Glyconectin Cell Adhesion Epitope, ß-d-GlcpNAc3S-(1→3)-α-l-Fucp, Is Involved in Blastulation of Lytechinus pictus Sea Urchin Embryos.

Glycans, as the most peripheral cell surface components, are the primary candidates to mediate the initial steps of cell recognition and adhesion via glycan-glycan binding. This molecular mechanism was quantitatively demonstrated by biochemical and biophysical measurements at the cellular and molecular level for the glyconectin 1 ß-d-GlcpNAc3S-(1→3)-α-l-Fucp glycan structure (GN1). The use of adhesion blocking monoclonal antibody Block 2 that specifically recognize this epitope showed that, besides Porifera, human colon carcinoma also express this structure in the apical glycocalyx. Here we report that Block 2 selectively immune-precipitate a Mr 580 × 103 (g580) acidic non-glycosaminoglycan glycan from the total protein-free glycans of Lytechinus pictus sea urchin hatched blastula embryos. Immuno-fluorescence confocal light microscopy and immunogold electron microscopy localized the GN1 structure in the apical lamina glycocalyx attachments of ectodermal cells microvilli, and in the Golgi complex. Biochemical and immune-chemical analyses showed that the g580 glycan is carrying about 200 copies of the GN1 epitope. This highly polyvalent g580 glycan is one of the major components of the glycocalyx structure, maximally expressed at hatched blastula and gastrula. The involvement of g580 GN1 epitope in hatched blastula cell adhesion was demonstrated by: (1) enhancement of cell aggregation by g580 and sponge g200 glycans, (2) inhibition of cell reaggregation by Block 2, (3) dissociation of microvilli from the apical lamina matrix by the loss of its gel-like structure resulting in a change of the blastula embryonal form and consequent inhibition of gastrulation at saturating concentration of Block 2, and (4) aggregation of beads coated with the immune-purified g580 protein-free glycan. These results, together with the previous atomic force microscopy measurements of GN1 binding strength, indicated that this highly polyvalent and calcium ion dependent glycan-glycan binding can provide the force of 40 nanonewtons per single ectodermal cell association of microvilli with the apical lamina, and conservation of glycocalyx gel-like structure. This force can hold the weight of 160,000 cells in sea water, thus it is sufficient to establish, maintain and preserve blastula form after hatching, and prior to the complete formation of further stabilizing basal lamina.

Xenopus chip for single-egg trapping, in vitro fertilization, development, and tadpole escape.

Xenopus laevis is highly suitable as a toxicology animal model owing to its advantages in embryogenesis research. For toxicological studies, a large number of embryos must be handled simultaneously because they very rapidly develop into the target stages within a short period of time. To efficiently handle the embryos, a convenient embryo housing device is essential for fast and reliable assessment and statistical evaluation of malformation caused by toxicants. Here, we suggest 3D fabrication of single-egg trapping devices in which Xenopus eggs are fertilized in vitro, and the embryos are cultured. We used manual pipetting to insert the Xenopus eggs inside the trapping sites of the chip. By introducing a liquid circulating system, we connected a sperm-mixed solution with the chip to induce in vitro fertilization of the eggs. After the eggs were fertilized, we observed embryo development involving the formation of egg cleavage, blastula, gastrula, and tadpole. After the tadpoles grew inside the chip, we saved their lives by enabling their escape from the chip through reverse flow of the culture medium. The Xenopus chip can serve as an incubator to induce fertilization and monitor normal and abnormal development of the Xenopus from egg to tadpole.

Cytokinetic abscission is part of the midblastula transition in early zebrafish embryogenesis.

Animal cytokinesis ends with the formation of a thin intercellular membrane bridge that connects the two newly formed sibling cells, which is ultimately resolved by abscission. While mitosis is completed within 15 min, the intercellular bridge can persist for hours, maintaining a physical connection between sibling cells and allowing exchange of cytosolic components. Although cell-cell communication is fundamental for development, the role of intercellular bridges during embryogenesis has not been fully elucidated. In this work, we characterized the spatiotemporal characteristics of the intercellular bridge during early zebrafish development. We found that abscission is delayed during the rapid division cycles that occur in the early embryo, giving rise to the formation of interconnected cell clusters. Abscission was accelerated when the embryo entered the midblastula transition (MBT) phase. Components of the ESCRT machinery, which drives abscission, were enriched at intercellular bridges post-MBT and, interfering with ESCRT function, extended abscission beyond MBT. Hallmark features of MBT, including transcription onset and cell shape modulations, were more similar in interconnected sibling cells compared to other neighboring cells. Collectively, our findings suggest that delayed abscission in the early embryo allows clusters of cells to coordinate their behavior during embryonic development.

