RhoA regulates initiation of invagination, but not convergent extension, during sea urchin gastrulation

During gastrulation, the archenteron is formed using cell shape changes, cell rearrangements, filopodial extensions, and convergent extension movements to elongate and shape the nascent gut tube. How these events are coordinated remains unknown, although much has been learned from careful morphological examinations and molecular perturbations. This study reports that RhoA is necessary to trigger archenteron invagination in the sea urchin embryo. Inhibition of RhoA results in a failure to initiate invagination movements, while constitutively active RhoA induces precocious invagination of the archenteron, complete with the actin rearrangements and extracellular matrix secretions that normally accompany the onset of invagination. Although RhoA activity has been reported to control convergent extension movements in vertebrate embryos, experiments herein show that RhoA activity does not regulate convergent extension movements during sea urchin gastrulation. Instead, the results support the hypothesis that RhoA serves as a trigger to initiate invagination, and once initiation occurs, RhoA activity is no longer involved in subsequent gastrulation movements.     

Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60-cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.     

Gastrulation in the Sea Urchin,Strongylocentrotus purpuratus,Is Disrupted by the Small Laminin Peptides YIGSR and IKVAV

Laminin is present on the apical and basolateral sides of epithelial cells of very early sea urchin blastulae. We investigated whether small laminin-peptides, known to have cell binding activities, alter the development of sea urchin embryos. The peptide YIGSR-NH₂ (850 μM) and the peptide PA22-2 (5 μM), which contains the peptide sequence IKVAV (Tashiro et al., J. Biol. Chem. 264, 16174, 1989), typically blocked archenteron formation when added to the sea water soon after fertilization. At lower doses, the YIGSR peptide allowed invagination of the archenteron but blocked archenteron extension and differentiation and evagination of the feeding arms. The effect of YIGSR and PA22-2 peptides declined when added to progressively older stages until no effect was seen when added at the mesenchyme blastula stage (24 hours after fertilization). Control peptides GRGDS, YIGSE, and SHA22, a dodeca-peptide with a scrambled IKVAV sequence, had no effect on development. The YIGSK peptide containing a conserved amino acid modification had only a small effect on gastrulation. The results suggest that YIGSR and IKVAV peptides specifically disrupt cell/extracellular matrix interactions required for normal development of the archenteron and feeding arms. Our recent finding that YTGIR is at the cell binding site of the B1 chain of S. purpuratus laminin supports this conclusion. Evidently, laminin or other laminin-like molecules are among the many extracellular matrix components needed for the invagination and extension of the archenteron during the gastrulation movements of these embryos.     

Regionalized Cell Division during Sea Urchin Gastrulation Contributes to Archenteron Formation and Is Correlated with the Establishment of Larval Symmetry

During gastrulation of the sea urchin, Lytechinus variegutus there is localized proliferation of cells in the vegetal plate region prior to its invagination. Cell counts show that during gastrulation the number of cells per embryo increases 60% from 1025 to 1640. Measurements of cell volumes suggest that some growth may follow these divisions. Feulgen staining shows that the greatest mitotic activity throughout gastrulation occurs in the vegetal plate region. Labelling embryos with 3H-thymidine reveals that incorporation in the vegetal plate is confined to cells that encircle the base of the archenteron. Pulse-chase experiments indicate that these labelled cells contribute descendants to the vegetal half of the archenteron. Additionally, 3-dimensional reconstructions of vegetal regions at different stages reveal that by the end of gastrulation two bilateral clusters of labelled cells lie at the future sites of the post-oral arms of the pluteus larva, thus marking the axes of bilateral and dorso-ventral symmetry. Our findings suggest that two of the principal events of sea urchin gastrulation — the formation of the archenteron and the establishment of symmetry in the larva — are accompanied by distinct patterns of cell division.     

