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.     

Ultrastructural localization of spicule matrix proteins in normal and metalloproteinase inhibitor-treated sea urchin primary mesenchyme cells

Studies of the sea urchin larval skeleton have contributed greatly to our understanding of the process of biomineralization. In this study we have undertaken an investigation of the morphology of skeleton formation and the localization of proteins involved in the process of spicule formation at the electron microscope level. Sea urchin primary mesenchyme cells undergo a number of morphological changes as they synthesize the larval skeleton. They form a large spicule compartment that surrounds the growing spicule and, as spicule formation comes to an end, the density of the cytoplasm decreases. Inhibition of spicule formation by specific matrix metalloproteinase inhibitors or serum deprivation has some subtle effects on the morphology of cells and causes the accumulation of specific classes of vesicles. We have localized proteins of the organic matrix of the spicule and found that one protein, SM30, is localized to the Golgi apparatus and transport vesicles in the cytoplasm as well as throughout the occluded protein matrix of the spicule itself. This localization suggests that SM30 is an important structural protein in the spicule. Another spicule matrix protein, SM50, has a similar cytoplasmic localization, but in the spicule much of it is localized at the periphery of the spicule compartment, and consequently it may play a role in the assembly of new material onto the growing spicule or in the maintenance of the integrity of the matrix surrounding the spicule.     

Microfilaments, cell shape changes, and the formation of primary mesenchyme in sea urchin embryos.

Primary mesenchyme formation in sea urchin embryos occurs when a subset of epithelial cells of the blastula move from the epithelial layer into the blastocoel. The role of microfilaments in producing the cell shape changes that characterize this process, referred to as ingression, was investigated in this study. f-Actin was localized by confocal microscopy using labeled phalloidin. The distribution of f-actin was observed before, during, and after ingression and was correlated with cellular movements. Prior to the onset of ingression, staining became intense in the apical region of putative primary mesenchyme and disappeared following the completion of mesenchyme formation. The apical end of these cells constricted coincidentally with the appearance of the intensified staining, indicating that f-actin may be involved in this constriction. In addition, papaverine, a smooth muscle cell relaxant that interferes with microfilament-based contraction, and that was shown in this study to inhibit cytokinesis, diminished apical constriction and delayed ingression. Despite this interference with apical constriction, the basal surface of ingressing cells protruded into the blastocoel. It is suggested that apical constriction, while not necessary for ingression, does contribute to the efficient production of mesenchyme and that protrusion of the basal surface results from changes that occur independent of apical constriction.     

Role of fibronectin in primary mesenchyme cell migration in the sea urchin

We studied the effect of fibronectin (FN) on the behavior of primary mesenchyme cells isolated from sea urchin mesenchyme blastulae in vitro using a time-lapse technique. The migration of isolated primary mesenchyme cells reconstituted in seawater and horse serum is dependent on the presence or absence of exogenous FN in the culture media. The cells in FN, 4 and 40 micrograms/ml, show a high percentage of migration and migrate long distances, whereas a higher concentration of FN at 400 micrograms/ml tends to inhibit migration.     

Dependence of sea urchin primary mesenchyme cell migration on xyloside- and sulfate-sensitive cell surface-associated components

The migration of sea urchin primary mesenchyme cells (PMC) is inhibited in embryos cultured in sulfate-free seawater and in seawater containing exogenous xylosides. In the present study, primary mesenchyme cells and extra-cellular matrix have been isolated from normal and treated Lytechinus pictus and Strongylocentrotus purpuratus embryos and recombined in an in vitro migration assay to determine whether the cells or the matrix are migration defective. Normal cells were found to migrate on either normal or treated matrix, whereas sulfate-deprived and xyloside-treated PMC failed to migrate in vitro on normal and treated substrata. Migratory ability can be restored to defective cells by returning the PMC to normal seawater, or by exposing the defective cells to materials removed from the surface of normal cells with 1 M urea. The similarity of the results obtained with sulfate-deprived and xyloside-treated PMC suggested that a common molecule may be affected by the two treatments. As a first test of this possibility, xyloside-treated S. purpuratus PMC were given the urea extract prepared from sulfate-deprived S. purpuratus PMC, and this extract did not restore migratory ability. These findings indicate that PMC normally synthesize a surface-associated molecule that is involved in cell migration, and the sensitivity to exogenous xylosides and sulfate deprivation suggests that a sulfated proteoglycan may be involved in primary mesenchyme cell migration.     

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.     