Segregation of brain and organizer precursors is differentially regulated by Nodal signaling at blastula stage.

The blastula Chordin- and Noggin-expressing (BCNE) center comprises animal-dorsal and marginal-dorsal cells of the amphibian blastula and contains the precursors of the brain and the gastrula organizer. Previous findings suggested that the BCNE behaves as a homogeneous cell population that only depends on nuclear ß-catenin activity but does not require Nodal and later segregates into its descendants during gastrulation. In contrast to previous findings, in this work, we show that the BCNE does not behave as a homogeneous cell population in response to Nodal antagonists. In fact, we found that chordin.1 expression in a marginal subpopulation of notochordal precursors indeed requires Nodal input. We also establish that an animal BCNE subpopulation of cells that express both, chordin.1 and sox2 (a marker of pluripotent neuroectodermal cells), and gives rise to most of the brain, persisted at blastula stage after blocking Nodal. Therefore, Nodal signaling is required to define a population of chordin.1+ cells and to restrict the recruitment of brain precursors within the BCNE as early as at blastula stage. We discuss our findings in Xenopus in comparison to other vertebrate models, uncovering similitudes in early brain induction and delimitation through Nodal signaling.

Interphase-arrested Drosophila embryos activate zygotic gene expression and initiate mid-blastula transition events at a low nuclear-cytoplasmic ratio.

Externally deposited eggs begin development with an immense cytoplasm and a single overwhelmed nucleus. Rapid mitotic cycles restore normality as the ratio of nuclei to cytoplasm (N/C) increases. A threshold N/C has been widely proposed to activate zygotic genome transcription and onset of morphogenesis at the mid-blastula transition (MBT). To test whether a threshold N/C is required for these events, we blocked N/C increase by down-regulating cyclin/Cdk1 to arrest early cell cycles in Drosophila. Embryos that were arrested two cell cycles prior to the normal MBT activated widespread transcription of the zygotic genome including genes previously described as N/C dependent. Zygotic transcription of these genes largely retained features of their regulation in space and time. Furthermore, zygotically regulated post-MBT events such as cellularization and gastrulation movements occurred in these cell cycle-arrested embryos. These results are not compatible with models suggesting that these MBT events are directly coupled to N/C. Cyclin/Cdk1 activity normally declines in tight association with increasing N/C and is regulated by N/C. By experimentally promoting the decrease in cyclin/Cdk1, we uncoupled MBT from N/C increase, arguing that N/C-guided down-regulation of cyclin/Cdk1 is sufficient for genome activation and MBT.

SPIN90 associates with mDia1 and the Arp2/3 complex to regulate cortical actin organization.

Cell shape is controlled by the submembranous cortex, an actomyosin network mainly generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in relative nucleator activity may alter cortical organization, mechanics and cell shape. Here we investigate how nucleation-promoting factors mediate interactions between nucleators. In vitro, the nucleation-promoting factor SPIN90 promotes formation of unbranched filaments by Arp2/3, a process thought to provide the initial filament for generation of dendritic networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our in vitro experiments reveal that this feature stems mainly from two mechanisms: efficient recruitment of mDia1 to SPIN90-Arp2/3 nucleated filaments and formation of a ternary SPIN90-Arp2/3-mDia1 complex that greatly enhances filament nucleation. Both mechanisms yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90-Arp2/3 at their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the proportion of long filaments elongated by mDia1.

Maternal contributions to gastrulation in zebrafish.

