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.     

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.     

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.     

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.     

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.     

Secondary mesenchyme of the sea urchin embryo: ontogeny of blastocoelar cells.

Secondary mesenchyme in sea urchin embryos is released into the blastocoel after primary mesenchyme, and although these cells have been recognized for some time, we lack knowledge about many fundamental aspects of their origin and fate. Here we documented the ontogeny of one of the principal, and least well-known, types of cells derived from secondary mesenchyme. The blastocoelar cells arise from mesenchyme released from the tip of the archenteron following the initial phase of gastrulation. The cells migrate with their cell bodies suspended in the blastocoel, rather than being apposed to the basal lamina like primary mesenchyme. The cells extend numerous fine filopodia to form a network of cytoplasmic processes around the gut, along the skeletal rods, and within the larval arms. Once the network is formed, the cells maintain their positions, although they actively translocate vesicles and cytoplasm along their filopodia. Cell counts indicate there is an initial recruitment of cells during gastrulation, followed by a more gradual increase in cell number after the larva begins to feed. Lineage studies in which 16-cell-stage macromeres were injected with horseradish peroxidase indicate that almost all of the macromere-derived mesenchyme forms pigment cells and blastocoelar cells. We propose that blastocoelar cells are a distinct subset of secondary mesenchyme that forms fibroblast-like cells in the blastocoel of sea urchin embryos.     

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.     

Effects of extracellular matrix components on the differentiation of chick embryo tail bud mesenchyme in culture

The mesenchymal cells of the chick tail bud comprise the remains of Hensen's node and the primitive streak after gastrulation. This mass of cells, situated at the caudal limit of the chick embryo, is morphologically homogeneous but pluripotent, with the ability to differentiate into a variety of tissues that are both ectoderm- and mesoderm-derived elsewhere in the embryo. These tissues include neuroectoderm, neurons, myoblasts and chondrocytes. As the factors regulating the differentiation of tail bud mesenchyme into so many cell types are unclear, and because the extracellular matrix (ECM) is known to have a profound effect on cellular differentiation in many embryonic systems, we studied the differentiation of tail bud mesenchyme explanted onto a variety of different ECM components as substrata. We report that the histogenetic potential of isolated tail buds in culture compares favourably with that in situ. Using various antibody markers, we have demonstrated that tail bud mesenchyme cultured upon different ECM components as substrata is able to differentiate into neurons, neuroepithelium, melanocytes, muscle and cartilage. Laminin and laminin-containing substrata (Matrigel) were found to promote the differentiation of neural crest derivatives (neurons and melanocytes) and neuroepithelial cells; type I collagen promoted both myogenesis and chondrogenesis; while type IV collagen promoted myogenesis only. We have therefore demonstrated that differentiation of tail bud mesenchyme in vitro is substratum-dependent.     

Cytoskeleton and thyroglobulin expression change during transformation of thyroid epithelium to mesenchyme-like cells

In considering the mechanism of transformation of epithelium to mesenchyme in the embryo, it is generally assumed that the ability to give rise to fibroblast-like cells is lost as epithelia mature. We reported previously that a definitive embryonic epithelium, that of the anterior lens, gives rise to freely migrating mesenchyme-like cells when suspended in type I collagen matrices. Here, we show that a highly differentiated epithelium that expresses cytokeratin changes to a vimentin cytoskeleton and loses thyroglobulin during epithelial-mesenchymal transformation induced by suspension in collagen gel. Using dispase and collagenase, we isolated adult thyroid follicles devoid of basal lamina and mesenchyme, and we suspended the follicles in 3D collagen gels. Cells bordering the follicle lumen retain epithelial polarity and thyroid phenotype, but basal cell surface organization is soon modified as a result of tissue multilayering and elongation of basal cells into the collagenous matrix. Cytodifferentiation, determined by thyroglobulin immunoreactivity, is lost as the basal epithelial cells move into the matrix after 3-4 days in collagen. By TEM, it can be seen that the elongating cells acquire pseudopodia, filopodia and mesenchyme-like nuclei and RER. Immunofluorescence examination of intermediate filaments showed that freshly isolated follicles and follicles cultured on planar substrata react only with anticytokeratin. However, all of the mesenchyme-like cells express vimentin and they gradually lose cytokeratin. These results suggest that vimentin may be necessary for cell functions associated with migration within a 3D matrix. The mesenchymal cells do not revert to epithelium when grown on planar substrata and the transformation of epithelium to mesenchyme-like cells does not occur within basement membrane gels. The results are relevant to our understanding of the initiation of epithelial-mesenchymal transformation in the embryo and the genetic mechanisms controlling cell shape, polarity and cytoskeletal phenotype.     

