 function are regulated by specific GTP exchange factors.     

Maternal-effect genes that alter the fate map of the Drosophila blastoderm embryo

The pattern of segmentation in the Drosophila embryo is controlled by at least 25 zygotically active genes and at least 20 maternally active genes. We have examined the pattern of expression of the protein product of the zygotically active segmentation gene fushi tarazu (ftz) at the cellular blastoderm stage in progeny of mutant females homozygous for each of six maternal-effect segmentation genes to observe the early effects of the maternal-effect genes on zygotic gene expression. The genes included exuperantia (a member of the anterior class of maternal-effect segmentation genes); staufen and vasa (members of the posterior class); and torso, trunk, and fs(1)N (members of the terminal class). Mutations in the genes caused a disruption of the normal pattern of ftz stripes in regions of the embryo where gene activity is known to be required. The ftz stripes provide a marker for segmental determination at the cellular blastoderm stage, making it possible to correlate aberrant patterns of ftz protein with defects in cuticle morphology at the end of embryogenesis. ftz protein expression in progeny of females mutant for combinations of the above genes was also examined. The changes in the ftz pattern in progeny of females doubly mutant for genes of the anterior and terminal classes or of the posterior and terminal classes can largely be understood as the result of the additive effects of the single mutations. In contrast, clearly nonadditive effects on the ftz pattern were seen when a mutation in a gene of the anterior class (exuperantia) was combined with mutations in posterior class genes.     

Electron microscopic analysis of chromatin replication in the cellular blastoderm drosophila melanogaster embryo

Transmission electron microscopic techniques were used to study the spatial distribution of replicons and the ultrastructure of chromatin in the S phase genome of cellular blastoderm Drosophila melanogaster embryos. We observed chromatin exhibiting distinct bifurcations along each fiber during the initial 20 min of the first cell cycle of blastulation. We interpreted the “bubble-like” configurations produced by adjacent bifurcations as intermediate structures in chromatin replication (that is, replicons). We observed homologous ribonucleoprotein (RNP) fiber arrays on both chromatid arms within some replicons, implying DNA sequence homology and reinforcing the identification of such arms as daughter chromatid fibers. We did not observe replicon configurations on chromatin obtained from embryos staged at more than 20 min into cellular blastulation. Daughter chromatid fibers, however, were identified by the presence of identical RNP fiber arrays on chromatid strands arranged in parallel on the electron microscope grid.We examined the distribution of replicon structures on the cellular blastoderm genome and compared it with electron microscopic data on DNA replication in cleavage embryos (Blumenthal, Kriegstein and Hogness, 1973). S phase is completed in slightly < 4 min during cleavage, or approximately one fifth the time required for DNA synthesis in cellular blastoderm embryos. The mean distance separating adjacent replication origins at cellularization was estimated to be 10.6 kilobases (kb), a value 35% greater than the 7.9 kb inter-origin average determined for cleavage embryos. In contrast to the near-simultaneous activation of replication origins during cleavage replication, we observed that replication origins are not activated synchronously at cellular blastulation. We concluded that the marked increase in the duration of S phase is effected by a reduction in the frequency of replication activation events which occur asynchronously during genome replication at cellularization.We found that the ultrastructure of newly replicated chromatin exhibited a morphology indistinguishable from nucleosomal chromatin. Unreplicated chromatin fibers separating adjacent replicons also exhibit spherical subunits. We inferred that the spherical structures on replicating chromatin are nucleosomes and concluded that histones are not disassociated from the DNA significantly prior to DNA replication, and that a very rapid reassociation of nucleosomes occurs on both daughter DNA molecules following replication.     

The 95F unconventional myosin is required for proper organization of the Drosophila syncytial blastoderm

The 95F myosin, a class VI unconventional myosin, associates with particles in the cytoplasm of the Drosophila syncytial blastoderm and is required for the ATP- and F-actin-dependent translocation of these particles. The particles undergo a cell cycle-dependent redistribution from domains that surround each nucleus in interphase to transient membrane invaginations that provide a barrier between adjacent spindles during mitosis. When 95F myosin function is inhibited by antibody injection, profound defects in syncytial blastoderm organization occur. This disorganization is seen as aberrant nuclear morphology and position and is suggestive of failures in cytoskeletal function. Nuclear defects correlate with gross defects in the actin cytoskeleton, including indistinct actin caps and furrows, missing actin structures, abnormal spacing of caps, and abnormally spaced furrows. Three- dimensional examination of embryos injected with anti-95F myosin antibody reveals that actin furrows do not invaginate as deeply into the embryo as do normal furrows. These furrows do not separate adjacent mitoses, since microtubules cross over them. These inappropriate microtubule interactions lead to aberrant nuclear divisions and to the nuclear defects observed. We propose that 95F myosin function is required to generate normal actin-based transient membrane furrows. The motor activity of 95F myosin itself and/or components within the particles transported to the furrows by 95F myosin may be required for normal furrows to form.     