Gastrulation is a critical early morphogenetic process of animal development, during which the three germ layers; mesoderm, endoderm and ectoderm, are rearranged by internalization movements. Concurrent epiboly movements spread and thin the germ layers while convergence and extension movements shape them into an anteroposteriorly elongated body with head, trunk, tail and organ rudiments. In zebrafish, gastrulation follows the proliferative and inductive events that establish the embryonic and extraembryonic tissues and the embryonic axis. Specification of these tissues and embryonic axes are controlled by the maternal gene products deposited in the egg. These early maternally controlled processes need to generate sufficient cell numbers and establish the embryonic polarity to ensure normal gastrulation. Subsequently, after activation of the zygotic genome, the zygotic gene products govern mesoderm and endoderm induction and germ layer patterning. Gastrulation is initiated during the maternal-to-zygotic transition, a process that entails both activation of the zygotic genome and downregulation of the maternal transcripts. Genomic studies indicate that gastrulation is largely controlled by the zygotic genome. Nonetheless, genetic studies that investigate the relative contributions of maternal and zygotic gene function by comparing zygotic, maternal and maternal zygotic mutant phenotypes, reveal significant contribution of maternal gene products, transcripts and/or proteins, that persist through gastrulation, to the control of gastrulation movements. Therefore, in zebrafish, the maternally expressed gene products not only set the stage for, but they also actively participate in gastrulation morphogenesis.

Molecular genetics of maternally-controlled cell divisions.

Forward genetic screens remain at the forefront of biology as an unbiased approach for discovering and elucidating gene function at the organismal and molecular level. Past mutagenesis screens targeting maternal-effect genes identified a broad spectrum of phenotypes ranging from defects in oocyte development to embryonic patterning. However, earlier vertebrate screens did not reach saturation, anticipated classes of phenotypes were not uncovered, and technological limitations made it difficult to pinpoint the causal gene. In this study, we performed a chemically-induced maternal-effect mutagenesis screen in zebrafish and identified eight distinct mutants specifically affecting the cleavage stage of development and one cleavage stage mutant that is also male sterile. The cleavage-stage phenotypes fell into three separate classes: developmental arrest proximal to the mid blastula transition (MBT), irregular cleavage, and cytokinesis mutants. We mapped each mutation to narrow genetic intervals and determined the molecular basis for two of the developmental arrest mutants, and a mutation causing male sterility and a maternal-effect mutant phenotype. One developmental arrest mutant gene encodes a maternal specific Stem Loop Binding Protein, which is required to maintain maternal histone levels. The other developmental arrest mutant encodes a maternal-specific subunit of the Minichromosome Maintenance Protein Complex, which is essential for maintaining normal chromosome integrity in the early blastomeres. Finally, we identify a hypomorphic allele of Polo-like kinase-1 (Plk-1), which results in a male sterile and maternal-effect phenotype. Collectively, these mutants expand our molecular-genetic understanding of the maternal regulation of early embryonic development in vertebrates.

Embryonic ethanol exposure alters expression of sox2 and other early transcripts in zebrafish, producing gastrulation defects.

Ethanol exposure during prenatal development causes fetal alcohol spectrum disorder (FASD), the most frequent preventable birth defect and neurodevelopmental disability syndrome. The molecular targets of ethanol toxicity during development are poorly understood. Developmental stages surrounding gastrulation are very sensitive to ethanol exposure. To understand the effects of ethanol on early transcripts during embryogenesis, we treated zebrafish embryos with ethanol during pre-gastrulation period and examined the transcripts by Affymetrix GeneChip microarray before gastrulation. We identified 521 significantly dysregulated genes, including 61 transcription factors in ethanol-exposed embryos. Sox2, the key regulator of pluripotency and early development was significantly reduced. Functional annotation analysis showed enrichment in transcription regulation, embryonic axes patterning, and signaling pathways, including Wnt, Notch and retinoic acid. We identified all potential genomic targets of 25 dysregulated transcription factors and compared their interactions with the ethanol-dysregulated genes. This analysis predicted that Sox2 targeted a large number of ethanol-dysregulated genes. A gene regulatory network analysis showed that many of the dysregulated genes are targeted by multiple transcription factors. Injection of sox2 mRNA partially rescued ethanol-induced gene expression, epiboly and gastrulation defects. Additional studies of this ethanol dysregulated network may identify therapeutic targets that coordinately regulate early development.

