Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development.

Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. Here, we show that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. We demonstrate that NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. We delineate a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. We finally show that this unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation.

Drosophila UNC-45 accumulates in embryonic blastoderm and in muscles, and is essential for muscle myosin stability.

UNC-45 is a chaperone that facilitates folding of myosin motor domains. We have used Drosophila melanogaster to investigate the role of UNC-45 in muscle development and function. Drosophila UNC-45 (dUNC-45) is expressed at all developmental stages. It colocalizes with non-muscle myosin in embryonic blastoderm of 2-hour-old embryos. At 14 hours, it accumulates most strongly in embryonic striated muscles, similarly to muscle myosin. dUNC-45 localizes to the Z-discs of sarcomeres in third instar larval body-wall muscles. We produced a dunc-45 mutant in which zygotic expression is disrupted. This results in nearly undetectable dUNC-45 levels in maturing embryos as well as late embryonic lethality. Muscle myosin accumulation is robust in dunc-45 mutant embryos at 14 hours. However, myosin is dramatically decreased in the body-wall muscles of 22-hour-old mutant embryos. Furthermore, electron microscopy showed only a few thick filaments and irregular thick-thin filament lattice spacing. The lethality, defective protein accumulation, and ultrastructural abnormalities are rescued with a wild-type dunc-45 transgene, indicating that the mutant phenotypes arise from the dUNC-45 deficiency. Overall, our data indicate that dUNC-45 is important for myosin accumulation and muscle function. Furthermore, our results suggest that dUNC-45 acts post-translationally for proper myosin folding and maturation.

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.

An early temperature-sensitive period for the plasticity of segment number in the centipede Strigamia maritima.

Geophilomorph centipedes show variation in segment number (a) between closely related species and (b) within and between populations of the same species. We have previously shown for a Scottish population of the coastal centipede Strigamia maritima that the temperature of embryonic development is one of the factors that affects the segment number of hatchlings, and hence of adults, as these animals grow epimorphically--that is, without postembryonic addition of segments. Here, we show, using temperature-shift experiments, that the main developmental period during which embryos are sensitive to environmental temperature is surprisingly early, during blastoderm formation and before, or very shortly after, the onset of segmentation.

Reversible chromosome condensation induced in Drosophila embryos by anoxia: visualization of interphase nuclear organization.

We have studied the morphology of nuclei in Drosophila embryos during the syncytial blastoderm stages. Nuclei in living embryos were viewed with differential interference-contrast optics; in addition, both isolated nuclei and fixed preparations of whole embryos were examined after staining with a DNA-specific fluorescent dye. We find that: (a) The nuclear volumes increase dramatically during interphase and then decrease during prophase of each nuclear cycle, with the magnitude of the nuclear volume increase being greatest for those cycles with the shortest interphase. (b) Oxygen deprivation of embryos produces a rapid developmental arrest that is reversible upon reaeration. During this arrest, interphase chromosomes condense against the nuclear envelope and the nuclear volumes increase dramatically. In these nuclei, individual chromosomes are clearly visible, and each condensed chromosome can be seen to adhere along its entire length to the inner surface of the swollen nuclear envelope, leaving the lumen of the nucleus devoid of DNA. (c) In each interphase nucleus the chromosomes are oriented in the "telophase configuration," with all centromeres and all telomeres at opposite poles of the nucleus; all nuclei at the embryo periphery (with the exception of the pole cell nuclei) are oriented with their centromeric poles pointing to the embryo exterior.

Three distinct distributions of F-actin occur during the divisions of polar surface caps to produce pole cells in Drosophila embryos.

