Enhancement of spicule formation and calcium uptake by monoclonal antibodies to fibronectin-binding acid polysaccharide in cultured sea urchin embryonic cells

The effect of monoclonal antibodies to fibronectin-binding acid polysaccharide (anti-FAPS) on differentiation of primary mesenchyme cells and spicule formation was examined in cultured embryonic cells isolated from the sea urchin, Hemicentrotus pulcherrimus. Spicule formation of micromere-derived cells was enhanced by anti-FAPS. The increase of spicule formation correlated with the increase of calcium uptake into micromere-derived cells and spicules. Furthermore, both spicule formation and calcium uptake were inhibited by calcium-channel blockers (verapamil, diltiazem and nicardipine) and divalent ions (manganese and cobalt). These results suggest that FAPS, a component of the blastocoelic extracellular matrix surrounding the primary mesenchyme cells, may regulate the level of calcium uptake and spicule formation.     

Spicule Formation-Inducing Substance in Sea Urchin Embryo

Isolated micromeres of sea urchin produced spicules in sea water containing blastocoelic fluid (BCF) taken from embryos, or in a medium in which embryos had previously been dissociated (dissociated solution, DS). When isolated micromeres were cultured in vitro , their descendants initiated spicule formation only when BCF was added to the culture medium by the time when, in normal development, primary mesenchyme cells form two aggregates in the vegetal region. After the initiation of spicule formation, growth of spicules occurred under the continuous influence of DS. Spicule formation-inducing (SFI) activity in DS was first detected at the mesenchme blastula stage. The activity in BCF was heat-labile and was inactivated by trypsin.     

Serum effects on the in vitro differentiation of sea urchin micromeres

When micromeres isolated from the 16-cell stage of Strongylocentrotus purpuratus are cultured in sea water containing 3.5% horse serum, they produce spicules at approximately the same time as in normal development. The serum requirement of the micromeres has been investigated by adding serum at varying intervals after isolation or by pulsing the cells with serum at specific times during their in vitro development. The optimum time of serum addition for spicule formation is 36 h after fertilization (AF). Further delay in the addition of serum results in a reduction in the number of spicules formed in culture and a delay in the time at which they appear. A 1-h pulse of serum at 36 h AF is sufficient to initiate a response in some of the micromere aggregates. A 12-h pulse at 36 h AF produces the maximum number of spicules per culture. The critical period for serum addition, 36-48 h AF, corresponds to the time in the normal embryo at which the syncytial primary mesenchyme ring is formed. Electron micrographs of cultured cells demonstrate that micromeres cultured without serum until 48 h AF fail to form pseudopodial extensions and remain as rosette-like clusters of cells. If serum is present, extensive pseudopodial networks form which resemble the primary ring syncytium. These results suggest that serum acts to stimulate fused pseudopodial networks in cultures of micromeres and that the resulting syncytium is necessary for spicule formation.     

Asymmetric inhibition of spicule formation in sea urchin embryos with low concentrations of gadolinium ion

As gastrulation proceeds during sea urchin embryogenesis, primary mesenchyme cells (PMCs) fuse to form syncytial cables, within which calcium is deposited as CaCO₃, and a pair of spicules is formed. Earlier studies suggested that calcium, previously sequestered by primary mesenchyme cells, is secreted and incorporated into growing spicules. We examined the effects of gadolinium ion (Gd³⁺), a Ca²⁺ channel blocker, on spicule formation. Gd³⁺ did not lead to a retardation of embryogenesis prior to the initiation of gastrulation and did not inhibit the ingression of PMCs from the blastula wall or their migration along the inner blastocoel surface. However, when embryos were raised in seawater containing submicromolar to a few micromolar Gd³⁺, of which levels are considered to be insufficient to block Ca²⁺ channels, a pair of triradiate spicules was formed asymmetrically. At 1–3 μmol/L Gd³⁺, many embryos formed only one spicule on either the left or right side, or embryos formed a very small second spicule. Induction of the spicule abnormality required the presence of Gd³⁺ specifically during late blastula stage prior to spicule formation. An accumulation or adsorption of Gd³⁺ was not detected anywhere in the embryos by X‐ray microanalysis, which suggests that Ca²⁺ channels were not inhibited. These results suggest that Gd³⁺ exerts an inhibitory effect on spicule formation through a mechanism that does not involve inhibition of Ca²⁺ channels.     