Archenteron elongation in the sea urchin embryo is a microtubule-independent process

Earlier studies using colchicine (L. G. Tilney and J. R. Gibbins, 1969, J. Cell Sci. 5, 195-210) had suggested that intact microtubules (MTs) are necessary for archenteron elongation during the second phase of sea urchin gastrulation (secondary invagination), presumably by allowing secondary mesenchyme cells (SMCs) to extend their long filopodial processes. In light of subsequently discovered effects of colchicine on other cellular processes, the role of MTs in archenteron elongation in the sea urchin, Lytechinus pictus, has been reexamined. Immunofluorescent staining of ectodermal fragments and isolated archenterons reveals a characteristic pattern of MTs in the ectoderm and endoderm during gastrulation. Ectodermal cells exhibit arrays of MTs radiating away from the region of the basal body/ciliary rootlet and extending along the periphery of the cell, whereas endodermal cells exhibit a similar array of peripheral MTs emanating from the region of the apical ciliary rootlet facing the lumen of the archenteron. MTs are found primarily at the bases of the filopodia of normal SMCs. beta-Lumicolchicine (0.1 mM), an analog of colchicine which does not bind tubulin, inhibits secondary invagination, indicating that the effects previously ascribed to the disruption of MTs are probably due to the effects of colchicine on other cellular processes. The MT inhibitor nocodazole (5-10 micrograms/ml) added prior to secondary invagination does not prevent gastrulation or spontaneous exogastrulation, even though indirect immunofluorescence indicates that cytoplasmic MTs are completely disrupted in drug-treated embryos. Transverse tissue sections indicate that a comparable amount of cell rearrangement occurs in nocodazole-treated and control embryos. Significantly, SMCs in nocodazole-treated embryos often detach prematurely from the tip of the gut rudiment and extend abnormally large broad lamellipodial protrusions but are also capable of extending long slender filopodia comparable in length to those of control embryos. These results indicate that cytoplasmic MTs are not essential for either filopodial extension by SMCs or for the active epithelial cell rearrangement which accompanies elongation during sea urchin gastrulation.     

Local cell interactions and the control of gastrulation in the sea urchin embryo

The sea urchin embryo is a good model system for studying the role of mechanical and cell-cell interactions during epithelial invagination, cell rearrangement and mesenchymal patterning in the gastrula. The mechanisms underlying the initial invagination of the archenteron have been surprisingly elusive; several possible mechanisms are discussed. In contrast to its initial invagination, the cellular basis for the elongation of the archenteron is better understood: both autonomous epithelial cell rearrangement and further rearrangement driven by secondary mesenchyme cells appear to be involved. Experiments indicate that patterning of freely migrating primary mesenchyme cells and secondary mesenchyme cells residing in the tip of the archenteron relies to a large extent on information resident in the ectoderm. Interactions between cells in the early embryo and later cell-cell interactions are both required for the establishment of ectodermal pattern information. Surprisingly, in the case of the oral ectoderm the fixation of pattern information does not occur until immediately prior to gastrulation.     

Determination and morphogenesis in the sea urchin embryo

The study of the sea urchin embryo has contributed importantly to our ideas about embryogenesis. This essay re-examines some issues where the concerns of classical experimental embryology and cell and molecular biology converge. The sea urchin egg has an inherent animal-vegetal polarity. An egg fragment that contains both animal and vegetal material will produce a fairly normal larva. However, it is not clear to what extent the oral-aboral axis is specified in embryos developing from meridional fragments. Newly available markers of the oral-aboral axis allow this issue to be settled. When equatorial halves, in which animal and vegetal hemispheres are separated, are allowed to develop, the animal half forms a ciliated hollow ball. The vegetal half, however, often forms a complete embryo. This result is not in accord with the double gradient model of animal and vegetal characteristics that has been used to interpret almost all defect, isolation and transplantation experiments using sea urchin embryos. The effects of agents used to animalize and vegetalize embryos are also due for re-examination. The classical animalizing agent, Zn2+, causes developmental arrest, not expression of animal characters. On the other hand, Li+, a vegetalizing agent, probably changes the determination of animal cells. The stability of these early determinative steps may be examined in dissociation-reaggregation experiments, but this technique has not been exploited extensively. The morphogenetic movements of primary mesenchyme are complex and involve a number of interactions. It is curious that primary mesenchyme is dispensable in skeleton formation since in embryos devoid of primary mesenchyme, the secondary mesenchyme cells will form skeletal elements. It is likely that during its differentiation the primary mesenchyme provides some of its own extracellular microenvironment in the form of collagen and proteoglycans. The detailed form of spicules made by primary mesenchyme is determined by cooperation between the epithelial body wall, the extracellular material and the inherent properties of primary mesenchyme cells. Gastrulation in sea urchins is a two-step process. The first invagination is a buckling, the mechanism of which is not understood. The secondary phase in which the archenteron elongates across the blastocoel is probably driven primarily by active cell repacking. The extracellular matrix is important for this repacking to occur, but the basis of the cellular-environmental interaction is not understood.(ABSTRACT TRUNCATED AT 400 WORDS)     

