Sea urchin primary mesenchyme cells: relation of cell polarity to the epithelial-mesenchymal transformation

In euechinoid sea urchin embryos, a subset of epithelial cells in the wall of the blastula become pulsatile, elongate, lose connections with their neighboring cells, and move into the blastocoel to form the primary mesenchyme cells. The Golgi apparatus and microtubule organizing center (MTOC) are located at the apical end of these epithelial cells. We show that as primary mesenchyme cells begin to move into the blastocoel, the Golgi apparatus and MTOC move to a new position adjacent to the apical side of the nucleus. They do not move to a position between the nucleus and the leading (i.e., basal) end of the cell as they do in cultured fibroblasts undergoing directed migration. In addition, we have inhibited the movement of membranous vesicles to the cell surface by incubating embryos in the ionophore monensin. We have used antibodies to msp130, a primary mesenchyme cell surface-specific glycoprotein, to demonstrate that monensin inhibits the movement of msp130-containing vesicles to the cell surface. Despite the inhibition of membrane shuttling by monensin, primary mesenchyme cells ingress on schedule and display normal cell-shape changes. We draw two conclusions from our data. First, the cellular elongation that characterizes ingression is not due to the local insertion of membrane at the leading (basal) end of the cell. Second, ingression does not depend upon establishment of the same cell polarity required for fibroblasts to carry out directed cell migration.     

Cell polarity and gastrulation in C. elegans.

Gastrulation in C. elegans embryos involves formation of a blastocoel and the ingression of surface cells into the blastocoel. Mutations in the par-3 gene cause abnormal separations between embryonic cells, suggesting that the PAR-3 protein has a role in blastocoel formation. In normal development, PAR proteins localize to either the apical or basal surfaces of cells prior to blastocoel formation; we demonstrate that this localization is determined by cell contacts. Cells that ingress into the blastocoel undergo an apical flattening associated with an apical concentration of non-muscle myosin. We provide evidence that ingression times are determined by genes that control cell fate, though interactions with neighboring cells can prevent ingression.     

Patterns of cells and extracellular material of the sea urchin Lytechinus variegatus (Echinodermata; Echinoidea) embryo,from hatched blastula to late gastrula

Scanning electron microscopy of six stages of Lytechinus variegatus embryos from hatching through gastrulation reveals changes in the shapes of the ectodermal cells and morphological changes in the extracellular material (ECM) in relation to the locations and migratory activities of mesenchyme cells. The classical optical patterns in the blastular wall (Okazaki patterns) are due to differential orientations of the cells, which bend and extend sheet-like lamellipodia over adjoining cells toward the eventual location of the primary mesenchymal ring. The blastocoelic surfaces of the blastomeres become covered with a thin basal lamina (BL) composed of fibers and nonfibrous material. During primary mesenchyme cell (PMC) ingression, a web-like ECM is located in the blastocoel overlying the amassed PMCs. This ECM becomes sparse in migratory mesenchyme blastulae, and is confined to the animal hemisphere. Localized regions of intertwining basal cell processes in the blastular wall are also present during PMC migration. While a distinct BL is present during early and midgastrulation, blastocoelic ECM is absent. Late gastrulae, on the other hand, have an abundance of blastocoelic ECM concentrated near secondary mesenchyme cell protrusive activity. ECM appearing at both the early mesenchyme and late gastrula stages are probably remnants of degraded BL and intercellular matrix preserved by fixation for SEM. Thus, early mesenchyme ECM is formed of BL material whose degradation is necessary for entry of PMCs into the blastocoel. Late gastrula ECM is apparently a degradation product of BL and intercellular material whose destruction is required for fusion of the gut with oral ectoderm in formation of the mouth.     