Extracellular matrix triggers a directed cell migratory response in sea urchin primary mesenchyme cells

The role of extracellular matrix in cell migration has generally been considered in terms of a substratum. However, when thin cell processes from migrating sea urchin primary mesenchyme cells contact small latex beads coated with extracellular matrix from the blastocoel, the cells migrate directly to the coated beads. Since the beads are not anchored, this result indicates that highly localized contact with the extracellular matrix can stimulate movement independently of any change in cell adhesion.     

Inhibitors of metalloendoproteases block spiculogenesis in sea urchin primary mesenchyme cells

Metalloendoproteases have been implicated in a variety of fusion processes including plasma membrane fusion and exocytosis. As a prerequisite to skeleton formation in the sea urchin embryo, primary mesenchyme cells undergo fusion via filopodia to form syncytia. The spicule is formed within the syncytial cable by matrix and mineral deposition. To investigate the potential involvement of a metalloendoprotease in spiculogenesis, the effect of inhibitors of this enzyme on skeleton formation was studied. Experiments with primary mesenchyme cells in vitro and in normal embryos revealed that skeleton formation was blocked by these inhibitors. These findings implicate a metalloendoprotease in spiculogenesis; such an enzyme has been demonstrated in homogenates of primary mesenchyme cells. The most likely site of action of the metalloendoprotease is at the cell membrane fusion stage and/or at subsequent events requiring membrane fusion.     

In vitro fusion and separation of sea urchin primary mesenchyme cells

Time-lapse videomicroscopy of cultured primary mesenchyme cells from mesenchyme blastulae of the sea urchin Lytechinus pictus demonstrates the dramatic ability of these cells to undergo cell fusion and cell separation. Although this plasticity of cell associations is presumed to play a role in the formation of the syncytial cables that secrete the larval skeleton, the surfaces of these cells must be specialized for fusion and cell separation.     

Determination of cell fate in sea urchin embryos

Classical embryological studies have provided a great deal of information on the autonomy and stability of cell fate determination in early sea urchin embryos. However, these studies were limited by the tools available at the time, and the interpretation of the results of these experiments was limited by the lack of information available at the molecular level. Recent studies which have re-examined classical experiments at the molecular level have provided important new insights into the mechanism of determination in sea urchins, and require us to re-evaluate some long standing theories on the process of differentiation.     

Acid mucopolysaccharide metabolism, the cell surface, and primary mesenchyme cell activity in the sea urchin embryo

The relationship between ³⁵SO₄ incorporation into acid mucopolysaccharides and the appearance and activity of the primary mesenchyme cells has been studied in the sea urchin, Lytechinus pictus. The ratio of the uptake of ³⁵SO₄ to its incorporation into cetylpyridinium chloride precipitable material varies over a wide range during early development, with the smallest ratio, therefore the greatest sulfation activity, being found at the early mesenchyme blastula stage. The types of mucopolysaccharides produced have not been identified, but are heterogeneous. At the mesenchyme blastula stage nearly 90% of the polysaccharides produced become sulfated. When embryos develop in sulfate-free sea water to the mesenchyme blastula stage there is a 70% decrease in the incorporation of ³H-acetate into polysaccharides and a 13-fold decrease in the ratio of sulfated to nonsulfated polysaccharides produced. Embryos raised in sulfate-free sea water develop normally to the mesenchyme blastula stage at which time there is an accumulation in the blastocoel of primary mesenchyme cells that do not migrate. The surface of the primary mesenchyme cells of sulfate-deficient embryos has a smooth appearance in the scanning electron microscope, while the surface of these cells in control embryos is rough, possibly reflecting the presence of an extracellular coat. It is suggested that there is a correlation between sulfated polysaccharide synthesis, cell surface morphology and cell movement.     

Characterization of sea urchin primary mesenchyme cells and spicules during biomineralization in vitro