Dynein anchors its mRNA cargo after apical transport in the Drosophila blastoderm embryo

Molecular motors actively transport many types of cargo along the cytoskeleton in a wide range of organisms. One class of cargo is localized mRNAs, which are transported by myosin on actin filaments or by kinesin and dynein on microtubules. How the cargo is kept at its final intracellular destination and whether the motors are recycled after completion of transport are poorly understood. Here, we use a new RNA anchoring assay in living Drosophila blastoderm embryos to show that apical anchoring of mRNA after completion of dynein transport does not depend on actin or on continuous active transport by the motor. Instead, apical anchoring of RNA requires microtubules and involves dynein as a static anchor that remains with the cargo at its final destination. We propose a general principle that could also apply to other dynein cargo and to some other molecular motors, whereby cargo transport and anchoring reside in the same molecule.     

Adult differentiation from partialDrosophila embryos after egg ligation during stages of nuclear multiplication and cellular blastoderm

When embryos are ligated during different stages of nuclear multiplication and cellular blastoderm they develop into partial larvae which never hatch. The partial larvae were injected into adult females for further development. During this in vivo culture the imaginal disk cells divide and achieve competence to differentiate into adult structures. We find that independent of the fragment size anterior embryonic tissues give rise to cranial adult structures and posterior fragments to adult caudal structures. This indicates that during the first nuclear divisions cranial versus caudal development is already determined. The two complementary fragments do not add up to a total embryo when separated very early; however, if separation of the two parts occurs at cellular blastoderm stage all adult structures of the fly can be found.     

Persistence of Hunchback in the terminal region of the Drosophila blastoderm embryo impairs anterior development

Anterior terminal development is controlled by several zygotic genes that are positively regulated at the anterior pole of Drosophila blastoderm embryos by the anterior (bicoid) and the terminal (torso) maternal determinants. Most Bicoid target genes, however, are first expressed at syncitial blastoderm as anterior caps, which retract from the anterior pole upon activation of Torso. To better understand the interaction between Bicoid and Torso, a derivative of the Gal4/UAS system was used to selectively express the best characterised Bicoid target gene, hunchback, at the anterior pole when its expression should be repressed by Torso. Persistence of hunchback at the pole mimics most of the torso phenotype and leads to repression at early stages of a labral (cap'n'collar) and two foregut (wingless and hedgehog) determinants that are positively controlled by bicoid and torso. These results uncovered an antagonism between hunchback and bicoid at the anterior pole, whereas the two genes are known to act in concert for most anterior segmented development. They suggest that the repression of hunchback by torso is required to prevent this antagonism and to promote anterior terminal development, depending mostly on bicoid activity.     

An antigen present in the Drosophila central nervous system only during embryonic and metamorphic stages

We report here about an antigen that is expressed in the central nervous system (CNS) of Drosophila only during the embryonic and metamorphic stages. In Drosophila, axonogenesis and synaptogenesis occur twice during the development: first in the embryonic and second in the metamorphic stages. We generated monoclonal antibodies (MAbs) in order to obtain molecular probes for analyzing axonogenesis or synaptogenesis in the CNS on the assumption that good candidates for molecules responsible for such phenomena must be present in the neuropil during those stages exclusively. As a result, we found MAb 66B2 whose intense immunoreactivity in the neuropil of the CNS was observed exclusively in the embryo and pupa, and not in the larva and adult. Immunoblot analyses showed that MAb 66B2 binds specifically to a protein with an apparent molecular weight of 350 K and neutral pl in the prepupal CNS. A significant amount of the antigen was isolated in forms that were soluble without detergent. Results of immunohistochemistry with MAb 66B2 in a primary culture of embryos showed that some live cells in the ganglion-like cluster were stained, and that neuronal cell bodies and neurites emanating from there were negative. These results strongly suggest that the 66B2 antigen observed in the CNS is an extracellular matrix component secreted from nonneuronal cells. These developmental changes in the 66B2 immuno-reactivity in the CNS presumably reflect dynamic changes of an extracellular matrix in the CNS that are accompanied by axonogenesis or synaptogenesis. © 1992 John Wiley & Sons, Inc.     

Measurement of intra-embryonic pH during the early stages of development in the chick embryo

Summary Measurements have been made of the pH in the extracellular space, adjacent to the neural tube, in 73 isolated chick embryos in vitro at stages from 4–22 somites. A pH of 7.8–8.4 was observed in the segmented region, while caudally, in the segmental plate, the pH was consistently lower falling by as much as 0.5 pH units at the regressing primitive streak. Variations were noted in the pH of embryos of the same age but the regional variation in pH was a consistent finding in all of the embryos examined. The buffering capacity of the extracellular space was found to be 12.9 mequiv/pH unit/1 in the segmented region and 13.9 mequiv/pH unit/1 in the segmental plate. Thus it is unlikely that the regional variations in pH result from local variations in the buffering power of the extracellular space. Varying the K⁺, Cl^-, Mg²⁺ or HCO ₃ ^- ion concentrations in the bathing medium caused little change in the intra-embryonic pH, while reducing the concentrations of Na⁺ or Ca²⁺ caused a small acidification. This suggests that the ectoderm and endoderm form an effective barrier between the embryo and the external environment. Exposure of the embryo to KCN reduced the intra-embryonic pH suggesting that the alkaline environment is maintained by active processes.