Tissue-specific, temporal changes in cell adhesion to echinonectin in the sea urchin embryo.

Echinonectin is a dimeric, glycoprotein found in the hyaline layer of the developing sea urchin embryo. It was found that echinonectin supports adhesion of embryonic cells in vitro. Previous studies have shown that the protein hyalin also supports adhesion. The purpose of this study was to examine the specificity of cell-echinonectin interactions during sea urchin development. Primary mesenchyme cells (PMCs) ingress into the blastocoel during gastrulation. In the process the PMCs lose contact with the hyaline layer. It was found experimentally that differentiating PMCs decreased their adhesion to hyalin at the time of ingression. It was of interest, therefore, to determine whether there was a coordinate loss of adhesion to echinonectin at ingression as well. When cell-echinonectin interactions were quantified using a centrifugal force-based adhesion assay, it was shown that micromeres adhered well to echinonectin. At the time of ingression, PMCs displayed reduced adhesion to echinonectin just as had been found when hyalin was tested as a substrate. There was no change in adhesion of presumptive ectoderm or endoderm to echinonectin over the same time period. Early in gastrulation presumptive ectoderm and endoderm adhered to echinonectin only half as strongly as to equimolar concentrations of hyalin. After gastrulation endoderm cells were observed to retain the same relative affinity to hyalin and echinonectin, while ectoderm cells became equally adhesive for both hyalin and echinonectin. Quantitatively, this represents an overall increase in the affinity of ectodermal cells for echinonectin. Adhesion to combined substrata of echinonectin and hyalin was reduced but not abolished by monoclonal antibodies specific for echinonectin. The antibodies did not cross-react with hyalin. We conclude that both echinonectin and hyalin independently act as adhesive substrata for the developing sea urchin embryo. PMCs lose an affinity for echinonectin and ectodermal cells later increase their affinity for this substrate.     

Lefty acts as an essential modulator of Nodal activity during sea urchin oral-aboral axis formation

Nodal is a key player in the process regulating oral–aboral axis formation in the sea urchin embryo. Expressed early within an oral organizing centre, it is required to specify both the oral and aboral ectoderm territories by driving an oral–aboral gene regulatory network. A model for oral–aboral axis specification has been proposed relying on the self activation of Nodal and the diffusion of the long-range antagonist Lefty resulting in a sharp restriction of Nodal activity within the oral field. Here, we describe the expression pattern of lefty and analyse its function in the process of secondary axis formation. lefty expression starts at the 128-cell stage immediately after that of nodal, is rapidly restricted to the presumptive oral ectoderm then shifted toward the right side after gastrulation. Consistently with previous work, neither the oral nor the aboral ectoderm are specified in embryos in which Lefty is overexpressed. Conversely, when Lefty's function is blocked, most of the ectoderm is converted into oral ectoderm through ectopic expression of nodal. Reintroducing lefty mRNA in a restricted territory of Lefty depleted embryos caused a dose-dependent effect on nodal expression. Remarkably, injection of lefty mRNA into one blastomere at the 8-cell stage in Lefty depleted embryos blocked nodal expression in the whole ectoderm consistent with the highly diffusible character of Lefty in other models. Taken together, these results demonstrate that Lefty is essential for oral–aboral axis formation and suggest that Lefty acts as a long-range inhibitor of Nodal signalling in the sea urchin embryo.     

The structure and activities of echinonectin: a developmentally regulated cell adhesion glycoprotein with galactose-specific lectin activity.

The extracellular matrix of the sea urchin embryo contains a 230 kD homodimeric glycoprotein known as echinonectin (EN). EN contains a cell attachment domain as well as a galactose-specific lectin activity. Cell attachment to EN is differentially regulated in the three primary germ layers, endoderm, ectoderm and mesoderm. Prior to gastrulation all embryonic cells adhere equally to EN-coated substrates, but during gastrulation primary mesenchyme cells lose affinity for EN, ectoderm cells increase their binding to the molecule, and cells of the endoderm maintain a similar or slightly lowered level of binding. The mechanisms governing these adhesive changes and the specific functions they serve in development are not currently understood. They are timed to coincide with distinct morphogenetic events such as primary mesenchyme cell ingression and archenteron formation, suggesting that regulated adhesion to EN plays at least a permissive role in early morphogenesis.     