Behavior and differentiation process of pigment cells in a tropical sea urchin Echinometra mathaei

The behavior and differentiation processes of pigment cells were studied in embryos of a tropical sea urchin Echinometra mathaei, whose egg volume was one half of those of well-known sea urchin species. Owing to earlier accumulation of pigments, pigment cells could be detected in the vegetal plate even before the onset of gastrulation, distributed dorsally in a hemi-circle near the center of the vegetal plate. Although some pigment cells left the archenteron during gastrulation, most of them remained at the archenteron tip. At the end of gastrulation, pigment cells left the archenteron and migrated into the blastocoele. Unlike pigment cells in typical sea urchins, however, they did not enter the ectoderm, and stayed in the blastocoele even at the pluteus stage. It is of interest that the majority of pigment cells were distributed in the vicinity of the larval skeleton. Aphidicolin treatment revealed that eight blastomeres were specific to pigment cell lineage after the eighth cleavage, one cell cycle earlier than that in well-known sea urchins. The pigment founder cells divided twice, and the number of pigment cells was around 32 at the pluteus stage. It was also found that the differentiation of pigment cells was blocked with Ni2+, whereas the treatment was effective only during the first division cycle of the founder cells.     

Cell movements during the initial phase of gastrulation in the sea urchin embryo.

The morphogenetic processes responsible for the initial phase of gastrulation in sea urchin embryos are not known. Here we report observations of the size and position of clones of cells derived from horseradish peroxidase (HRP)-injected mesomeres and macromeres. The displacement of these clones during the initial phase of gastrulation suggests that involution is a mechanism involved in primary invagination. Experiments with embryos marked with vital dyes indicate that movements occur only during a brief phase coincident with the invagination of the vegetal plate. Counts of cells derived from HRP-injected mesomeres and macromeres suggest it unlikely that localized growth in the vegetal plate is involved in gastrulation. An analysis of changes in cell shape during the initial phase of gastrulation indicates that there is a stage-dependent shift from cells being columnar to having their apices skewed toward the vegetal plate and an increase in the proportion of cells having basal processes during gastrulation. When embryos are grown in the presence of monoclonal antibodies to the apical lamina or monovalent fragments of these antibodies, the initial phase of gastrulation is delayed and they form partial exogastrulae. Analysis of embryos marked with HRP indicate that the antibody treatments interfere with the cellular movements observed in untreated embryos. We conclude that directed movements of cells within the blastoderm, probably employing tractoring on components of the hyaline layer, cause the buckling of the vegetal plate and displacement of presumptive endoderm cells seen during the initial phase of gastrulation.     

A cyto-embryological study of gastrulation in the sand dollar, Scaphechinus mirabilis

Processes of gastrulation in the sand dollar Scaphechinus mirabilis were compared with those in the sea urchin Hemicentrotus pulcherrimus , which seemed to show a typical pattern of gastrulation. Measurement of the archenteron length clearly demonstrated that invagination processes in H. pulcherrimus are divided into two phases, the primary and secondary invagination. On the other hand, invagination in S. mirabilis was revealed to continue at a constant rate. To see the movement of cells during gastrulation, embryos were labeled with Nile blue. In H. pulcherrimus embryos, labeled cells were observed along the full length of the archenteron, if the embryos had been labeled before and during the primary invagination. Labeled cells were never observed in the embryos stained after the primary invagination. In contrast, labeled cells were always discerned at the basal part of the archenteron in S. mirabilis , even if the embryos were stained after invagination had undergone considerable progress. The number of cells in the archenteron of S. mirabilis embryos increased with the advancement of gastrulation, while the numbers were almost constant in H. pulcherrimus . These results suggest that the cellular basis of gastrulation in S. mirabilis is quite different from that in well-known species of sea urchins.     