Embryonic and early larval development of two marine angelfish, Centropyge bicolor and Centropyge bispinosa.

Marine angelfish (family: Pomacanthidae) are among the most sought-after fish species in the saltwater aquarium trade. However, there is a lack of information in the literature on their early ontogeny. The objective of this study was to describe the embryonic and early larval development of two dwarf angelfish, the bicolour angelfish, Centropyge bicolor and the coral beauty angelfish, Centropyge bispinosa. The eggs of these two species were obtained from spontaneous spawning of the broodstock fish in captivity and incubated at 26.0 ± 0.2°C throughout the study. Fertilized eggs (n = 15) of both species are transparent, pelagic and spherical; the mean diameters of the eggs were measured at 703.6 ± 7.8 µm for C. bicolor and 627.6 ± 7.8 µm for C. bispinosa. The eggs of both species possessed a narrow perivitelline space, smooth and thin chorion, a homogenous and non-segmented yolk as well as a single oil globule. Overall, the observed embryonic development pattern of C. bicolor and C. bispinosa was very similar, and the main difference was the embryonic pigmentation pattern, which only became evident close to hatching. Larvae of both species started hatching at 13 h 30 min after fertilization, and the larval characteristics of both species also showed high levels of similarities. However, the mouth opening time for C. bicolor was 72 h after hatching (AH) and 96 AH for C. bispinosa. In general, the observed early ontogeny of C. bicolor and C. bispinosa also resembled that of other Centropyge species documented in the literature.

Blastula stage specification of avian neural crest.

Cell fate specification defines the earliest steps towards a distinct cell lineage. Neural crest, a multipotent stem cell population, is thought to be specified from the ectoderm, but its varied contributions defy canons of segregation potential and challenges its embryonic origin. Aiming to resolve this conflict, we have assayed the earliest specification of neural crest using blastula stage chick embryos. Specification assays on isolated chick epiblast explants identify an intermediate region specified towards the neural crest cell fate. Furthermore, low density culture suggests that the specification of intermediate cells towards the neural crest lineage is independent of contact mediated induction and Wnt-ligand induced signaling, but is, however, dependent on transcriptional activity of ß-catenin. Finally, we have validated the regional identity of the intermediate region towards the neural crest cell fate using fate map studies. Our results suggest a model of neural crest specification within a restricted epiblast region in blastula stage chick embryos.

Histone concentration regulates the cell cycle and transcription in early development.

The early embryos of many animals, including flies, fish and frogs, have unusually rapid cell cycles and delayed onset of transcription. These divisions are dependent on maternally supplied RNAs and proteins including histones. Previous work suggests that the pool size of maternally provided histones can alter the timing of zygotic genome activation (ZGA) in frogs and fish. Here, we examine the effects of under- and overexpression of maternal histones in Drosophila embryogenesis. Decreasing histone concentration advances zygotic transcription, cell cycle elongation, Chk1 activation and gastrulation. Conversely, increasing histone concentration delays transcription and results in an additional nuclear cycle before gastrulation. Numerous zygotic transcripts are sensitive to histone concentration, and the promoters of histone-sensitive genes are associated with specific chromatin features linked to increased histone turnover. These include enrichment of the pioneer transcription factor Zelda, and lack of SIN3A and associated histone deacetylases. Our findings uncover a crucial regulatory role for histone concentrations in ZGA of Drosophila.

Self-Similar Dynamics of Nuclear Packing in the Early Drosophila Embryo.

Embryonic development starts with cleavages, a rapid sequence of reductive divisions that result in an exponential increase of cell number without changing the overall size of the embryo. In Drosophila, the final four rounds of cleavages occur at the surface of the embryo and give rise to ∼6000 nuclei under a common plasma membrane. We use live imaging to study the dynamics of this process and to characterize the emergent nuclear packing in this system. We show that the characteristic length scale of the internuclear interaction scales with the density, which allows the densifying embryo to sustain the level of structural order at progressively smaller length scales. This is different from nonliving materials, which typically undergo disorder-order transition upon compression. To explain this dynamics, we use a particle-based model that accounts for density-dependent nuclear interactions and synchronous divisions. We reproduce the pair statistics of the disordered packings observed in embryos and recover the scaling relation between the characteristic length scale and the density both in real and reciprocal space. This result reveals how the embryo can robustly preserve the nuclear-packing structure while being densified. In addition to providing quantitative description of self-similar dynamics of nuclear packings, this model generates dynamic meshes for the computational analysis of pattern formation and tissue morphogenesis.

