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.

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.

Building quantitative, three-dimensional atlases of gene expression and morphology at cellular resolution.

Animals comprise dynamic three-dimensional arrays of cells that express gene products in intricate spatial and temporal patterns that determine cellular differentiation and morphogenesis. A rigorous understanding of these developmental processes requires automated methods that quantitatively record and analyze complex morphologies and their associated patterns of gene expression at cellular resolution. Here we summarize light microscopy-based approaches to establish permanent, quantitative datasets-atlases-that record this information. We focus on experiments that capture data for whole embryos or large areas of tissue in three dimensions, often at multiple time points. We compare and contrast the advantages and limitations of different methods and highlight some of the discoveries made. We emphasize the need for interdisciplinary collaborations and integrated experimental pipelines that link sample preparation, image acquisition, image analysis, database design, visualization, and quantitative analysis.

Clustering and protein dynamics of Drosophila melanogaster telomeres.

Telomeres are obligatory chromosomal landmarks that demarcate the ends of linear chromosomes to distinguish them from broken ends and can also serve to organize the genome. In both budding and fission yeast, they cluster at the periphery of the nucleus, potentially to establish a compartment of silent chromatin. To gain insight into telomere organization in higher organisms, we investigated their distribution in interphase nuclei of Drosophila melanogaster. We focused on the syncytial blastoderm, an excellent developmental stage for live imaging due to the synchronous division of the nuclei at this time. We followed the EGFP-labeled telomeric protein HOAP in vivo and found that the 16 telomeres yield four to six foci per nucleus, indicative of clustering. Furthermore, we confirmed clustering in other somatic tissues. Importantly, we observed that HOAP signal intensity in the clusters increases in interphase, potentially due to loading of HOAP to newly replicated telomeres. To determine the rules governing clustering, we used in vivo imaging and fluorescence in situ hybridization to test several predictions. First, we inspected mutant embryos that develop as haploids and found that clustering is not mediated by associations between homologs. Second, we probed specifically for a telomere of novel sequence and found strong evidence against DNA sequence identity and homology as critical factors. Third, we ruled out predominance of intrachromosomal interactions by marking both ends of a chromosome. Based on these results, we propose that clustering is independent of sequence and is likely maintained by an as yet undetermined factor.

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.

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.

[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.

Ultrastructure of normal and JHA-treated eggs of the soft tick Argas persicus (Oken).

The ultrastructure of normal development and JHA-treated egg was examined with the transmission electron microscope. In newly oviposited egg, the chorion consists of exo- and endochorion. Between the chorion and plasma membrane lies vitelline (envelope) membrane, a thin non cellular membrane. The periplasm is free of the yolk spheres with contains vesicles of various sizes and lipid inclusions. The endoplasm is rich in yolk spheres, mitochondria, lipid inclusions, dense vesicles and vacuoles. In 48 h-old egg, cleavage nuclei was seen migrating to the periphery of the egg. The ooplasm around the nuclei contains vesicles, endoplasmic reticulum and few mitochondria. The blastoderm cells contain many organelles and inclusions. Secondary vitellophages and pole cells were observed. In 96 h-old egg, the germ band is formed. It consists of two layers, ectoderm and mesoderm. Amnion and serosa were visible. At (0-1 hour) eggs post-treatment with JHA (Admiral), no real effects was observed except for vacuolation of endoplasm; while at 48 h & 96 h-old post-treatment of eggs, great deterioration were seen which ended by degeneration and lysis of cell components leaving cell debris, vacuolation of nuclei, iregular shape of nuclear membrane and dispersed chromatin material.

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.

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.

Stages and other aspects of the embryology of the parthenogenetic Marmorkrebs (Decapoda, Reptantia, Astacida).

The early development of the parthenogenetic Marmorkrebs (marbled crayfish) is described with respect to external morphology, cell lineage, and segment formation. Due to its parthenogenetic reproduction mode, the question arises whether or not the marbled crayfish is a suitable model organism for developmental approaches. To address this question, we describe several aspects of the embryonic development until hatching. We establish ten stages based on characteristic external changes in the living eggs such as blastoderm formation, gastrulation process, formation and differentiation of the naupliar and post-naupliar segments, limb bud differentiation, and eye differentiation. The study of the post-naupliar cell division patterns, segment formation, and engrailed expression reveals distinct similarities to that of other freshwater crayfish. On this basis, we evaluate the possibility of a generalization of ontogenetic processes in the Marmorkrebs for either freshwater crayfish or other crustacean developmental systems.

Gastrulation in the sea anemone Nematostella vectensis occurs by invagination and immigration: an ultrastructural study.