Limited left-right cell migration across the midline of the gastrulating avian embryo

During avian development the earliest phase in which the avian embryo expresses axial features of a left-right axis is at the primitive streak stage. Until the stage of definitive primitive streak (streak 4 H&H), the axis seems to possess morphological bilateral symmetry. Morphological asymmetry begins only during the next few hours of incubation, with development of overt morphological and molecular asymmetry within Hensen's node (stage 5 H&H). In this report, we present an experimental study aimed at following the pattern of cell movements during primitive streak formation and gastrulation of specific left-right regions from earlier stages of the avian embryo. To determine the origin of cells contributing to each side of the primitive streak, we applied the dye Lysinated-Rodamine-Dextran (LRD) to one half, either left or right, of the pre-streak blastoderm (stages X–XIII, EG&K). We tried to estimate the relative cell contribution to primitive streak formation, and to the three germ layers evolving during gastrulation in the context of the left-right axis. Moreover, we asked whether the midline serves as a border, that is, as a physiological barrier preventing cell passing during gastrulation. Our results demonstrate that on each side of the axis, either the right or the left, most of the cells originate from the same half of a pre-streak blastoderm, populate the same half of the PS and contribute to tissues largely confined to that particular side. However, along the primitive streak, a few cells were detected on the opposite side of the midline. Moreover, variation in the number of cells crossing the midline at specific regions along the primitive streak was found. Most crossing cells were located near the mid rostrocaudal extent of the primitive streak, from 25–85% of its length. At the posterior end of the primitive streak, fewer crossing cells were detected. At the anterior region of the PS, that is, within Hensen's node, cells do not cross the midline. These results suggest that differences occur in the process of ingression along the rostrocaudal extent of the PS. Dev. Genet. 23:175–184, 1998. © 1998 Wiley-Liss, Inc.     

Cellular Basis of Neuralization of Induced Neurectoderm in Amphibian Embryogenesis: Changes of Cell Shape, Cell Size, and Cytodifferentiation of the Neurectoderm after Neural Induction

Cellular alterations of the neurectoderm after primary embryonic induction were examined by measuring several indices of shape, volume, and cytodifferentiation of the neurectodermal cells of Cynops embryos during gastrulation and early neurulation.
Results showed that cellular alterations occurs just after the 18 hr embryo stage (stage 13b). The thickness of the neurectoderm layer decreases like that of the epidermal ectoderm during early and middle gastrulation. After the 18 hr embryo stage, however, the neurectoderm thickens, mainly due to formation of columnar cells. Measurement of cell volume indicates that the neurectoderm of the early and middle gastrulae consists of a cell population of heterogeneous size. The heterogeneity diminishes sharply after the 18 hr embryo stage and the neural plate of the 36 hr embryo (stage 18) consists of cells of homogeneous size.
Stages before the 12 hr embryo (stage 12b) and after the 18 hr embryo (stage 13b) also showed differed in cell adhesion to the culture flask and in cytodifferentiating potency. Single cells dissociated from the neurectoderm of 18 hr embryos could adhere to the substratum and differentiate into both nerve-like cells and pigment cells. Both capacities increase during further development.
These results are discussed in relation to the neuralizing determination of neurectoderm after primary embryonic induction.     

The effect of larval age on developmental changes in the polyp prepattern of a hydrozoan planula

The larvae of many marine organisms including hydrozoans are lecithotrophic and will not feed until after metamorphosis. In hydrozoans the aboral region of the planula becomes the holdfast and stolon, while the oral region becomes the stalk and hydranth that grows out of the holdfast following metamorphosis. If metamorphosis is delayed, the portion of the planula allocated to form holdfast and stolon shrinks and the region that forms the hydranth increases in size. Planulae also have the ability to regenerate their polyp prepattern. When the aboral region of the planula that does not normally form a hydranth is isolated and metamorphosis is delayed, it acquires the capacity to form a hydranth from the holdfast. A relatively high proportion of entodermal cells of young planulae engage in DNA synthesis (BrdU labeling index); as planulae age, the labeling index falls close to zero. When the polyp prepattern is modified during planula regeneration, entodermal cells are induced to engage in DNA synthesis. If DNA synthesis is inhibited in planulae, the polyp prepattern changes during regeneration and age-related developmental changes in planula are inhibited, suggesting that DNA synthesis is a necessary part of the pattern respecification process.     

Collective Epithelial and Mesenchymal Cell Migration During Gastrulation

Gastrulation, the process that puts the three major germlayers, the ectoderm, mesoderm and endoderm in their correct topological position in the developing embryo, is characterised by extensive highly organised collective cell migration of epithelial and mesenchymal cells. We discuss current knowledge and insights in the mechanisms controlling these cell behaviours during gastrulation in the chick embryo. We discuss several ideas that have been proposed to explain the observed large scale vortex movements of epithelial cells in the epiblast during formation of the primitive streak. We review current insights in the control and execution of the epithelial to mesenchymal transition (EMT) underlying the formation of the hypoblast and the ingression of the mesendoderm cells through the streak. We discuss the mechanisms by which the mesendoderm cells move, the nature and dynamics of the signals that guide these movements, as well as the interplay between signalling and movement that result in tissue patterning and morphogenesis. We argue that instructive cell-cell signaling and directed chemotactic movement responses to these signals are instrumental in the execution of all phases of gastrulation.     