Embryonic and larval development of the northern pike: An emerging fish model system for evo-devo research.

The northern pike, Esox lucius, is one of the largest temperate freshwater apex predators with a characteristic morphology: an elongated body with pelvic, dorsal, and anal fins located at the rear as a functional feature to sprint predation. However, the typical pike character is its head, which is characterized by a long, flattened snout, a well-armed mouth with numerous teeth, and large eyes characteristic of shallow water visual predators. Although the northern pike is becoming increasingly popular as a model system for ecology and evolutionary research, a detailed staging table has not yet been reported. In this study, we report the first comprehensive staging table for the northern pike, spanning from the one-cell stage to the freely-swimming juvenile stage. In addition to classical embryological descriptions, we use a DAPI staining to distinguish individual cells and embryonic structures during the early development. This dataset, in combination with the genomic and transcriptomic resources already available, serves as a foundation for in-depth mechanistic studies dealing with development using this species.

The cleavage pattern of calanoid copepods-a case study.

Many crustacean groups show stereotyped cleavage patterns during early ontogeny. However, these patterns differ between the various major crustacean taxa, and a general mode is difficult to extract. Previous studies suggested that also copepods undergo an early cleavage with a more or less stereotyped pattern of blastomere divisions and fates. Yet, copepod embryology has been largely neglected. The last investigation of this kind dates back more than a century and the results are somewhat contradictory when compared with those of other researchers. To overcome these problems, we studied the early development of a so far undescribed calanoid copepod species, Skistodiaptomus sp., applying histochemical staining, confocal laser scanning microscopy, and bifocal 4D microscopy. The blastomere arrangement of the four-cell stage of this species varies to a large degree. It can either form a typical radial pattern with the four blastomeres lying in one plane or a tilted orientation of the axes connecting the sister cells of the previous division. In both cases, a stereotyped division pattern is maintained inside each quadrant during subsequent cleavages. In addition, we found two types of blastomere arrangements with a mirror symmetry. Most divisions within the quadrants follow the perpendicularity rule until the eighth cleavage. Deviations from this rule occur only in the narrow regions where the different quadrants touch and near the site of gastrulation. Gastrulation is initiated around the descendants of one individually identifiable blastomere of the 16-cell stage. This cell divides in a specific manner forming a characteristic cell arrangement, the gastrulation triangle. This gastrulation triangle initiates the internalization process of the gastrulation and it is encircled by another characteristic cell type, the crown cells. Our observations reveal several similarities to the early development of Calanus finmarchicus, another calanoid species. These relate to blastomere arrangements and divisions and the pattern of gastrulation. As Calanoida represent a basal or near basal branch of the copepod tree, this description will provide the ground for reconstruction of the cleavage pattern of the last common ancestor of Copepoda.

Embryonic development of the bullseye puffer Sphoeroides annulatus (Tetraodontidae): A morphofunctional approach to ontogenetic steps.

The embryonic development of the bullseye puffer, Sphoeroides annulatus, was characterized on the basis of the theory of saltatory ontogeny. This theory predicts a correlative relationship between the ontogeny-type in an altricial-precocial spectrum and the habitat that a species occupies within an unstable-stable environmental spectrum. Because S. annulatus inhabits a variety of unstable environments along a wide latitudinal range, the hypothesis that this species presents one of the most altricial embryonic developments among tetraodontids was tested. Based on major developmental events that marked the ontogenetic thresholds nine embryonic steps were identified. Developmental features such as small adhesives eggs, lack of vitelline circulation, small free embryos swimming up at hatching guided by positive phototaxis, and small first-feeding larvae actively swam in the water column, suggest that S. annulatus belongs to the reproductive guild of the nonguarders-lithopelagophils. Moreover, a comparative analysis of the developmental sequences, egg size, and first-feeding larvae size between tetraodontids confirms the hypothesis of this study and supports the evolutionary principle of the altricial-precocial spectrum postulated in the theory of saltatory ontogeny.