The regulation of primary mesenchyme cell patterning

The primary mesenchyme cells (PMCs) of the sea urchin embryo undergo a dramatic sequence of morphogenetic behaviors that includes migration, localization at specific sites within the embryo, and synthesis of the larval skeleton. To gain information about how these processes are regulated, PMC migration and patterning were analyzed in embryos with experimentally altered numbers of PMCs. PMC movements were followed by labeling the cells with a fluorescent dye, rhodamine B isothiocyanate, or with the PMC-specific monoclonal antibody 6a9. These methods show that individual PMCs have the capacity to join any position in the pattern, and rule out the possibility that PMC morphogenesis involves a sorting out of discrete subpopulations of cells to predetermined sites. All sites in the PMC pattern have the capacity to accept more cells than they normally do, and PMCs do not appear to compete with one another for preferred sites in the pattern. Even in embryos with 2-3 times the normal complement of PMCs, all these cells take part in spiculogenesis and the resultant skeleton is normal in size and configuration. Two special sites along the basal lamina (those corresponding to the positions of the PMC ventrolateral clusters) promote spicule elongation, an effect that is independent of the numbers of PMCs at these sites. These observations emphasize the role of the basal lamina, blastocoel matrix, and embryonic epithelium in regulating key aspects of PMC morphogenesis. The PMCs remain highly flexible in their ability to respond to patterning cues in the blastocoel, since postmigratory PMCs will repeat their patterning process if microinjected into the blastocoel of young recipient embryos.     

Probable Contribution of Protein Phosphorylation by Protein Kinase C to Spicule Formation in Sea Urchin Embryos

The formation of spicules and development of pluteus arms in sea urchin embryos were strongly blocked by H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride) but were not affected by HA1004 ( N -(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride). Archenteron formation occurred normally in the presence of these compounds. Late gastrulae (28 hr after fertilization) were exposed to ³²Pi for 30 min at 20°C, and then dissociated and their primary mesenchyme cells with spicules, embryo-wall cells and archenteron cells were separated. Then, the radioactivities in the protein fractions of these separated cells were measured. Results showed that culture of embryos with H-7 strongly inhibited ³²p incorporation into proteins in primary mesenchyme cells but caused little inhibition of its incorporations in embryo-wall cells and archenteron cells. The effective concentrations of H-7 for inhibition of ³²p incorporation were within the range that blocked spicule formation and growth of pluteus arms in embryos. HA1004 only slightly inhibited ³²p incorporation into protein in mesenchyme cells, embryo-wall cells and archenteron cells of embryos exposed to ³²Pi. Protein kinase C activity was detectable only in isolated primary mesenchyme cells associated with spicule structures. These suggest that phosphorylation of proteins by protein kinase C contributes to the formation of spicule structures.     

Study of larval and adult skeletogenic cells in developing sea urchin larvae

The larval skeleton of sea urchin embryos is formed by primary mesenchyme cells (PMCs). Thereafter, the larvae start feeding and additional arms develop. An adult rudiment that contains spines, tube feet, tests, and other parts of the adult body is formed in the eight-armed larva. The cellular mechanism of the later skeletogenesis and the lineage of the adult skeletogenic cells are not known. In this study, the morphogenesis of larval and adult skeletons during larval development of the sea urchin Hemicentrotus pulcherrimus was investigated by immunostaining cells with PMC-specific monoclonal antibodies, which are useful markers of skeletogenic cells. All spicules and the associated cells in the later larvae were stained with the antibodies. We could observe the initiation of skeletal morphogenesis at each developmental stage and visualize the cellular basis of skeleton formation in whole-mount embryos that possessed an intact morphology. There were some similarities between PMCs and the later skeletogenic cells. Both had a rounded shape with some filopodia, and the antigen expression started just before overt spicule formation. In the later-stage embryos, cells with filopodia and faint antigen expression were observed migrating in the blastocoel or aggregating in the presumptive location of new skeletogenesis.     

Spicule matrix protein LSM34 is essential for biomineralization of the sea urchin spicule.

Biomineralized skeletal structures are composite materials containing mineral and matrix protein(s). The cell biological mechanisms that underlie the formation, secretion, and organization of the biomineralized materials are not well understood. Although the matrix proteins influence physical properties of the structures, little is known of the role of these matrix proteins in the actual formation of the biomineralized structure. We present here results using an antisense oligonucleotide directed against a spicule matrix protein, LSM34, present in spicules of embryos of Lytechinus pictus. After injection of anti-LSM34 into the blastocoel of a sea urchin embryo, LSM34 protein in the primary mesenchyme cells decreases and biomineralization ceases, demonstrating that LSM34 function is essential for the formation of the calcareous endoskeletal spicule of the embryo. Since LSM34 is found primarily in a specialized extracellular matrix surrounding the spicule, it is probable that this matrix is important for the biomineralization process.     