The F-actin distribution was studied during pole cell formation in Drosophila embryos using the phalloidin derivative rhodaminyl-lysine-phallotoxin. Nuclei were also stained with 4'-6 diamidine-2-phenylindole dihydrochloride to correlate the pattern seen with the nuclear cycle. The precursors of the pole cells, the polar surface caps, were found to have an F-actin-rich cortex distinct from that of the rest of the embryo surface and an interior cytoplasm that was less intensely stained but brighter than the cytoplasm deeper in the embryo. They were found to divide once without forming true cells and then a second time when cells formed as a result of a meridional and a basal cleavage. Three distinct distributions of the cortical F-actin have been identified during these cleavages. It is concluded that the first division, which cleaves the polar caps but does not separate them from the embryo, involves very different processes from those that lead to the formation of the pole cells. A contractile-ring type of F-actin organization may not be present during the first cleavage but is suggested to occur during the second.

Direct cell lineage analysis in Drosophila melanogaster by time-lapse, three-dimensional optical microscopy of living embryos.

One of the first signs of cell differentiation in the Drosophila melanogaster embryo occurs 3 h after fertilization, when discrete groups of cells enter their fourteenth mitosis in a spatially and temporally patterned manner creating mitotic domains (Foe, V. E. and G. M. Odell, 1989, Am. Zool. 29:617-652). To determine whether cell residency in a mitotic domain is determined solely by cell position in this early embryo, or whether cell lineage also has a role, we have developed a technique for directly analyzing the behavior of nuclei in living embryos. By microinjecting fluorescently labeled histones into the syncytial embryo, the movements and divisions of each nucleus were recorded without perturbing development by using a microscope equipped with a high resolution, charge-coupled device. Two types of developmental maps were generated from three-dimensional time-lapse recordings: one traced the lineage history of each nucleus from nuclear cycle 11 through nuclear cycle 14 in a small region of the embryo; the other recorded nuclear fate according to the timing and pattern of the 14th nuclear division. By comparing these lineage and fate maps for two embryos, we conclude that, at least for the examined area, the pattern of mitotic domain formation in Drosophila is determined by the position of each cell, with no effect of cell lineage.

Homeo boxes in the study of development.

The body plan of Drosophila is determined to a large extent by homeotic genes, which specify the identity and spatial arrangement of the body segments. Homeotic genes share a characteristic DNA segment, the homeo box, which encodes a defined domain of the homeotic proteins. The homeo domain seems to mediate the binding to specific DNA sequences, whereby the homeotic proteins exert a gene regulatory function. By isolating the normal Antennapedia gene, fusing its protein-coding sequences to an inducible promoter, and reintroducing this fusion gene into the germline of flies, it has been possible to transform head structures into thoracic structures and to alter the body plan in a predicted way. Sequence homologies suggest that similar genetic mechanisms may control development in higher organisms.

Induction by soluble factors of organized axial structures in chick epiblasts.

Inductive action of soluble factors was tested on isolated chick epiblasts. An assay was developed wherein conditioned medium derived from the Xenopus XTC cell line induced the formation of a full-length notochord and rows of bilaterally symmetric somites. Basic fibroblast growth factor, epidermal growth factor, retinoic acid, and transforming growth factor type B1 and B2 were not capable of inducing axial structures. Thus, soluble factors can elicit the development of polarity stored in the epiblast and behave as true morphogens since they can induce the formation of the organized complex structures that constitute the embryonic axis.

Mutations of the Drosophila zinc finger-encoding gene vielfältig impair mitotic cell divisions and cause improper chromosome segregation.

We describe the molecular characterization and function of vielfältig (vfl), a X-chromosomal gene that encodes a nuclear protein with six Krüppel-like C2H2 zinc finger motifs. vfl transcripts are maternally contributed and ubiquitously distributed in eggs and preblastoderm embryos, excluding the germline precursor cells. Zygotically, vfl is expressed strongly in the developing nervous system, the brain, and in other mitotically active tissues. Vfl protein shows dynamic subcellular patterns during the cell cycle. In interphase nuclei, Vfl is associated with chromatin, whereas during mitosis, Vfl separates from chromatin and becomes distributed in a granular pattern in the nucleoplasm. Functional gain-of-function and lack-of-function studies show that vfl activity is necessary for normal mitotic cell divisions. Loss of vfl activity disrupts the pattern of mitotic waves in preblastoderm embryos, elicits asynchronous DNA replication, and causes improper chromosome segregation during mitosis.