Frizzled5/8 is required in secondary mesenchyme cells to initiate archenteron invagination during sea urchin development

Wnt signaling pathways play key roles in numerous developmental processes both in vertebrates and invertebrates. Their signals are transduced by Frizzled proteins, the cognate receptors of the Wnt ligands. This study focuses on the role of a member of the Frizzled family, Fz5/8, during sea urchin embryogenesis. During development, Fz5/8 displays restricted expression, beginning at the 60-cell stage in the animal domain and then from mesenchyme blastula stage, in both the animal domain and a subset of secondary mesenchyme cells (SMCs). Loss-of-function analyses in whole embryos and chimeras reveal that Fz5/8 is not involved in the specification of the main embryonic territories. Rather, it appears to be required in SMCs for primary invagination of the archenteron, maintenance of endodermal marker expression and apical localization of Notch receptors in endodermal cells. Furthermore, among the three known Wnt pathways, Fz5/8 appears to signal via the planar cell polarity pathway. Taken together, the results suggest that Fz5/8 plays a crucial role specifically in SMCs to control primary invagination during sea urchin gastrulation.     

Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages

The entry of beta-catenin into vegetal cell nuclei beginning at the 16-cell stage is one of the earliest known molecular asymmetries seen along the animal-vegetal axis in the sea urchin embryo. Nuclear beta-catenin activates a vegetal signaling cascade that mediates micromere specification and specification of the endomesoderm in the remaining cells of the vegetal half of the embryo. Only a few potential target genes of nuclear beta-catenin have been functionally analyzed in the sea urchin embryo. Here, we show that SpWnt8, a Wnt8 homolog from Strongylocentrotus purpuratus, is zygotically activated specifically in 16-cell-stage micromeres in a nuclear beta-catenin-dependent manner, and its expression remains restricted to the micromeres until the 60-cell stage. At the late 60-cell stage nuclear beta-catenin-dependent SpWnt8 expression expands to the veg2 cell tier. SpWnt8 is the only signaling molecule thus far identified with expression localized to the 16-60-cell stage micromeres and the veg2 tier. Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. These results establish SpWnt8 as a critical component of the endomesoderm regulatory network in the sea urchin embryo.     

Behavior of pigment cells in gastrula-stage embryos of Hemicentrotus pulcherrimus and Scaphechinus mirabilis

The behavior of pigment cells in sea urchin embryos, especially at the gastrula stage, is not well understood, due to the lack of an appropriate method to detect pigment cells. We found that pigment cells emanated autofluorescence when they were fixed with formalin and irradiated with ultraviolet or green light. In Hemicentrotus pulcherrimus, fluorescent pigment cells became visible at the archenteron tip at the mid-gastrula stage. The cells detached from the archenteron slightly before the initiation of secondary invagination and migrated toward the apical plate. Most pigment cells entered the apical plate. This entry site seemed to be restricted, because pigment cells could not enter the ectoderm and remained in the blastocoele at the vegetal pole side when elongation of archenteron was blocked. Pigment cells that had entered the apical plate soon began to migrate in the aboral ectoderm toward the vegetal pole. In contrast, pigment cells of Scaphechinus mirabilis embryos were first detected in the vegetal plate before the onset of gastrulation. Without entering the blastocoele, these cells began to migrate preferentially in the aboral ectoderm toward the animal pole. When the archenteron tip reached the apical plate, pigment cells had already distributed throughout the aboral ectoderm. Thus, the behavior of pigment cells was quite different between H. pulcherrimus and S. mirabilis.     

Pattern of Brachyury gene expression in starfish embryos resembles that of hemichordate embryos but not of sea urchin embryos.