Fibronectin-binding acid polysaccharide in the sea urchin embryo

Summary A novel fibronectin-binding acid polysaccharide (FAPS) was isolated from embryos of the sea urchin. Binding of FAPS to fibronectin was quantitatively measured at physiological pH and ionic strength by two different assay systems. Immunofluorescent studies revealed that FAPS is localized in the extracellular matrix surrounding the mesenchyme cells and primitive gut of middle gastrula. Sea urchin fibronectin was also detected in the extracellular matrix surrounding mesenchyme cells and the cells surrounding the blastopore. When a monoclonal antibody to FAPS (anti-FAPS) was microinjected into the blastocoel, more than one pair of triradiate spicular rudiments was formed and the malformation of spicules was induced. Armless and deformed larvae were also induced by anti-FAPS. FAPS may regulate the number, length, position and direction of spicules. These results implicate the extracellular matrix of the blastocoel in the complex process of differentiation of mesenchyme and the formation of spicules.     

Probable Role of Allylisothiocyanate-Sensitive H+, K+-ATPase in Spicule Calcification in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

In embryos of the sea urchin, Hemicentrotus pulcherrimus , as well as in cultured cells derived from isolated micromeres, spicule formation was inhibited by allylisothiocyanate, an inhibitor of H⁺, K⁺-ATPase, at above 0.5 μM and was almost completely blocked at above 10 μM. Amiloride, an inhibitor of Na⁺, H⁺ antiporter, at above 100 μM exerted only slight inhibitory effect, if any, on spicule formation. Intravesicular acidification, determined using [ dimethylamine -¹⁴C]-aminopyrine as a pH probe, was observed in the presence of ATP and 200 mM KCl in microsome fraction obtained from embryos at the post gastrula stage, at which embryos underwent spicule calcification. Intravesicular acidification and K⁺-dependent ATPase activity were almost completely inhibited by allylisothiocyanate at 10 μM. Allylisothiocyanate-sensitive ATPase activity was found mainly in the mesenchyme cells with spicules isolated from prisms. H⁺, K⁺-ATPase, an H⁺ pump, probably mediates H⁺ release to accelerate CaCO₃ deposition from Ca²⁺, CO₂ and H₂O in the primary mesenchyme cells. Intravesicular acidification was stimulated by valinomycin at the late gastrula and the prism stages but not at the pluteus stage. K⁺ permeability probably increases after the prism stage to activate H⁺ release.     

Differential distribution of spicule matrix proteins in the sea urchin embryo skeleton

Spicule matrix proteins are the products of primary mesenchyme cells, and are present in calcite spicules of the sea urchin embryo. To study their possible roles in skeletal morphogenesis, monoclonal antibodies against SM50, SM30 and another spicule matrix protein (29 kDa) were obtained. The distribution of these proteins in the embryo skeleton was observed by immunofluorescent staining. In addition, their distribution inside the spicules was examined by a 'spicule blot' procedure, direct immunoblotting of proteins embedded in crystallized spicules. Our observations showed that SM50 and 29 kDa proteins were enriched both outside and inside the triradiate spicules of the gastrulae, and also existed in the corresponding portions of growing spicules in later embryos and micromere cultures. The straight extensions of the triradiate spicules and thickened portions of body rods in pluteus spicules were also rich in these proteins. The SM30 protein was only faintly detected along the surface of spicules. By examination using the spicule blot procedure, however, SM30 was clearly detectable inside the body rods and postoral rods. These results indicate that SM50 and 29 kDa proteins are concentrated in radially growing portions of the spicules (normal to the c-axis of calcite), while SM30 protein is in the longitudinally growing portions (parallel to the c-axis). Such differential distribution suggests the involvement of these proteins in calcite growth during the formation of three-dimensionally branched spicules.     

Analysis of competence in cultured sea urchin micromeres.