An in vitro culture system for primary mesenchyme cells of the sea urchin embryo has been used to study the cellular characteristics of skeletal spicule formation. As judged initially by light microscopy, these cells attached to plastic substrata, migrated and fused to form syncytia in which mineral deposits accumulated in the cell bodies and in specialized filopodial templates. Subsequent examination by scanning electron microscopy revealed that the cell bodies and the filopodia and lamellipodia formed spatial associations similar to those seen in the embryo and indicated that the spicule was surrounded by a membrane-limited sheath derived by fusion of the filopodia. The spicules were dissolved from living or fixed cells by a chelator of divalent cations or by lowering the pH of the medium. However, granular deposits found in the cell bodies appeared relatively refractory to such treatments, indicating that they were inaccessible to agents that dissolved the spicules. Use of rapid freezing and an anhydrous fixative to preserve the syncytia for transmission electron microscopy and X-ray microprobe analysis, indicated that electron-dense deposits in the cell bodies contain elements (Ca, Mg and S) common to the spicule. Examination of the spicule cavity after dissolution of the spicule mineral revealed openings in the filopodia-derived sheath, coated pits within the limiting membrane and a residual matrix that stained with ruthenium red. Concanavalin A--gold applied exogenously entered the spicule cavity and bound to matrix glycoproteins. Based on these observations, we conclude that components of the spicule initially are sequestered intracellularly and that spicule elongation occurs in an extracellular cavity. Ca2+ and associated glycoconjugates may be routed in this cavity via a secretory pathway.     

Occurrence of fibronectin on the primary mesenchyme cell surface during migration in the sea urchin embryo

The distribution of fibronectin in situ in the sea urchin embryo was examined by using indirect immunofluorescence with an antibody raised against human plasma fibronectin. Fibronectin was detected on the surfaces of primary mesenchyme cells in the mid-mesenchyme blastula stage, when these cells are migratory. However, it was not detected on these cells at the early mesenchyme blastula or early gastrula stages. Also, it was not detected in the blastocoel nor on the basal surface of the blastular wall. The migration of the primary mesenchyme cells is therefore correlated with a stage-dependent occurrence of cell surface-associated fibronectin.     

HpEts, an ets-related transcription factor implicated in primary mesenchyme cell differentiation in the sea urchin embryo

The mechanism of micromere specification is one of the central issues in sea urchin development. In this study we have identified a sea urchin homologue of ets 1 + 2. HpEts, which is maternally expressed ubiquitously during the cleavage stage and which expression becomes restricted to the skeletogenic primary mesenchyme cells (PMC) after the hatching blastula stage. The overexpression of HpEts by mRNA injection into fertilized eggs alters the cell fate of non-PMC to migratory PMC. HpEts induces the expression of a PMC-specific spicule matrix protein, SM50, but suppresses of aboral ectoderm-specific arylsulfatase and endoderm-specific HpEndo16. The overexpression of dominant negative delta HpEts which lacks the N terminal domain, in contrast, specifically represses SM50 expression and development of the spicule. In the upstream region of the SM50 gene there exists an ets binding site that functions as a positive cis-regulatory element. The results suggest that HpEts plays a key role in the differentiation of PMCs in sea urchin embryogenesis.     

In situ screening for genes expressed preferentially in secondary mesenchyme cells of sea urchin embryos

In sea urchin embryos, four types of non-skeletogenic mesodermal cells are derived from secondary mesenchyme cells (SMCs). Although determining the complete lineage of SMCs is currently a high-priority goal, specific markers for each type of SMC-derived cell in Hemicentrotus pulcherrimus are unavailable. To identify genes preferentially expressed in the various types of SMC-derived cells, we constructed a cDNA library of the archenteron isolated from late gastrulae. Both the 5' and 3' ends of 1,050 cDNAs randomly selected from 7,500 picked clones were sequenced. Based on the sequence at the 3' end, the cDNAs were grouped into 671 independent clusters. Among these, 605 clusters were analysed by whole-mount in situ hybridisation; 28% (170 clusters) exhibited differential expression patterns, while 24% were ubiquitously expressed and 48% did not show any staining. Of 170 clusters showing differential expression patterns, 33 clusters were differentially expressed in SMC-derived cells. From these clusters, several genes were obtained that were specifically or predominantly expressed in each type of SMCs, including coelomic pouch cells in which specific expression patterns have not been reported previously, and hence will be useful for lineage studies. Furthermore, in situ hybridisation revealed the existence of a new type or subpopulation of SMCs distributed sparsely in the blastocoel.     

Immunofluorescence studies of developmental changes in sea urchin eggs and embryos

By means of indirect immunofluorescence techniques, distinct sea urchin antigens were localized in eggs and embryos (Paracentrotus lividus). The specificity of the method was ascertained from controls in which the specific rabbit anti-sea-urchin sera were substituted by rabbit antiserum to an unrelated antigen (human serum albumin), by normal rabbit serum or by phosphate-buffered saline. The specificity of staining was also evaluated by comparing the different staining patterns obtained either with antisera to whole homogenates of eggs and embryos or with antisera to distinct antigens.     

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.