Cleavage Blastomere Explant Culture in Xenopus.

The individual blastomeres of Xenopus two- to 32-cell embryos have been fate mapped. This work identified the precursors of most of the embryonic cell types, tissues and organs; however, the maps do not reveal the cell interactions or signaling pathways that are required for establishing cell fates. This protocol describes an explant culture approach for culturing blastomeres in isolation to test whether a cell's fate has been determined. Cleavage blastomeres can be cultured in a simple salt medium without added factors because they contain intracellular yolk platelets, which provide an intrinsic energy source. This method allows one to test whether an isolated blastomere can produce specific cell types or express tissue-specific genes independent of interactions with other cells or specific signaling pathways. The role of cell-cell interactions can be revealed by co-culturing different sets of blastomeres. One can identify the molecules that are required for those cell fates by applying knockdown approaches to the isolated cell. One also can determine the developmental time at which cell fates are committed by explanting blastomere lineages at different stages.

Analysis of Cell Fate Commitment in Xenopus Embryos.

The fates of individual cleavage-stage blastomeres and of groups of cells at the blastula through gastrula stages of Xenopus embryos have been mapped in great detail. These studies identified the major contributors of the three germ layers as well as a variety of tissues and organs and several specific cell types. One can use these fate maps to test the commitment of single cells or groups of cells to produce their normal repertoire of descendants, to identify the genes that regulate fate commitment, and to modulate the levels of gene expression in specific lineages to determine gene function in a variety of developmental processes. Here we introduce methods that include how to identify specific blastomeres, inject them with lineage tracers, and alter gene expression levels. We also discuss methods for assaying protein and mRNA expression in situ and for providing novel embryonic environments to test fate commitment. These techniques draw upon classical approaches that are quite easy to perform in the versatile Xenopus embryo.

Cleavage Blastomere Deletion and Transplantation to Test Cell Fate Commitment in Xenopus.

Fate maps identify the precursors of an organ, and tracing the members of a blastomere lineage over time shows how its descendants come to populate that organ. The fates of the individual blastomeres of the two- to 32-cell Xenopus embryo have been fully mapped to reveal which cells are the major contributors to various cell types, tissues, and organs. However, because these fate maps were produced in the normal embryo, they do not reveal whether a precursor blastomere is competent to give rise to additional tissues or is already committed to its fate-mapped repertoire of descendants. To identify the mechanisms by which a cell's fate is committed, one needs to expose the cell to different experimental environments. If the cell's fate is determined, it will express its normal fate or gene expression profile in novel environments, whereas if it is not yet determined it will express different fates or gene expression profiles when exposed to novel external factors or neighboring cells. This protocol describes two techniques for testing cell fate commitment: single cell deletion and single cell transplantation. Deleting a blastomere allows one to test whether the deleted cell is required for the remaining cells to produce their normal, specific cell fates. Transplanting a blastomere to a novel location in a host embryo allows one to test whether the transplanted cell is committed to produce its normal fate-mapped repertoire, or whether it is still competent to respond to novel cell-cell interactions.

Generation of Ectopic Morphogen Gradients in the Zebrafish Blastula.

In the zebrafish embryo, cells of the early blastula animal pole are all equivalent and are fully pluripotent until the midblastula transition that occurs at the tenth cell cycle (512 to 1K cells). This naive territory of the embryo is therefore perfectly suited to assay for morphogen activity. Here we describe different methods to generate ectopic morphogen gradients, either in vivo at the animal pole of the embryo, or in vitro in animal pole explants or in aggregates of animal pole blastomeres (also named embryoid bodies). These methods include injection of mRNA coding for growth factor(s) into animal pole blastomere(s), transplantation of growth factor(s) secreting cells, implantation of beads coated with purified protein(s), and various combinations of these different approaches. Our comparative study reveals that all these methods allow to generate morphogen gradient(s) that are able to induce, both in vivo and in vitro, the formation of a well-patterned embryonic axis.