Characterization of Vegetal Plate Cells Separated from Cytochalasin B-Treated Blastulae of the Sea Urchin, Clypeaster japonicus

Vegetal plate forming cells (VPCs) of the vegetal plate blastulae of the sea urchin, Clypeaster japonicus , had a layer of microfilaments on the basal side. The VPCs specifically protruded from the embryos after a treatment with 1 μg/ml of cytochalasin B (CB). Based on scanning electron microscopy, unlike other epithelial cells the protruded VPCs possessed neither cilium nor microvilli on their surface. The protruded VPCs were easily separated from the embryos by stirring the embryo suspension with pipette. An in vitro immunohistochemistry using a primary mesenchyme cell (PMC) surface-specific monoclonal antibody (MAb) raised against PMCs of Strongylocentrotus purpuratus showed that the MAb also specifically bound to the PMCs in mesenchyme blastulae of C. japonicus . The MAb bound in 81% of the separated VPCs in C. japonicus vegetal plate blastulae examined. However, the MAb binding occurred only after the separated VPCs were incubated in artificial sea water (ASW) for at least 1 hr. In the VPC-deprived embryos, gastrulation occurred after they were transferred to normal ASW. However, the PMCs and the spicules were not formed in these embryos.
In conclusion, a majority of the VPCs separated from the CB-treated blastulae were presumptive PMCs. These VPCs provide an excellent source of presumptive PMCs.     

Collagen modulates cell shape and cytoskeleton of embryonic corneal and fibroma fibroblasts: Distribution of actin, α-actinin,and myosin

In the embryo, fibroblasts migrating through extracellular matrices (ECM) are generally elongate in shape, exhibiting a leading pseudopodium with filopodial extensions, and a trailing cell process. Little is known about the mechanism of movement of embryonic cells in ECM, for studies of fibroblast locomotion in the past have been largely confined to observations of flattened cells grown on planar substrata. We confirm here that embryonic avian corneal fibroblasts migrating within hydrated collagen gels in vitro have the bipolar morphology of fibroblasts in vivo, and we show for the first time that highly flattened gerbil fibroma fibroblasts, grown as cell lines on planar substrata, can also respond to hydrated collagen gels by becoming elongate in shape. We demonstrate that the collagen-mediated change in cell shape is accompanied by dramatic rearrangement of the actin, α-actinin, and myosin components of the cytoskeleton. By immunofluorescence, the stress fibers of the flattened corneal fibroblasts grown on glass are seen to stain with antiactin, anti-α-actinin, and antimyosin, as has been reported for fibroma and other fibroblasts grown on glass. Stress fibers, adhesion plaques, and ruffles do not develop when the corneal or fibroma fibroblast is grown in ECM; these features seem to be a response to strong attachment of the cell underside to a planar substratum. When the fibroblasts are grown in ECM, antimyosin staining is distributed diffusely through the cytoplasm. Antiactin and anti-α-actinin stain the microfilamentous cell cortex strongly. We suggest that locomotion of the fibroblast in ECM is accompanied by adhesion of the cell to the collagen fibrils and may involve an interaction of the myosin-rich cytosol with the actin-rich filamentous cell cortex. Interestingly, the numerous filopodia that characterize the tips of motile pseudopodia of cells in ECM are very rich in actin and α-actinin, but seem to lack myosin; if filopodia use myosin to move, the interaction must be at a distance. Soluble collagen does not convert flattened fibroblasts on planar substrata to bipolar cells. Thus, the effect of collagen on the fibroblast cytoskeleton seems to depend on the presence of collagen fibrils in a gel surrounding the cell.     

