Protease-insensitive sea urchin embryo cell adhesions become protease sensitive in the presence of azide or cytochalasin B

To understand the nature of the cell adhesions that must be modified during sea urchin embryo primary mesenchyme formation, we are studying the adhesive components of the hatched blastula stage embryo of Strongylocentrotus purpuratus. Pronase treatment conditions have been defined that leave the cells intact and able to recover from the effects of the protease upon its removal. Under these conditions, adhesion of the cells to tissue culture plates is totally eliminated, but cell-cell adhesion formation is only partially inhibited. Analysis of iodinated cell surface proteins indicates that most are affected by thepronase. Further studies of pronase effects found that sodium azide-treated cells are slightly adhesive and that pronase treatment of azidc-treated cells totally eliminates cell-cell adhesions.     

Sulfated polysaccharides and cell differentiation in the sea urchin embryo

The synthesis of sulfated polysaccharides during the embryonic development of Paracentrotus lividus has been investigated by incorporation of radioactive sulfate, glucose, glucosamine and fucose. The following substances become labelled: fucan sulfate (approximately 60%), heparan sulfate (approximately 20%) and dermatan sulfate (approximately 20%), and possibly a very slight amount of chondroitin sulfate. In animalized and vegetalized embryos, the rate of incorporation is significantly reduced, and furthermore dermatan sulfate is almost absent in animalized embryos. It is concluded that this substance is associated with the differentiation of vegetative cells, possibly the mesenchyme cells.     

Changes of cell and embryo volume during development of vitelline membrane-free sea urchin embryos

The rearing in large batches of sea urchin embryos stripped of their fertilization membranes is described. The developmental stages are electronically characterized and compared with untreated, coated embryos. The volumes of disaggregated cells from early stages are determined. The total number of cells per embryo can be evaluated reliably. The conclusions drawn from the volume distribution curves—such as the volume increase of fertilized eggs preceding the first cleavage, the constancy of the embryo volume between the first and fifth cleavage and the constancy of the cellular volumes from the onset of the blastula stage around 15 h up to 30 h are discussed.     

Fibronectin in the developing sea urchin embryo

The presence of fibronectin in developing sea urchin embryos was studied uing immunofluorescence staining. The fluorescence pattern indicates that fibronectin is found on the cell surfaces and between cells in the blastula and gastrula stages, indicating that it plays a role in cell adhesion. Its presence on invaginating cells also suggests its involvement in morphogenesis during early development.     

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

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

Activator of G-protein signaling in asymmetric cell divisions of the sea urchin embryo

An asymmetric fourth cell division in the sea urchin embryo results in formation of daughter cells, macromeres and micromeres, with distinct sizes and fates. Several lines of functional evidence presented here, including pharmacological interference and dominant negative protein expression, indicate that heterotrimeric G protein Gi and its interaction partner, activator of G-protein signaling (AGS), are necessary for this asymmetric cell division. Inhibition of Gi signaling by pertussis toxin interferes with micromere formation and leads to defects in embryogenesis. AGS was isolated in a yeast two-hybrid screen with G alpha i as bait and was expressed in embryos localized to the cell cortex at the time of asymmetric divisions. Introduction of exogenous dominant-negative AGS protein, containing only G-protein regulatory (GPR) domains, selectively prevented the asymmetric division in normal micromere formation. These results support the growing evidence that AGS is a universal regulator of asymmetric cell divisions in embryos.     

Cell lineage conversion in the sea urchin embryo

The mesoderm of the sea urchin embryo conventionally is divided into two populations of cells; the primary mesenchyme cells (PMCs), which produce the larval skeleton, and the secondary mesenchyme cells (SMCs), which differentiate into a variety of cell types but do not participate in skeletogenesis. In this study we examine the morphogenesis of embryos from which the PMCs have been removed microsurgically. We confirm the observation of Fukushi (1962) that embryos lacking PMCs form a complete skeleton, although in a delayed fashion. We demonstrate by microsurgical and cell marking experiments that the appearance of skeletogenic cells in such PMC-deficient embryos is due exclusively to the conversion of other cells to the PMC phenotype. Time-lapse video recordings of PMC-deficient embryos indicate that the converting cells are a subpopulation of late-ingressing SMCs. The conversion of these cells to the skeletogenic phenotype is accompanied by their de novo expression of cell surface determinants normally unique to PMCs, as shown by binding of wheat germ agglutinin and a PMC-specific monoclonal antibody. Cell transplantation and cell marking experiments have been carried out to determine the number of SMCs that convert when intermediate numbers of PMCs are present in the embryo. These experiments indicate that the number of converting SMCs is inversely proportional to the number of PMCs in the blastocoel. In addition, they show that PMCs and converted SMCs cooperate to produce a skeleton that is correct in both size and configuration. This regulatory system should shed light on the nature of cell-cell interactions that control cell differentiation and on the way in which evolutionary processes modify developmental programs.     

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.     

Determination and morphogenesis in the sea urchin embryo

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

Cell movements in the sea urchin embryo.

Recent studies show that gastrulation in the sea urchin embryo involves movement of cells over the blastopore lip (involution). Some cells in the vegetal plate of the late blastula become bottle-shaped but they play a limited role in gastrulation. The functions of specific integrins, regulators of cell-cell adhesion, and extracellular matrix components in gastrulation are currently being analyzed. In addition, light-microscopic studies continue to provide a unique picture of dynamic cell behavior in vivo.     