Sea urchin embryo micromeres form the primary mesenchyme, the skeleton-producing cells of the embryo. Almost nothing is known about nature and timing of the embryonic cues which induce or initiate spicule formation by these cells. A related question concerns the competence of the micromeres to respond to the cues. To examine competence in this system we have exposed cultured sea urchin micromeres to an inducing medium containing horse serum for various periods of time and have identified a period when micromeres are competent to respond to serum and form spicules. This window, between 30 and 50 h after fertilization, corresponds to the time when mesenchyme cells in vivo are aggregating and beginning to form the syncytium in which the spicule will be deposited. The loss of competence after 50 h is not due to impaired cell health since protein synthesis at this time is not significantly different from controls. Likewise the accumulation of a spicule matrix mRNA (SM 50) and a cell surface glycoprotein (msp 130), both indices of micromere/mesenchyme differentiation, still occurs in cells that have lost competence to respond to serum by forming spicules. These experiments demonstrate that the acquisition and loss of competence in these cells are regulated developmental events and establish an in vitro system for the identification of the molecular basis for inductive signal recognition and signal transduction.     

Immunogold detection of glycoprotein antigens in sea urchin embryos

Four developmental stages of sea urchin embryos were labeled with colloidal gold in an attempt to elucidate the intracellular trafficking patterns within the cells that produce the glycoprotein matrix of the embryonic spicule. The primary mesenchyme cells (PMCs) form a syncytium and secrete an organic matrix on which calcium carbonate is laid down to form an endoskeletal spicule. The organic matrix has been isolated and characterized as glycoprotein consisting of four major bands. Polyclonal antibodies to these glycoproteins were used to label embryos from the mesenchyme blastula, early gastrula, late gastrula, and plutei stages of development. The label is concentrated in the Golgi complex and associated vesicles, in secretory vesicles, and in the organic matrix. The density of the labeling increases as development proceeds.     

Commitment and response to inductive signals of primary mesenchyme cells of the sea urchin embryo

In the sea urchin embryo, primary mesenchyme cells (PMC) are committed to produce the larval skeleton, although their behavior and skeleton production are influenced by signals from the embryonic environment. Results from our recent studies showed that perturbation of skeleton development, by interfering with ectoderm-extracellular matrix (ECM) interactions, is linked to a reduction in the gene expression of a transforming growth factor (TGF)-beta growth factor, Pl-univin, suggesting a reduction in the blastocoelic amounts of the protein and its putative involvement in signaling events. In the present study, we examined PMC competence to respond to environmental signals in a validated skeleton perturbation model in Paracentrotus lividus. We found that injection of blastocoelic fluid (BcF), obtained from normal embryos, into the blastocoelic cavity of skeleton-defective embryos rescues skeleton development. In addition, PMC from skeleton-defective embryos transplanted into normal or PMC-less blastula embryos are able to position in correct regions of the blastocoel and to engage spicule elongation and patterning. Taken together, these results demonstrate that PMC commitment to direct skeletogenesis is maintained in skeleton perturbed embryos and confirm the role played by inductive signals in regulating skeleton growth and shape.     

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.     

Socket cells mediate spicule morphogenesis in Caenorhabditis elegans males.

Caenorhabditis elegans male spicule morphogenesis requires the coordinated cellular behaviors of several types of cells. We found that the spicule neurons and sheath cells, although important for spicule function, are dispensable for spicule morphology. In contrast, the spicule socket cells are essential for both spicule elongation and formation of spicule cuticle. The socket cells are not only necessary but also sufficient to produce spicule cuticle. This functional aspect of socket cells is genetically separable from their function in mediating spicule elongation: elongated spicules with defective spicule cuticle can be formed. During spicule morphogenesis, the expression of an egl-17::GFP reporter gene is found in the spicule socket cells and its expression appears to be regulated in the socket cells. Mutants defective in TGF-beta signaling display a crumpled spicules phenotype as a result of failure of socket cell movement during spicule morphogenesis. These observations suggest that both the FGF and the TGF-beta signaling pathways might be involved in spicule elongation.     

Role of LSM34/SpSM50 proteins in endoskeletal spicule formation in sea urchin embryos

Abstract. Sea urchin embryos form an endoskeletal spicule composed of calcium carbonate and occluded matrix proteins. The accumulation of the LSM34 spicule matrix protein in embryos of Lytechinus pictus (and its ortholog, SpSM50, in Strongylocentrotus purpuratus ) has been inhibited using morpholino antisense oligonucleotides. The inhibition, using relatively high levels of antisense reagent, can result in the complete absence of spicules, and the complete loss of immunoreactive LSM34/SpSM50, as judged by immunostaining and Western blotting. Primary mesenchyme cells (PMCs) do form and express PMC-specific cell surface antigens despite this inhibition. However, these anti-LSM34/SpSM50-treated embryos do not accumulate SM30 protein, another major matrix protein. Hence, both the initiation of spicule formation and subsequent morphogenesis require LSM34 accumulation in L. pictus , and the accumulation of its ortholog, SpSM50, in S. purpuratus .     

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.