Primary mesenchyme cells of the sea urchin embryo require an autonomously produced, nonfibrillar collagen for spiculogenesis

A collagen molecule in the sea urchin embryo was characterized by analysis of a 2.7-kb cDNA clone. This clone, Spcoll, was obtained by screening a gastrula stage Strongylocentrotus purpuratus cDNA library with a 237-bp genomic clone encoding a collagen-like sequence previously isolated by Venkatesan et al. (1986). DNA sequence analysis of the cDNA clone demonstrated the nonfibrillar nature of the encoded molecule--13 interruptions of the Gly-X-Y repeat motif were found in the 85-kDa open reading frame. The mRNA of approximately 9 kb accumulated specifically in mesenchyme cells of the embryo through development to the pluteus larva. Polyclonal antibodies generated against a Spcoll-beta-galactosidase fusion protein were utilized to identify and localize the native Spcoll. This collagen molecule of approximately 210 kDa was deposited into the blastocoel by the primary mesenchyme cells. When primary mesenchyme cells were cultured in vitro, Spcoll was secreted into the media and accumulated at sites of cell-substrate interaction. Addition of anti-Spcoll antibodies to primary mesenchyme cell cultures selectively inhibited spiculogenesis, whereas other antibodies had no inhibitory effect. Since collagen is not a component of the organic matrix of spicules (Benson et al., 1986), these results suggest that the autonomous production of Spcoll by differentiating mesenchyme cells in turn influences the point in differentiation at which these cell initiate biomineralization.     

Extracellular materials and determination of neuroectoblast in amphibian gastrula

The biological activity of the extracellular material (ECM) located in the dorsal and ventral regions of Bufo arenarum is assayed. Ectoblast cells were isolated and cultured with ECM taken from the embryonal regions. The epiblastic cells treated with dorsal ECM, mainly located at the interphase between the invaginating blastoporal lip and the overlying ectoblast differentiated morphologically into neural, mesenchyme and pigment cells. In contrast, control epiplastic cells, cultured either in salt solution or in ventral region ECM, differentiated into ciliated and secretory cell types. The results reported here provide evidence that the dorsal region ECM of the embryo can induce neural differentiation in indeterminate epiblastic cells.     

Cell adhesion during gastrulation : A new approach

In gastrulating sea urchin embryos, secondary mesenchyme cells at the tip of the advancing archenteron extend long narrow filopodia which probe the inner surface of the blastocoele wall, rejecting some surface contacts before adhering to other cells. After specific cell adhesions are made, contractions of the filopodia pull the leading tip of the archenteron to the opposite wall of the blastocoele with an accompanying elongation of the archenteron. A study was made of the biochemistry and morphology of the specific adhesions of filopodial extensions by injecting a variety of compounds into the blastocoele of living sea urchin gastrulae and observing their effects on filopodia and cell movements. A number of agents (proteases, lectins) caused specific filopodial detachment and subsequent archenteron regression. Fluorescein-conjugated lectins, including concanavalin A (conA) and wheat germ agglutinin (WGA) exhibited marked specificity of cell surface binding to specific regions (primary mesenchyme cells, blastocoele wall, etc.) of the embryo.     

Histological distribution of FR-1, a cyclic RGDS-peptide, binding sites during early embryogenesis, and isolation and initial characterization of FR-1 receptor in the sand dollar embryo

A fibronectin-related synthetic cyclic H-Cys-Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Cys-OH (RGDSPASS) peptide (FR-1) binding site in the embryo of the sand dollar Clypeaster japonicus was specified using dansyl-labeled FR-1 (Dns-FR-1) and horseradish peroxidase-labeled FR-1, and an FR-1 receptor was isolated using FR-1-affinity column chromatography. The FR-1 introduced to the blastocoel of blastulae inhibited primary mesenchyme cell (PMC) migration in mesenchyme blastulae, and complete gastrulation and spicule differentiation in gastrulae. The Dns-FR-1 bound to the entire basal side of the ectoderm in mesenchyme blastulae, and then restricted to the basal side of the ectoderm at the apical tuft region and the vegetal hemisphere in early gastrulae. The cytoplasm of the archenteron also bound to Dns-FR-1. In PMC, Dns-FR-1 bound to the nucleus and cytoplasmic reticular features. In unfertilized eggs, Dns-FR-1 bound to the entire cytoplasm, particularly to the oval-shaped granules and the nuclear envelope, but only to the cytoplasm after fertilization. Relative molecular mass ( Mr ) of the FR-1 -binding protein was 240 kDa under non-reducing conditions and 57 kDa under reducing conditions. The FR-1 receptor protein bound anti-sea urchin integrin (Spl) βL subunit antibodies raised against the embryos of Strongylocentrotus purpuratus . Immunohistochemistry showed that the antibody binding site was similar to the histochemical distribution of Dns-FR-1. However, Mr of the FR-1 receptor is distinctively larger than that of the Spl βL subunit.     