[Dynamics of the annulate lamellae in Drosophila syncytial embryos].

In eukaryotic cells, mitotic events are controlled by evolutionarily conserved cyclin-dependent kinases (cdk): these kinases phosphorylate cell proteins, which causes structural reorganization of the entire cell. Our recent studies of Drosophila syncytial embryos have demonstrated that cdk1 activity is a key factor that controls nuclear pore complex assembly/disassembly and affects the structure of cytoplasmic pores in the annulate. In this paper, we report a comparative analysis of these cytoplasmic organelles throughout the cell-cycle and throughout the development of Drosophila syncytial embryos. Based on the results obtained, it was presupposed that distribution of annulate lamellae containing cytoplasmic pores could reflect the inactivation of the mitotic kinase cdk1 in Drosophila syncytial embryos.

Cryopreservation of chicken blastodermal cells and their quality assessment by flow cytometry and transmission electron microscopy.

The goal of this study was to evaluate effect of slow freezing and vitrification methods on the viability of chicken blastodermal cells (BCs). Proper aliquot of isolated BCs were diluted in the freezing medium composed of 10% DMSO and frozen in the freezing vessel BICELL to reach desired temperature up to -80°C. Then samples were immersed in liquid nitrogen. Other cell aliquot was vitrified in solution containing 10% DMSO and samples were immediately immersed in the liquid nitrogen. The viability of fresh and frozen/thawed BCs was evaluated using Trypan blue method and flow cytometry. Flow cytometry analysis was provided by DRAQ5 dye in combination with Live-Dead kit. Overall, this technique provides both quantitative and qualitative information about BCs. Results obtained from Trypan blue method showed significant differences (P < 0.05) between control (8.37 ± 1.04%) slow freezing (83.73 ± 2.72%) and vitrification group (84.39 ± 1.77%) in the percentage of Trypan blue positive (necrotic) BCs. Moreover, differences (P < 0.05) between control and slow freezing (5.08 ± 1.94%, 73.31 ± 3.90%) and control and vitrification group (2.97 ± 0.30%, 79.02 ± 1.56%) in results on portion of necrotic cells (DRAQ5+ /LD+ ) analyzed by flow cytometry were also observed. The large percentage of necrotic BCs was found in all freezing methods. However, based on ultrastructural analysis, our study showed, that BCs contain lipid granules which prevent successful freezing even though different methods of cryopreservation were used. Thus, freezing of BCs probably required subsequent culture to eliminate lipid droples and yolk granules in the cells, which could possibly improve the success. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:778-783, 2018.

Inductive signals. Revolving vertebrates.

An old idea about the relationship between arthropod and vertebrate body plans has been given new life by studies of the signalling genes controlling dorsal and ventral development in Drosophila and Xenopus.

Functional coordination of three mitotic motors in Drosophila embryos.

It is well established that multiple microtubule-based motors contribute to the formation and function of the mitotic spindle, but how the activities of these motors interrelate remains unclear. Here we visualize spindle formation in living Drosophila embryos to show that spindle pole movements are directed by a temporally coordinated balance of forces generated by three mitotic motors, cytoplasmic dynein, KLP61F, and Ncd. Specifically, our findings suggest that dynein acts to move the poles apart throughout mitosis and that this activity is augmented by KLP61F after the fenestration of the nuclear envelope, a process analogous to nuclear envelope breakdown, which occurs at the onset of prometaphase. Conversely, we find that Ncd generates forces that pull the poles together between interphase and metaphase, antagonizing the activity of both dynein and KLP61F and serving as a brake for spindle assembly. During anaphase, however, Ncd appears to have no effect on spindle pole movements, suggesting that its activity is down-regulated at this time, allowing dynein and KLP61F to drive spindle elongation during anaphase B.

Probability distributions and compartment boundaries in the development of Drosophila.