Isolation of sea urchin embryo cell surface membranes on polycationic beads

Summary Blastula cell surface membranes of the sea urchin, Strongylocentrotus purpuratus, were isolated on polycationic beads by a method modified from Jacobson and Branton (1977) and Jacobson (1980). This study represents the first application of this procedure to an embryonic system. Embryo cells were attached to polylysine-coated polyacrylamide beads and lysed, leaving the embryo cell surface membranes still attached to the beads, and cytoplasmic particles were washed free of the exposed inner surfaces of the membranes. Cell surface membrane sheets were desorbed from the beads and collected by centrifugation. Approximately 8% and 5% of the cell surface membranes of dissociated embryo cells were recovered on the beads and in the membrane pellet, respectively. Specific activities of [³H]concanavalin A-binding and of the cell surface marker enzymes, alkaline phosphatase and Na⁺/K⁺ ATPase, were 16-, 19-, and 32-fold higher, respectively, in the cell surface membrane fraction than in the embryo cell homogenate. Membranes were relatively free of cytoplasmic contaminants as judged from electron micrographs and enzyme analysis. Activities in the membrane fraction of the cytoplasmic marker enzymes, cytochrome c oxidase, catalase, acid phosphatase, NADP- and NADPH-cytochrome c reductase, and acetylcholinesterase, were substantially less than homogenate levels. The entire procedure can be completed in 4 h. Since this cell surface membrane isolation technique relies only on the tendency of a negatively charged cell to adhere to a positively charged surface, it is less likely than most other methods to exhibit species and developmental stage specificity and should prove useful in the study of the developmental role of embryonic stage-specific membrane components.     

Matrix metalloproteases of the developing sea urchin embryo

Abstract. A distinct group of metalloproteases has been identified in the developing sea urchin embryo by gelatin substrate gel zymography, a highly sensitive protease detection assay. The developing Arbacia embryo exhibited four prominent bands of gelatinase activity with apparent molecular masses of 55, 50, 42 and 38 kDa. The activity of the 55, 42 and 38 kDa tissue gelatinases increased and that of the 50 kDa tissue gelatinase decreased during embryonic development. All four enzymes were EDTA- and 1,10-phenanthroline sensitive and phenyl methyl sulphonyl fluoride (PMSF) insensitive. None of the enzymes had detectable caseinolytic activity in casein substrate gels. Although the Arbacia enzymes possessed a number of properties that are characteristic of the mammalian matrix metalloprotease family, they did not appear to be converted to lower molecular weight forms by organomercurial treatment and are distinct in this aspect. The Arbacia metalloproteases are candidate enzymes for the tissue and matrix remodeling that occurs during sea urchin embryo development.     

Matrix metalloproteases of the developing sea urchin embryo

Abstract. A distinct group of metalloproteases has been identified in the developing sea urchin embryo by gelatin substrate gel zymography, a highly sensitive protease detection assay. The developing Arbacia embryo exhibited four prominent bands of gelatinase activity with apparent molecular masses of 55, 50, 42 and 38 kDa. The activity of the 55, 42 and 38 kDa tissue gelatinases increased and that of the 50 kDa tissue gelatinase decreased during embryonic development. All four enzymes were EDTA- and 1,10-phenanthroline sensitive and phenyl methyl sulphonyl fluoride (PMSF) insensitive. None of the enzymes had detectable caseinolytic activity in casein substrate gels. Although the Arbacia enzymes possessed a number of properties that are characteristic of the mammalian matrix metalloprotease family, they did not appear to be converted to lower molecular weight forms by organomercurial treatment and are distinct in this aspect. The Arbacia metalloproteases are candidate enzymes for the tissue and matrix remodeling that occurs during sea urchin embryo development.     

Ontogeny of the basal lamina in the sea urchin embryo

The patterns of expression for several extracellular matrix components during development of the sea urchin embryo are described. An immunofluorescence assay was employed on paraffin-sectioned material using (i) polyclonal antibodies against known vertebrate extracellular matrix components: laminin, fibronectin, heparan sulfate proteoglycan, collagen types I, III, and IV; and (ii) monoclonal antibodies generated against sea urchin embryonic components. Most extracellular matrix components studied were found localized within the unfertilized egg in granules (0.5-2.0 micron) distinct from the cortical granules. Fertilization initiated trafficking of the extracellular matrix (ECM) components from within the egg granules to the basal lamina of the developing embryo. The various ECM components arrived within the developing basal lamina at different times, and not all components were unique to the basal lamina. Two ECM components were not found within the egg. These molecules appeared de novo at the mesenchyme blastula stage, and remained specific to the mesoderm through development. The reactivity of antibodies to vertebrate ECM antigens with components of the sea urchin embryo suggests the presence of immunologically similar ECM molecules between the phyla.     

Calcium-dependent self-aggregation of toposome,a sea urchin embryo cell adhesion molecule

Summary— Sea urchin embryos can be easily dissociated into single cells by exposure to Ca²⁺- and Mg²⁺-free seawater. When transferred back to normal seawater, isolated cells spontaneously form aggregates capable of development. Here, the Ca²⁺-dependent self-aggregation of toposome, a 22S glycoprotein complex which mediates cell-cell adhesion in sea urchin embryos, has been investigated using the purified molecule. Results show that the 22S complex is completely converted to 15S particles by sedimentation on sucrose isokinetic gradients in the presence of EDTA. Reconstitution of the 22S complex is achieved by readdition of Ca²⁺. We propose that the 15S particle constitutes the toposome functional unit on the cell surface.