Elongated Microvilli on Vegetal Pole Cells in Sea Urchin Embryos

The ultrastructure of cells in the vegetal pole region of sea urchin embryos during early development to the mesenchyme blastula stage was examined by scanning electron microscopy. Vegetal pole cells in the ectoderm with longer microvilli than those of neighboring cells were first detectable at the early blastula stage just before hatching. These cells with elongated microvilli remained in the central region of the vegetal plate when most vegetal plate cells ingressed into the blastocoel to form primary mesenchyme. When first detectable in the sea urchin, Anthocidaris crassispina , four vegetal pole cells had elongated microvilli, but at the time of primary mesenchyme cell ingression, the number of cells with elongated microvilli had increased to eight, apparently by cell division. These vegetal pole cells were wedge-shaped with a broad surface adhering to the hyaline layer at the time of primary mesenchyme cell ingression. SEM observation of the outer surface of embryos showed that the microvilli extended into the hyaline layer. The reinforced attachment of vegetal pole cells to the hyaline layer through their elongated microvilli may explain why these cells could remain at the vegetal pole when the surrounding cells ingressed into the blastocoel as primary mesenchyme cells.     

Three cell recognition changes accompany the ingression of sea urchin primary mesenchyme cells

At gastrulation the primary mesenchyme cells of sea urchin embryos lose contact with the extracellular hyaline layer and with neighboring blastomeres as they pass through the basal lamina and enter the blastocoel. This delamination process was examined using a cell-binding assay to follow changes in affinities between mesenchyme cells and their three substrates: hyalin, early gastrula cells, and basal lamina. Sixteen-cell-stage micromeres (the precursors of primary mesenchyme cells), and mesenchyme cells obtained from mesenchyme-blastula-stage embryos were used in conjunction with micromeres raised in culture to intermediate ages. The micromeres exhibited an affinity for hyalin, but the affinity was lost at the time of mesenchyme ingression in vivo. Similarly, micromeres had an affinity for monolayers of gastrula cells but the older mesenchyme cells lost much of their cell-to-cell affinity. Presumptive ectoderm and endoderm cells tested against the gastrula monolayers showed no decrease in binding over the same time interval. When micromeres and primary mesenchyme cells were tested against basal lamina preparations, there was an increase in affinity that was associated with developmental time. Presumptive ectoderm and endoderm cells showed no change in affinity over the same interval. Binding measurements using isolated basal laminar components identified fibronectin as one molecule for which the wandering primary mesenchyme cells acquired a specific affinity. The data indicate that as the presumptive mesenchyme cells leave the vegetal plate of the embryo they lose affinities for hyalin and for neighboring cells, and gain an affinity for fibronectin associated with the basal lamina and extracellular matrix that lines the blastocoel.     

Electron microscopic studies on primary mesenchyme cell ingression and gastrulation in relation to vegetal pole cell behavior in sea urchin embryos

Early morphogenetic events of primary mesenchyme cell (PMC) ingression and gastrulation were examined by scanning and transmission electron microscopy, with special attention directed to changes in the shape of vegetal pole cells, the length of their microvilli, and interactions between microvilli and the hyaline layer (HL). Eight cells (vegetal pole cells) with elongated microvilli remained in the vegetal pole region while surrounding cells ingressed into the blastocoel to form the primary mesenchyme. These vegetal pole cells indented with the surrounding cells at the stage of gastrulation. The outer surface area with elongated microvilli of vegetal pole cells expanded at the stage of PMC ingression, but was considerably reduced at gastrulation. Microvilli on vegetal pole cells continued to adhere to the HL up to the stage of PMC ingression, but ceased to do so at the time of gastrulation. Thus, the area with separated HL, which is restricted to the region of the PMC released at the stage of PMC ingression, spreads almost entirely throughout the area of the indenting vegetal plate at gastrulation. The apical lamina, apparently consisting of fibrous material intertwinning the stalks of the microvilli, filled the space between the HL and ectodermal cells. The cells surrounding those of the vegetal pole and indenting with those at the stage of gastrulation appeared to behave in the same way as ingressing PMCs in both cell-shape and loss of adhesion of microvilli to HL. The role of vegetal pole cells in early morphogenetic events is discussed.     