Clone analysis and fate mapping probe several properties of development. Here it is shown that data on fate mapping support a probabilistic model of cell commitment in Drosophila blastoderms. Adult cells have a distribution of possible ancestors, as W. Baker (1978b) inferred from the theory of compartment-boundary development (Garcia-Bellido, Ripoll and Morata 1973). Fate-map data are used here to describe quantitatively the ancestry distributions on the blastoderm fate map. The properties of the distributions are sensitive to, and probes of, developmental events, such as relative time of cellularization and time of commitment. The theory of this analysis shows first how the meaningful interpretation of the stage represented by a fate map depends on the assumptions made in mapping. A general mapping model described below makes it possible to evaluate several interpretations. Interestingly, the data require a 3-dimensional map, and it is argued that this must be due to an effect of the preblastoderm nuclear synctial stage. Second, the theory shows how compartment boundaries affect ancestry distribution and why they have no observable effect on mapping. Third, the variability implied by ancestry variance does not create too much "noise" to make meaningful maps of small areas; rather, oddly enough, it tends to magnify the apparent distances within small areas to make them more resolvable. Empirical results include probabilistic maps of the Drosophila blastoderm. These results argue that time of commitment varies even for cells in the same compartment, demonstrating the need for a more complex model of early development than that proposed in the compartment model. The results also help to evaluate the significance of compartment boundaries in respect to developmental commitment.

Mechanism of closure of experimental excision-wounds in the bare upper layer of the chick blastoderm.

The closure of experimental excision-wounds in the upper layer of the gastrulating chick blastoderm was studied by time-lapse videography and videomicrography and by light microscopy, transmission electron microscopy and scanning electron microscopy. One experimental excision-wound was made in the upper layer of stages 4V to 6V blastoderms (Vakaet, Arch. Biol. (Liège) 81:387-426, 1970), in the proamnion where no middle layer cells are present. The deep layer was previously discarded, so that the wounds were made in the bare upper layer. They closed within 2 to 6 hours and further development was normal by in vitro standards. With videography, global movements of the upper layer towards the wound were observed. With videomicrography, the wound submarginal region cells were seen to move like sheep in a flock: individual cells in different directions, the whole flock towards the wound. During closure the shape of the wound edge was irregular. The structure of the epithelium of the wound submarginal region was unchanged throughout closure: a pseudostratified columnar epithelium in which cell divisions occur at its dorsal side and are parallel to its surface. The basal lamina was absent below the edge of the wounds. We propose that the cells of the upper layer are mobile against one another and are not confined to a specific part of the basal lamina. During wound closure the movements of the cells on the basal lamina would be driven by mitotic pressure. This is the horizontal pressure exerted by the addition of daughter cells and their parting during anaphase and telophase.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical analysis of the segregation process of the quail germ cell lineage.

An antiserum against quail 7 day gonadal germ cells was found to react specifically with gonadal germ cells of both sexes. Transverse sections from a range of early quail developmental stages were submitted to the antibody PAP reaction. Blastodiscs from the earliest uterine stages (II to X E.G. & K) reacted very strongly, while the overall reaction gradually decreased in older blastoderms. At stage XIII both epiblast and hypoblast were weakly stained, but some large, PGC-like cells stained intensively. During gastrulation (PS formation) the reaction of the epiblast disappears quicker than that of the hypoblast. The newly formed mesoderm and entoderm do not react at all and the reaction gradually becomes limited mainly to the PGCs and somewhat to the primary hypoblast which is moving into the germinal crescent. The widely spread reaction at the early stages is thus gradually being restricted to the PGCs.

Animal model: dysmorphogenesis and death in a chicken embryo model.