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.     

Fine structure of the down feather during its early development

The down feather of the chick embryo has been examined by electron microscopy during three distinct stages of its early development; the presumptive stage, represented by dorsal skin of an area from which the feather organ will arise; the thickening stage, during which areas of the basal epidermis form spurs projecting into the mesenchyme, and the latter condenses under a thickened area of the epidermis; the elevation stage, at which time the basal epidermis flattens, the entire epidermis increases in thickness, and the underlying mesenchyme becomes more compact. As development proceeds the rough endoplasmic reticulum of the epidermal cells dilates, but during the elevation stage begins to flatten, and Golgi is observed with increasing frequency. The mitochondria do not appear to differ except for those in the periderm during the presumptive stage, in which case they reveal a vacant matrix and irregular cristae. Evidence is presented for actual contact between basal epidermal spurs and filopodia of cells within the mesenchyme, some of which contain numerous vesicles. The basal epidermal spurs are also seen in intimate association with collagen and anchor filaments and a network of reticulin. Evidence is also presented for the presence of neuronal elements within the mesenchyme during the thickening stage. Cross sections of cell processes within the condensations of the mesenchyme resemble unmyelinated nerve fibers, and cross sections of filopodia similar to arborizing axons abound at and within the basal lamina of both the thickening and elevation stages. Further support for the presence of nerve fibers within the mesenchyme comes from positive staining results with Bodian's and Ungewitter's methods. This comparative study of three stages of early development of the feather organ serves as a basis for more detailed investigations of each stage.     

Fibronectin and laminin in the extracellular matrix and basement membrane of sea urchin embryos

Fibronectin and laminin have been found in the extracellular matrix and in the basement membrane of sea urchin embryos during early development. These glycoproteins are also found on the cell surfaces of the outer epithelial layer and on the secondary mesenchyme cells within the blastocoel. The similarity of functions of the extracellular matrix and basement membrane is discussed, as is the similarity of their molecular components. These observations suggest the possibility that fibronectin and laminin form a continuous matrix surrounding the cells which links the outer ECM (hyaline layer) to the inner ECM (basement membrane). Such a network could coordinate the various activities of the embryo during early morphogenesis.     

Developmental distribution of a cell surface glycoprotein in the sea urchin Strongylocentrotus purpuratus

Recent studies from this laboratory have shown that an antigen recognized by a monoclonal antibody (MAb 1223) displays a bimodal distribution of expression in development of the embryo of Strongylocentrotus purpuratus. This molecule is specifically localized to the primary mesenchyme cells of the embryo, but is also found within the egg. In the current study, immunoelectron microscopy was used to determine the subcellular distribution of the antigen and to determine its fate during early stages of development of the embryo. In eggs, the epitope recognized by MAb 1223 was localized to the cortical vesicles. Immunoblot analysis of an isolated cell surface complex (CSC) that contained the cortical vesicles revealed the presence of a 130-kDa protein, as well as immunoreactive components of higher molecular weight. Upon fertilization, the antigen was exocytosed from the cortical vesicles and became associated with the hyaline layer, the fertilization envelope, and the plasma membrane. Subsequently, the epitope could be detected within small vesicles and yolk platelets. By 60 min postfertilization, the amount of epitope detected intracellularly or in the perivitelline compartment was greatly reduced. At later stages of development, when formation of the embryonic skeleton occurred, the 1223 antigen was principally localized to the Golgi complex and to the syncytial cell surface of the primary mesenchyme cells. Thus, the results of this study suggest that in S. purpuratus the 1223 antigen is stored and secreted from the cortical vesicles of the egg, degraded after fertilization, and then later expressed on the surface of the primary mesenchyme cells.