Focal adhesion kinase (FAK) expression and phosphorylation in sea urchin embryos

We have cloned three cDNA isoforms of focal adhesion kinase (FAK) from the sea urchin, Lytechinus variegatus. The sea urchin FAK is more closely related to FAK from other deuterostomes than from invertebrate protostomes or to cell adhesion kinase beta (CAKbeta/Pyk2/FAK2). FAK is expressed in all cells of sea urchin embryos by the 120-cell stage and strongly in blastulae. Phospho-FAK concentrates on basal surfaces of epithelial cells in early blastulae and occurs in syncytial cables of primary mesenchyme cells (PMC). Inhibition of FAK by constructs of FAK-related non-kinase delays blastocoel expansion and early PMC ingression. These results suggest that FAK has roles in cell adhesion and in the shape and integrity of the epithelial cells in sea urchin embryos.     

Gastrulation: PARtaking of the bottle

Gastrulation in C. elegans embryos involves ingression of individual cells, but is driven by apical constriction of the kind that promotes migration of epithelial cell sheets. Recent work shows that PAR proteins, known for their role in polarization and unequal cell division, are also associated with the polarization that establishes this apical constriction.     

Isolation,culture, and differentiation of echinoid primary mesenchyme cells

Summary Methods are described for isolation and culture of primary mesenchyme cells from echinoid embryos. Ninety-five percentpure primary mesenchyme cells were isolated from early gastrulae ofStrongylocentrotus purpuratus, exploiting the biological segregation of these cells within the blastocoel. When cultured, more than 90% of the isolated cells reached the differentiated state, spicule formation, in synchrony with in vivo controls. Isolated primary mesenchyme cells were cultured with and without various cellular and acellular components of normal embryos in order to study the potential involvement of these components in the morphogenesis of the primary mesenchyme. Our data indicate that: 1. primary mesenchyme cells lack the ability to form the annular pattern of the primary mesenchymal ring autonomously; 2. they autonomously produce spicules of a characteristic morphology that differs from that of embryonic spicules; 3. morphogenesis of the primary mesenchyme is not affected by association with embryonic basal lamina, blastocoel matrix, or loosely aggregated epithelial cells, or by close confinement of each set of primary mesenchyme cells within the blastocoelar space; and 4. reaggregated, tightly associated epithelial cells can promote normal primary mesenchyme ring formation, and modify the primary mesenchyme-intrinsic spicule pattern to produce more normal spicule forms.     

Inhibition of cell migration in sea urchin embryos by beta-D-xyloside

This investigation examines the effect of exogenous xylosides on primary mesenchyme cell behavior in Strongylocentrotus purpuratus embryos. In confirmation of studies in some other species the addition of 2 mM p-nitrophenyl-beta-D-xylopyranoside blocks the migration but not the initial ingression of primary mesenchyme cells. The blastocoel matrix of treated embryos appears deficient in a 15- to 30-nm-diameter granular component that is observed extensively on the basal lamina and on filopodia of migrating primary mesenchyme cells in untreated embryos. Other blastocoel components appear unaffected by ultrastructural criteria. The incorporation of 35SO4(2-) per embryo into ethanol precipitates of isolated blastocoel matrices was reduced significantly after xyloside treatment but the distribution of 35SO4(2-) after polyacrylamide gel electrophoresis or the glycosaminoglycan composition was unaffected. Chromatography on Sepharose CL-2B demonstrates a reduction in size of sulfated components of the blastocoel. While over 60% of the 35S-labeled material from the blastocoel of normal mesenchyme blastulae is voided from a Sepharose CL-2B column run in a dissociative solvent, only 10% from xyloside treated embryos is voided. Instead, there is a large included peak with Kav of 0.33. This material is acid soluble but cetylpyridinium chloride precipitable. It apparently consists largely of free glycosaminoglycan chains. Based on analysis of chondroitinase ABC digestion products this material consists of 41% chondroitin-6-sulfate and 58% dermatan sulfate. These results are consistent with a role in cell migration for intact chondroitin sulfate/dermatan sulfate proteoglycans in the sea urchin blastocoel matrix.     