The chicken embryo is a useful animal model for investigating problems in developmental biology and teratology. Here we report data that further define the causes of 2 different patterns of malformation (one associated with amnion abnormalities, the other with isolated neural tube defects) and death induced by making a window in the shell and subshell membranes during the first day of incubation. The interpretation of these data suggests to us the following hypotheses. An early amnion deficit spectrum or syndrome (EADS) in chicken embryos is caused by a brief (less than 10 sec) perturbation that occurs during the windowing procedure. This perturbation results in an acute increase in mechanical tension to the developing embryo and support structures, dehydration localized to the area of the blastoderm, and/or increased friction between the blastoderm and overlying vitelline and shell membranes. Isolated neural tube defects (NTDs) are caused by a longer perturbation (greater than 3 hr) consisting of increased mechanical stress across the blastoderm. The mechanical stress is associated with the introduction of a new air space over the animal pole of the yolk during windowing. The new air space causes the shape of the yolk to change (ie, to be deformed), resulting in an increase in mechanical tension across the vitelline membrane and blastoderm. NTDs involving the head are associated with significant early embryonic mortality, whereas those involving the trunk are not. Death may also be caused by cardiovascular anomalies observed in EADS. It is concluded that disturbances in morphogenesis and death in this model are, therefore, the result of extrinsic forces (eg, mechanical stress, localized dehydration, or friction) acting on different tissue types at various critical times in development. Intensity and duration of these forces on the developing blastoderm are important variables.

Molecular genetic control of axis patterning during early embryogenesis of vertebrates.

Formation of the axis and its subsequent patterning to establish the tube-within-a-tube body plan characteristic of vertebrates are initiated during gastrulation. In higher vertebrates (i.e., birds and mammals), gastrulation involves six key events: establishment of the rostrocaudal/mediolateral axis; formation and progression of the primitive streak and organizer; epiboly of the epiblast, ingression of prospective mesodermal and endodermal cells through the primitive streak, and migration of cells away from the primitive streak; regression of the primitive streak; establishment of the right-left axis; and formation of the tail bud. Over 50 years of study of these processes have provided a morphological framework for understanding how these events occur, and recent advances in imaging, microsurgical intervention, and cell tracking are beginning to elucidate the underlying cell behaviors that drive morphogenetic movements. Moreover, homotopic transplantation and dye microinjection studies are being used to generate high-resolution fate maps, and heterotopic transplantation studies are revealing the cell-cell interactions that are sufficient as well as required for mesodermal and ectodermal commitment. Additionally, the roles of the organizer and secondary signaling centers in establishing the body plan are being defined. With the advent of the molecular/genetic age, the molecular basis for axis formation is beginning to become understood. Thus, it is becoming clear that secreted growth factors/signaling molecules produced by localized signaling centers induce and pattern the axis, presumably through downstream activation of signal-transduction proteins and cascades of transcription factors.

From egg to pole cells: ultrastructural aspects of early cleavage and germ cell determination in insects.

Insect eggs are giant and very complex cells covered by an extremely resistant shell. Both the egg cell and surrounding eggshell express anteroposterior and ventrodorsal polarity. The molecular and cytoplasmic organization of both axes originates during oogenesis and leads to the production of an ooplasmic system which consists of euplasm and deutoplasm (yolk) and contains a nucleus as well as extranuclear determinants of maternal origin. Both are part of the store of information for early embryogenesis. In addition, the deutoplasm serves as raw material and early nutrient supply for building the embryo. The insect egg cell, which is arrested in the first maturation division when released from the ovary during oviposition, will be activated by different stimuli among different species to complete meiosis and start embryogenesis. The zygote nucleus undergoes a number of synchronous mitotic divisions leading to cleavage energids which initially form a syncytial blastoderm and subsequently the cellular blastoderm. In many insects, prior to blastoderm formation, polar granules (or oosome material) are incorporated in a single cell or a small number of cells which bud off at the posterior pole. These so called pole cells give rise to the primordial germ cells. Therefore, polar granules or the oosome material mark the germ line, and while structural counterparts of determinants of body pattern formation have so far not been found, the polar granules or oosome serve as an autonomous ooplasmic determinant for the pole or germ cells. Anteroposterior body polarity can arise independent of the germ plasm.