Gastrulation in Halocordyle disticha (Hydrozoa,Athecata)

Summary

Previous reports of development in Halocordyle disticha have described gastrulation as occurring by gradual differentiation of the inner and outer cells of the stereoblastula. In 1984, however, Martin and Thomas described an indentation on the surface of the embryo at the time of gastrulation. They hypothesized from morphological data that the indentation represented a blastopore. Here we provide results of marking studies which demonstrate that the indentation is in fact a site of cellular ingression. This is the first example known of gastrulation that involves unipolar ingression in a form with a stereoblastula. Possible functions of gastrulation by unipolar ingression are discussed, and the possible phylogenetic significance of the occurrence of such a mode of gastrulation in H. disticha is considered.     

Dynamic changes in the distribution of cytoplasmic myosin during Drosophila embryogenesis

Dramatic changes in the localization of conventional non-muscle myosin characterize early embryogenesis in Drosophila melanogaster. During cellularization, myosin is concentrated around the furrow canals that form the leading margin of the plasma membrane as it plunges inward to package each somatic nucleus into a columnar epithelial cell. During gastrulation, there is specific anti-myosin staining at the apical ends of those cells that change shape in regions of invagination. Both of these localizations appear to result from a redistribution of a cortical store of maternal myosin. In the preblastoderm embryo, myosin is localized to the egg cortex, sub-cortical arrays of inclusions, and, diffusely, the yolk-free periplasm. At the syncytial blastoderm stage, myosin is found within cytoskeletal caps associated with the somatic nuclei at the embryonic surface. Following the final syncytial division, these myosin caps give rise to the myosin rings observed during cellularization. These distributions are observed with both whole immune serum and affinity-purified antibodies directed against Drosophila non-muscle myosin heavy chain. They are not detected in embryos stained with anti-Drosophila muscle myosin antiserum or with preimmune serum. Although immunolocalization can only suggest possible function, these myosin localizations and the coincident changes in cell morphology are consistent with a key role for non-muscle myosin in powering cellularization and gastrulation during embryogenesis.     

Epithelial type, ingression, blastopore architecture and the evolution of chordate mesoderm morphogenesis

Chordate embryos show an evolutionary trend in the mechanisms they use to internalize presumptive mesoderm, relying predominantly on invagination in the basal chordates, varying combinations of involution and ingression in the anamniote vertebrates and reptiles, and predominantly on ingression in birds and mammals. This trend is associated with variations in epithelial type and changes in embryonic architecture as well as variations in the type of blastopore formed by an embryo. We also note the surprising conservation of the involution, during gastrulation, of at least a subset of the notochordal cells throughout the chordates, and suggest that this indicates a constraint on morphogenic evolution based on a functional linkage between architecture and patterning. Finally, we propose a model for the evolutionary transitions from gastrulation through a urodele amphibian-type blastopore to gastrulation through a primitive streak, as in chick or mouse.     

A cell-based model of Nematostella vectensis gastrulation including bottle cell formation, invagination and zippering

The gastrulation of Nematostella vectensis, the starlet sea anemone, is morphologically simple yet involves many conserved cell behaviors such as apical constriction, invagination, bottle cell formation, cell migration and zippering found during gastrulation in a wide range of more morphologically complex animals.In this article we study Nematostella gastrulation using a combination of morphometrics and computational modeling. Through this analysis we frame gastrulation as a non-trivial problem, in which two distinct cell domains must change shape to match each other geometrically, while maintaining the integrity of the embryo. Using a detailed cell-based model capable of representing arbitrary cell-shapes such as bottle cells, as well as filopodia, localized adhesion and constriction, we are able to simulate gastrulation and associate emergent macroscopic changes in embryo shape to individual cell behaviors.We have developed a number of testable hypotheses based on the model. First, we hypothesize that the blastomeres need to be stiffer at their apical ends, relative to the rest of the cell perimeter, in order to be able to hold their wedge shape and the dimensions of the blastula, regardless of whether the blastula is sealed or leaky. We also postulate that bottle cells are a consequence of cell strain and low cell–cell adhesion, and can be produced within an epithelium even without apical constriction. Finally, we postulate that apical constriction, filopodia and de-epithelialization are necessary and sufficient for gastrulation based on parameter variation studies.     