Active reinforcement of externally imposed folding in amphibians embryonic tissues

Although the folding of epithelial layers is one of the most common morphogenetic events, the underlying mechanisms of this process are still poorly understood. We aimed to determine whether an artificial bending of an embryonic cell sheet, which normally remains flat, is reinforced and stabilized by intrinsic cell transformations. We observed both reinforcement and stabilization in double explants of blastocoel roof tissue from Xenopus early gastrula embryos. The reinforcement of artificial bending occurred over the course of a few hours and was driven by the gradual apical constriction and radial elongation of previously compressed cells situated at the bending arch of the concave layer of explant. Apical constriction was associated with actomyosin contraction and endocytosis-mediated engulfing of the apical cell membranes. Cooperative apical constrictions of the concave layer of cells produced a tensile force that extended over the entire surface of the explant and correlated with apical contraction of the concave side cells. In the explants taken from the anterior regions of the embryo, this reinforcement was more stable and the bending better expressed than in those taken from suprablastoporal areas. The morphogenetic role of cell responses to the bending force is discussed.     

The origin of pigment cells in embryos of the sea urchin Strongylocentrotus purpuratus

A monoclonal antibody (SP1/20.3.1) that recognizes a cell surface epitope expressed by pigment cells in the pluteus larva of Strongylocentrotus purpuratus has been produced. Using indirect immunofluorescence, the epitope is first detected in nonpigmented cells of the vegetal plate after primary mesenchyme ingression. Between the beginning of gastrulation, and when the archenteron is one-third the distance across the blastocoel, SP1/20.3.1-positive cells are free within the blastocoel, at the tip of the archenteron, and dispersed within the blastoderm. Cells at the tip of the archenteron, and mesenchyme near the tip in later stages of gastrulation (secondary mesenchyme), do not express the SP1/20.3.1 antigen. By the completion of gastrulation all SP1/20.3.1-positive cells are dispersed throughout the epidermis. It has been concluded that in S. purpuratus pigment cell precursors are released from the vegetal plate during the initial phase of gastrulation. The cells migrate first to the vegetal ectoderm, and subsequently disperse throughout the ectoderm and develop pigment granules.     

Behavior of Primary Mesenchyme Cells In situ Associated with Ultrastructural Alteration of the Blastocoelic Material in the Sea Urchin, Anthocidaris crassispina

In the blastula of the sea urchin, Anthocidaris crassispina , a small number of primary mesenchyme cells (PMCs) ingressed from the blastocoel wall taking a bottle shape. The majority of the PMCs followed the first group of PMCs. These ingressed without taking the bottle shape, and became round within the blastocoel wall. After ingression, the PMCs migrated as single cells retaining their round cell contour. The average velocity of their migration was 13.3 μm/hr.
The blastocoel contained Alcian blue (pH 1.0)-positive material which changed its light microscopic configuration from being amorphous in the hatched and mesenchyme blastulae to being fibrous in the early gastrulae. Ultrastructurally, the blastocoelic material in the hatched blastulae was composed of 27 nm diameter granules. In the mesenchyme blastulae and the early gastrulae relatively long 15 nm diameter fibers were seen in addition to the 27 nm diameter granules. The 27 nm diameter granules bound the ruthenium red while the 15 nm diameter fibers did not. The 27 nm diameter granules formed aggregates in the hatched blastulae, and were bound to the 15 nm diameter fibers in the mesenchyme blastulae and early gastrulae to form a fibrous network which was observed by a light microscope.     

Sea urchin primary mesenchyme cells: ingression occurs independent of microtubules

The formation of primary mesenchyme cells in euechinoid sea urchin embryos involves the transformation of 32 epithelial cells into mesenchymal cells in a process referred to as ingression. The mechanism that drives this epithelial-mesenchymal transformation has yet to be identified. Previous studies (J. R. Gibbins, L. G. Tilney, and K. R. Porter, 1969, J. Cell Biol. 41, 201-226; L. G. Tilney and J. R. Gibbins, 1969, J. Cell Biol. 41, 227-250) implicated that microtubules are essential components for the normal development, including ingression, of the mesenchymal cells. In the present study I have reinvestigated the role of microtubules in ingression by using the microtubule-disrupting drugs colchicine and nocodazole, and the microtubule-stabilizing drug taxol. The effect of these drugs on microtubules was monitored by indirect immunofluorescence using monoclonal antibodies specific for alpha- and beta-tubulins. The microtubule array seen in control embryos disappeared in colchicine- and nocodazole-treated embryos, while it was enhanced in taxol-treated embryos. When premesenchyme blastulae of Strongylocentrotus purpuratus were treated with any of these reagents the primary mesenchyme cells ingressed on schedule and appeared to undergo cell-shape changes identical to those observed in untreated embryos. The conclusion of this study is that the mechanism of primary mesenchyme cell ingression does not include an essential role for microtubules; ingression occurs regardless of the presence or absence of microtubules.     