Echinoderms, hemichordates and chordates are deuterostomes and share a number of developmental features. The Brachyury gene is responsible for formation of the notochord, the most defining feature of chordates, and thus may be a key to understanding the origin and evolution of the chordates. Previous studies have shown that the ascidian Brachyury (As-T and Ci-Bra) is expressed in the notochord and that a sea urchin Brachyury (HpTa) is expressed in the secondary mesenchyme founder cells. A recent study by [Tagawa et al. (1998)], however, revealed that a hemichordate Brachyury (PfBra) is expressed in a novel pattern in an archenteron invagination region and a stomodaeum invagination region in the gastrula. The present study demonstrated that the expression pattern of Brachyury (ApBra) of starfish embryos resembles that of PfBra in hemichordate embryos but not of HpTa in sea urchin embryos. Namely, ApBra is expressed in an archenteron invagination region and a stomodaeum invagination region.     

Cellular basis of gastrulation in the sand dollar Scaphechinus mirabilis

The processes of gastrulation in the sand dollar Scaphechinus mirabilis are quite different from those in regular echinoids. In this study, we explored the cellular basis of gastrulation in this species with several methods. Cell-tracing experiments revealed that the prospective endodermal cells were convoluted throughout the invagination processes. Histological observation showed that the ectodermal layer remained thickened, and the vegetal cells retained an elongated shape until the last step of invagination. Further, most of the vegetal ectodermal cells were skewed or distorted. Wedge-shaped cells were common in the vegetal ectoderm, especially at the subequatorial region. In these embryos, unlike the embryos of regular echinoids, secondary mesenchyme cells did not seem to exert the force to pull up the archenteron toward the inner surface of the apical plate. In fact, the archenteron cells were not stretched along the axis of elongation and were in close contact with each other. Here we found that gastrulation was completely blocked when the embryos were attached to a glass dish coated with poly-L-lysine, in which the movement of the ectodermal layer was inhibited. These results suggest that a force generated by the thickened ectoderm, rather than rearrangement of the archenteron cells, may play a key role in the archenteron elongation in S. mirabilis embryos.     

Behavior of pigment cells closely correlates the manner of gastrulation in sea urchin embryos

To know whether behavior of pigment cells correlates the process of gastrulation or not, gastrulating embryos of several species of regular echinoids (Anthocidaris crassispina, Mespilia globulus and Toxopneustes pileolus) and irregular echinoids (Clypeaster japonicus and Astriclypeus manni) were examined. In M. globulus and A. crassispina, the archenteron elongated stepwise like in well-known sea urchins. In the embryos of both species, fluorescent pigment cells left the archenteron tip and migrated into the blastocoel during gastrulation. In T. pileolus, C. japonicus and A. manni, on the other hand, the archenteron elongated at a constant rate throughout gastrulation. In these species, no pigment cell was observed at the archenteron tip during invagination processes; pigment cells began to migrate in the ectoderm from the vegetal pole side toward the apical plate without entering the blastocoel. These results clearly indicate that the behavior of pigment cells closely correlated the manner of gastrulation. Further, it was examined whether the archenteron cells are rearranged during invagination, by comparing the number of cells observed on cross sections of the archenteron at the early and late gastrula stages. The rearrangement was not conspicuous in A. crassispina and M. globulus, in which archenteron elongated stepwise. In contrast, the archenteron cells were remarkably rearranged in C. japonicus, alothough the archenteron elongated continuously. Thus, neither the behavior of pigment cells nor the manner of gastrulation matches the current taxonomic classification of echinoids.     

The effects of aphidicolin on morphogenesis and differentiation in the sea urchin embryo

Aphidicolin, an inhibitor of DNA polymerase alpha, blocks DNA synthesis and cell division in sea urchin embryos. The effects of this inhibition appear to be stage dependent. Blastulae treated with aphidicolin before the thickening of the vegetal plate undergo developmental arrest prior to gastrulation. The extent of inhibition of DNA synthesis varies from 60 to 93% in these embryos. However, when aphidicolin is added after the vegetal plate has thickened, development continues normally through pluteus formation, even though DNA synthesis is inhibited by greater than or equal to 90% and cell division has ceased. These observations indicate that, from the vegetal plate stage onward, morphogenesis and overt differentiation are independent of DNA synthesis and cell division.