C. elegans PAR-3 and PAR-6 are required for apicobasal asymmetries associated with cell adhesion and gastrulation

PAR proteins distribute asymmetrically across the anterior-posterior axis of the 1-cell-stage C. elegans embryo, and function to establish subsequent anterior-posterior asymmetries. By the end of the 4-cell stage, anteriorly localized PAR proteins, such as PAR-3 and PAR-6, redistribute to the outer, apical surfaces of cells, whereas posteriorly localized PAR proteins, such as PAR-1 and PAR-2, redistribute to the inner, basolateral surfaces. Because PAR proteins are provided maternally, distinguishing apicobasal from earlier anterior-posterior functions requires a method that selectively prevents PAR activity after the 1-cell stage. In the present study we generated hybrid PAR proteins that are targeted for degradation after the 1-cell stage. Embryos containing the hybrid PAR proteins had normal anterior-posterior polarity, but showed defects in apicobasal asymmetries associated with gastrulation. Ectopic separations appeared between lateral surfaces of cells that are normally tightly adherent, cells that ingress during gastrulation failed to accumulate nonmuscle myosin at their apical surfaces and ingression was slowed. Thus, PAR proteins function in both apicobasal and anterior-posterior asymmetry during the first few cell cycles of embryogenesis.     

Initial analysis of immunochemical cell surface properties, location and formation of the serotonergic apical ganglion in sea urchin embryos

In the present study it was found that serotonergic apical ganglion (SAG)-forming cells in plutei of the sea urchin, Hemicentrotus pulcherrimus, possessed a characteristic pear shape with broad apical sides and a pointed basal side in the acron epithelium. The basal side extended axons through the space between the epithelium and the basal lamina toward the midline of the embryo that aligned parallel to the embryonic anteroposterior axis. Serotonergic apical ganglion-forming cells had epithelial cell surface-specific proteins on their entire surface. The SAG in 4-arm plutei was composed of a 4-cell trunk region that aligned at right angles to the embryonic anteroposterior axis, and forked into two branches of one to two cells at both ends. Two branches extended toward the oral and the other two toward the aboral region, respectively. Double-stained immunohistochemistry using antiserotonin antibodies and oral ectoderm-specific anti-Ecto V monoclonal antibody or aboral ectoderm-specific anti-Ars antibodies indicated that SAG was in the aboral ectoderm region. Serotonergic apical ganglion cells were first detected in late gastrulae and increased in number rapidly between 36 and 48 h after fertilization, and then slowly afterwards. A 5-bromo-2-deoxyuridine incorporation study indicated that none of the increased SAG cells were in the S phase during the aforementioned period, suggesting that SAG cells do not proliferate by cell division, but acquire the property in particular cells by transdifferentiation using a mechanism that has yet to be elucidated.     

Non-canonical Wnt signaling through Wnt5a/b and a novel Wnt11 gene, Wnt11b, regulates cell migration during avian gastrulation

Knowledge of the molecular mechanisms regulating cell ingression, epithelial–mesenchymal transition and migration movements during amniote gastrulation is steadily improving. In the frog and fish embryo, Wnt5 and Wnt11 ligands are expressed around the blastopore and play an important role in regulating cell movements associated with gastrulation. In the chicken embryo, although Wnt5a and Wnt5b are expressed in the primitive streak, the known Wnt11 gene is expressed in paraxial and intermediate mesoderm, and in differentiated myocardial cells, but not in the streak. Here, we identify a previously uncharacterized chicken Wnt11 gene, Wnt11b, that is orthologous to the frog Wnt11 and zebrafish Wnt11 (silberblick) genes. Chicken Wnt11b is expressed in the primitive streak in a pattern similar to chicken Wnt5a and Wnt5b. When non-canonical Wnt signaling is blocked using a Dishevelled dominant-negative protein, gastrulation movements are inhibited and cells accumulate in the primitive streak. Furthermore, disruption of non-canonical Wnt signaling by overexpression of full-length or dominant-negative Wnt11b or Wnt5a constructions abrogates normal cell migration through the primitive streak. We conclude that non-canonical Wnt signaling, mediated in part by Wnt11b, is important for regulation of gastrulation cell movements in the avian embryo.     