The sea anemone Nematostella vectensis has recently been established as a new model system for the understanding of the evolution of developmental processes. In particular, the evolutionary origin of gastrulation and its molecular regulation are the subject of intense investigation. However, while molecular data are rapidly accumulating, no detailed morphological data exist describing the process of gastrulation. Here, we carried out an ultrastructural study of different stages of gastrulation in Nematostella using transmission electron microscope and scanning electron microscopy techniques. We show that presumptive endodermal cells undergo a change in cell shape, reminiscent of the bottle cells known from vertebrates and several invertebrates. Presumptive endodermal cells organize into a field, the pre-endodermal plate, which undergoes invagination. In parallel, the endodermal cells decrease their apical cell contacts but remain loosely attached to each other. Hence, during early gastrulation they display an incomplete epithelial-mesenchymal transition (EMT). At a late stage of gastrulation, the cells eventually detach and fill the interior of the blastocoel as mesenchymal cells. This shows that gastrulation in Nematostella occurs by a combination of invagination and late immigration involving EMT. The comparison with molecular expression studies suggests that cells expressing snailA undergo EMT and become endodermal, whereas forkhead/brachyury expressing cells at the ectodermal margin of the blastopore retain their epithelial integrity throughout gastrulation.

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.

Ultrastructural and immunohistochemical characterization of the bovine epiblast.

The epiblast represents the final embryonic founder cell population with the potential for giving rise to all cell types of the adult body. The pluripotency of the epiblast is lost during the process of gastrulation. Large animal species have a lack of specific markers for pluripotency. The aim of the present study was to characterize the bovine epiblast cell population and to provide such markers. Bovine Day 12 and Day 14 embryos were processed for transmission-electron microscopy or immunohistochemistry. In Day 12 embryos, two cell populations of the epiblast were identified: one constituting a distinctive basal layer apposing the hypoblast, and one arranged inside or above the former layer, including cells apposing the Rauber layer. Immunohistochemically, staining for the octamer-binding transcription factor 4 (OCT4, also known as POU5F1), revealed a specific and exclusive staining of nuclei of the complete epiblast. Colocalization of vimentin and OCT4 was demonstrated. Only trophectodermal cells stained for alkaline phosphatase. Staining for the proliferation marker Ki-67 was localized to most nuclei throughout the epiblast. A continuous staining for zonula occludens-1 protein was found between cells of the trophectoderm and hypoblast but was not evident in the epiblast. A basement membrane, detected by staining for laminin, formed a "cup-like" structure in which the epiblast was located. The ventrolateral sides of the cup appeared to be incomplete. In conclusion, the bovine epiblast includes at least two cell subpopulations, and OCT4 was shown, to our knowledge for the first time, to be localized exclusively to epiblast cells in this species.

Induction and improved embryonic development by the nucleus of Pander in associated avian blastoderm parts: influence of delta or gamma ooplasm.

After placing in vitro, central subgerminal ooplasm (containing a central nucleus of Pander) from a quail germ disc of a prelaid egg (before symmetrization) on the upper layer of an isolated chicken antisickle, we observed the induction of a radially oriented preneural plate (without interference of chordamesoblast). This observation suggests the primary existence during the period of symmetrization in utero of an until now unknown temporospatially linked "vertical" effect, emanating from the nucleus of Pander, on the parallel (pre)neural plate anlage forming part of the area centralis in the overlying blastoderm. For comparison, we "sandwiched" in vitro a quail sickle endoblast fragment between the deep side of the upper layer of an isolated chicken antisickle region and a central subgerminal ooplasmic mass. This resulted in a colonization of the subgerminal ooplasmic mass by quail sickle endoblast cells followed by improved neurulation and/or gastrulation phenomena. The latter never occurs in the absence of central subgerminal ooplasm. In both types of experiments there seems to exist a common link between the observed induction phenomena: the presence of delta ooplasm in the involved deep structures. Indeed, the nucleus of Pander contains delta ooplasm as well as the structures derived from it, i.e., endophyll with primordial germ cells and sickle endoblast-derived cells after colonization of the neighboring central ooplasm (present study). Therefore, we think that the preneural plate-inducing effect observed after placing a nucleus of Pander on the antisickle region is due to the presence of a factor in the delta ooplasm that diffuses in the neighborhood. The appearance of gastrulation phenomena in the second type of experiment seems to be due to colonization of the more peripheral part of the central subgerminal ooplasm containing the more superficial and peripheral gamma ooplasm in which Rauber's sickle material can develop. This suggests that the kind of involved ooplasm (delta or gamma) can predetermine the inductive activity of the deep structures that contain it: the central part of the nucleus of Pander and/or endophyll for preneurulation phenomena and sickle endoblast (in the presence of central subgerminal ooplasm) for gastrulation and/or neurulation phenomena.

Early development and segment formation in the centipede, Strigamia maritima (Geophilomorpha).