Localization and expression of msp130, a primary mesenchyme lineage-specific cell surface protein in the sea urchin embryo

In this report, we use a monoclonal antibody (B2C2) and antibodies against a fusion protein (Leaf et al. 1987) to characterize msp130, a cell surface protein specific to the primary mesenchyme cells of the sea urchin embryo. This protein first appears on the surface of these cells upon ingression into the blastocoel. Immunoelectronmicroscopy shows that msp130 is present in the trans side of the Golgi apparatus and on the extracellular surface of primary mesenchyme cells. Four precursor proteins to msp130 are identified and we show that B2C2 recognizes only the mature form of msp130. We demonstrate that msp130 contains N-linked carbohydrate groups and that the B2C2 epitope is sensitive to endoglycosidase F digestion. Evidence that msp130 is apparently a sulphated glycoprotein is presented. The recognition of the B2C2 epitope of msp130 is disrupted when embryos are cultured in sulphate-free sea water. In addition, two-dimensional immunoblots show that msp130 is an acidic protein that becomes substantially less acidic in the absence of sulphate. We also show that two other independently derived monoclonal antibodies, IG8 (McClay et al. 1983; McClay, Matranga & Wessel, 1985) and 1223 (Carson et al. 1985), recognize msp130, and suggest this protein to be a major cell surface antigen of primary mesenchyme cells.     

Human disease-associated extracellular matrix orthologs ECM3 and QBRICK regulate primary mesenchymal cell migration in sea urchin embryos

Sea urchin embryos have been one of model organisms to investigate cellular behaviors because of their simple cell composition and transparent body. They also give us an opportunity to investigate molecular functions of human proteins of interest that are conserved in sea urchin. Here we report that human disease-associated extracellular matrix orthologues ECM3 and QBRICK are necessary for mesenchymal cell migration during sea urchin embryogenesis. Immunofluorescence has visualized the colocalization of QBRICK and ECM3 on both apical and basal surface of ectoderm. On the basal surface, QBRICK and ECM3 constitute together a mesh-like fibrillar structure along the blastocoel wall. When the expression of ECM3 was knocked down by antisense-morpholino oligonucleotides, the ECM3-QBRICK fibrillar structure completely disappeared. When QBRICK was knocked down, the ECM3 was still present, but the basally localized fibers became fragmented. The ingression and migration of primary mesenchymal cells were not critically affected, but their migration at later stages was severely affected in both knock-down embryos. As a consequence of impaired primary mesenchymal cell migration, improper spicule formation was observed. These results indicate that ECM3 and QBRICK are components of extracellular matrix, which play important role in primary mesenchymal cell migration, and that sea urchin is a useful experimental animal model to investigate human disease-associated extracellular matrix proteins.     

Passive Mechanical Forces Control Cell-Shape Change during Drosophila Ventral Furrow Formation

During Drosophila gastrulation, the ventral mesodermal cells constrict their apices, undergo a series of coordinated cell-shape changes to form a ventral furrow (VF) and are subsequently internalized. Although it has been well documented that apical constriction is necessary for VF formation, the mechanism by which apical constriction transmits forces throughout the bulk tissue of the cell remains poorly understood. In this work, we develop a computational vertex model to investigate the role of the passive mechanical properties of the cellular blastoderm during gastrulation. We introduce to our knowledge novel data that confirm that the volume of apically constricting cells is conserved throughout the entire course of invagination. We show that maintenance of this constant volume is sufficient to generate invagination as a passive response to apical constriction when it is combined with region-specific elasticities in the membranes surrounding individual cells. We find that the specific sequence of cell-shape changes during VF formation is critically controlled by the stiffness of the lateral and basal membrane surfaces. In particular, our model demonstrates that a transition in basal rigidity is sufficient to drive VF formation along the same sequence of cell-shape change that we observed in the actual embryo, with no active force generation required other than apical constriction.