The fine structure of the embryo during the gastrula stage of Comanthus japonica (Echinodermata: Crinoidea)

The fine structure of the embryo of Comanthus has been described by scanning and transmission electron microscopy at two-hourly intervals throughout the gastrula stage (from the fifth through the fifteenth hours of development). At 5 hr, gastrulation has occurred in the absence of any structure comparable to the echinoid hyaline layer; therefore, at least one important mechanism proposed for echinoid gastrulation cannot occur in this crinoid. At 7 hr, the blastocoelic basal lamina has formed, and all ectodermal and entodermal nuclei contain dense aggregates, which are probably perichromatin fibrils. At 9 hr, the blastocoel contains mesenchyme cells, presumably of entodermal origin. At 11 hr, ciliogenesis has started at the apical surfaces of the ectoderm cells and at the archenteral surfaces of the entoderm cells; many of the newly formed cilia are swollen subterminally. At 13 hr, a conspicuous glycocalyx is beginning to cover the apical ends of the ectoderm cells, and the fertilization membrane is beginning to dissolve from its inner surface. Between 5 and 13 hr, there is a gradual development of a junctional complex associating the apicolateral margins of the ectoderm cells ; the zonula adherens part of the complex appears at 5 hr and is well developed by 7 hr, and then the septate junction part of the complex appears at 9 hr and is well developed by 13 hr. At 15 hr, the blastopore has closed, the ectodermal glycocalyx is fully developed, some mesenchyme cells appear to be differentiating into skeleton forming cells, and the fertilization membrane is in the last stages of dissolution.     

Requirements for zygotic gene activity during gastrulation in Drosophila melanogaster

Mutations at the folded gastrulation (fog) and twisted gastrulation (tsg) loci interfere with early morphogenetic movements in Drosophila melanogaster. fog embryos do not form a normal posterior midgut and although their germbands do elongate, they do not extend dorsally. As a result, when normal embryos have fully extended germbands, the germbands in mutant embryos are folded into the interior on the ventral side of the embryo. tsg embryos have abnormally deep dorsal folds during early gastrulation, associated with the failure of dorsal cells to slip laterally to make way for the expanding germband. Both fog and tsg embryos continue to develop, but form disorganized first instar larvae. fog and tsg are zygotically active genes expressed at least by 10 and 20 min after the onset of gastrulation. Both mutations are viable in homozygous germ cells and the wild-type genes need not be expressed during oogenesis for survival of heterozygous progeny. Elimination of fog+ gene product from maternal germ cells does, however, affect the extent of folding observed during gastrulation in viable heterozygotes. Analysis of fog adult and larval gynandromorphs indicates that normal folded gastrulation gene function is only required at the posterior region of the embryo, most probably in the cells giving rise to the posterior midgut or proctodeum. The relative survival of fog mosaics suggests that embryos with mosaic "lethal foci" also die during embryogenesis, although the typical fog phenotype is only produced when the entire focus is mutant. In contrast to the fog focus, no particular cell must be wild type in tsg mosaics for survival. Wild-type cells on the dorsal side of the embryo, however, are most effective in rescuing the embryo. This indicates that normal tsg gene product may be required only on the dorsal side of the embryo, potentially in the region which gives rise to the amnion serosa.     

Localization of sensory mechanisms utilized by coral planulae to detect settlement cues

Coral planulae are induced to settle and metamorphose by contact with either crustose coralline algae or marine bacterial biofilms. Larvae of two coral species, Pocillopora damicornis and Montipora capitata, which respond to different metamorphic cues, were utilized to investigate the sensory mechanisms used to detect metamorphic cues. Because the aboral pole of the coral planula is the point of attachment to the substratum, we predicted that it is also the point of detection for cues. To determine where sensory cells for cues are localized along the body, individual larvae were transversely cut into oral and aboral portions at various levels along the oral–aboral axis, and exposed to settlement‐inducing substrata. Aboral ends of M. capitata metamorphosed, while oral ends continued to swim. However, in larvae of P. damicornis, ¾ oral ends (i.e., lacking the aboral pole) were also able to metamorphose, indicating that the cells that detect cues may be distributed along the sides of the body. These cells do not correspond to FMRFamide‐immunoreactive cells that are present throughout the body. Cesium ions induced both aboral and oral ends of larvae of both species to settle, suggesting that oral ends have not lost their capacity to metamorphose, despite lacking sensory cells to detect natural cues. To determine whether sensory cells in larvae of P. damicornis are restricted to one side of the body, swimming behavior over substrata was observed in larvae labeled with diI, a red fluorescent lipophilic membrane stain. The larvae were found to rotate around the oral–aboral axis, with their surface against the substratum, not favoring a particular side for detecting cues. While clarifying the regions of the larval body important for settlement and metamorphosis in coral planulae, we conclude that significant differences between coral species may be due to differences in the distribution of sensory structures in relation to different planular sizes.