Geophilomorph centipedes exhibit a number of unique characteristics that make them of particular developmental and evolutionary interest. Segment numbers in geophilomorphs are higher than in any other centipedes, ranging from 27 to 191. They may be constant within a species, presenting in extreme form the "counting" problem in development, or they may vary--a situation that provides us with the opportunity to study naturally occurring variation in segment numbers. All their segments are generated during embryogenesis, a situation unlike that in the more basal centipede orders, which generate only a fraction of their 15 trunk segments in the embryo and develop the rest postembryonically. Here we provide a foundation for further developmental studies of the Geophilomorpha, building on the one study that has been conducted to date, on the coastal species Strigamia maritima. Development begins with the migration of nuclei to the surface of the egg, which then condense to form an embryonic rudiment of more than 20,000 cells, covering an entire hemisphere. During early development, the embryo can be divided into two distinct areas: a large terminal disc of apparently undifferentiated tissue and the germ-band, which has a clear anteroposterior axis and differentiated segments. The germ-band forms from the anterior of the terminal disc and extends anteriorly as the disc contracts. New segments are formed at the posterior margin of the germ-band. Once the process of segmentation ends, the germ-band folds and sinks into the yolk. We note that the classic description of centipede development, by Heymons more than a century ago, contains a fundamental error in the identification of the axes and hence in the interpretation of early segmentation.

Florence Sabin and the mechanism of blood vessel lumenization during vasculogenesis.

The notion that blood vessel lumina and primordial blood plasma are linked by a single mechanism, intracellular vacuolation of angioblasts, has, for the most part, been overlooked since it was first described in the early decades of the last century. That vacuolation may play a major role in blood vessel formation during vasculogenesis is revisited from the perspective of Florence Sabin's seminal studies in the nascent mesoderm of living chick blastoderms.

In the absence of Rauber's sickle material, no blood islands are formed in the avian blastoderm.

Using the quail-chick chimera technique, we followed the fate of Rauber's sickle cells in older whole blastoderms (cultured for approximately 2 days): after removal of the autochthonous Rauber's sickle from an unincubated chicken blastoderm, a quail Rauber's sickle was grafted isotopically and isochronically in its place. In transverse sections through these chimeras, the grafted quail Rauber's sickle cells were seen to have transformed into a broad row or ridge of quail junctional endoblast cells extending at the inner border of the area containing blood islands. After unilateral removal of the junctional endoblast from an intermediate streak chicken blastoderm (Stage 3; Hamburger and Hamilton [1951] J Morphol 88:49-92), we observed during further in vitro culture that at the operated side, in the area previously occupied by this junctional endoblast, blood islands no longer developed. If after such a unilateral removal of the chicken junctional endoblast quail junctional endoblast was apposed in its place, then blood islands reappeared in the operated area. The intimate contact between the apposed quail junctional endoblast and the recently formed blood islands, derived from peripherally migrating mesoderm, was very obvious on sections through such chimeras. We further demonstrate that Rauber's sickle vs. junctional endoblast is indispensable for the anlage of blood islands in avian blastoderms. Indeed, in the absence of Rauber's sickle material no blood islands develop (even when mesoderm is present after ingression of the upper layer via a primitive streak) in the isolated central region of the area centralis of unincubated chicken blastoderms after culture in vitro. Also, no junctional endoblast and no sickle canal appear in these explants. By contrast, if a Rauber's sickle fragment is placed on such an isolated central blastoderm region, then blood islands develop. These blood islands start to develop from peripherally migrating mesoderm in the neighborhood of the Rauber's sickle-derived junctional endoblast.

Utilization of central disk of blastoderm and germinal crescent region for production of interspecific germline chimera between chicken and quail.

AIM: The production of interspecific germline chimeras between chicken and quail were attempted employing the dissociated cells derived from the blastodermal central disk (stage X) and the germinal crescent region of embryo (stage 7-8). METHODS: The central disk (CD) of the area pellucida in chicken blastoderm (stage X) and the germinal crescent region (GCR) of embryo (stage 7-8) were dispersed and injected into the subgerminal cavity of quail blastoderm (stage X). Injected eggs were incubated for 7 days or to hatching. The donor chicken DNA was detected by the polymerase chain reaction. RESULTS: In day-7 embryos, chicken DNA was detected in 5 gonads and 9 brains from 53 survived embryos received chicken CD cells, and 1 gonads and 6 brains from 27 survived embryos received chicken GCR. Chicken DNA was also detected from the semen of one adult male hatched from eggs received chicken GCR cells. CONCLUSION: CD and GCR cells as the donors showed the possibility to produce the interspecific germline chimera, but further studies are needed to make necessary improvement.

Post-nodal mesoblast caudalizes the host axis and inhibits cell population growth, and induces new primitive streaks in chick embryos.

One to eight post-nodal fragments (PN) or Hensen's nodes (HN) from full primitive streak stage chick embryos were transplanted onto the area pellucida or area opaca of stage 4 embryos and cultured for 20 h. Thirteen morphological and numerical parameters were affected in the host embryos and analyzed by multiple logistic regression for parametric hierarchy. In the area pellucida, both PN and HN transplants inhibited cell population growth while only PN caudalized the host axis and induced supernumerary primitive streaks expressing the mesoderm-specific gene Brachyury. In the area opaca, neither grafts influenced host axis morphogenesis, but PN inhibited the cell population growth significantly. Tracking [(3)H]TdR labeled grafts showed that PN cells migrated towards the host axis and participated in the formation of supernumerary somites and hearts. When placed near the host axis, PN caudalized it and inhibited cell population growth.