The "lecithotrophic" sea urchin Heliocidaris erythrogramma lacks typical yolk platelets and yolk glycoproteins

The sea urchin Heliocidaris tuberculata undergoes typical development, forming an echinoid pluteus larva, whereas H. erythrogramma undergoes direct development via a highly modified, nonfeeding larva. Using a polyclonal antibody prepared against yolk glycoproteins from the typical developer Stronglyocentrotus purpuratus, we found that H. tuberculata contains cross-reactive proteins in abundance, but H. erythrogramma does not. In addition, we used immunoelectron microscopy to demonstrate that unfertilized eggs of H. tuberculata contain yolk platelets, but those of H. erythrogramma do not.     

Proteolysis of the major yolk glycoproteins is regulated by acidification of the yolk platelets in sea urchin embryos

The precise function of the yolk platelets of sea urchin embryos during early development is unknown. We have shown previously that the chemical composition of the yolk platelets remains unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and total protein content after fertilization and early development. However, the platelet is not entirely static because the major 160-kD yolk glycoprotein YP-160 undergoes limited, step-wise proteolytic cleavage during early development. Based on previous studies by us and others, it has been postulated that yolk platelets become acidified during development, leading to the activation of a cathepsin B-like yolk proteinase that is believed to be responsible for the degradation of the major yolk glycoprotein. To investigate this possibility, we studied the effect of addition of chloroquine, which prevents acidification of lysosomes. Consistent with the postulated requirement for acidification, it was found that chloroquine blocked YP-160 breakdown but had no effect on embryonic development. To directly test the possibility that acidification of the yolk platelets over the course of development temporally correlated with YP-160 proteolysis, we added 3-(2,4-dinitroanilo)-3-amino-N-methyldipropylamine (DAMP) to eggs or embryos. This compound localizes to acidic organelles and can be detected in these organelles by EM. The results of these studies revealed that yolk platelets did, in fact, become transiently acidified during development. This acidification occurred at the same time as yolk protein proteolysis, i.e., at 6 h after fertilization (64-cell stage) in Strongylocentrotus purpuratus and at 48 h after fertilization (late gastrula) in L. pictus. Furthermore, the pH value at the point of maximal acidification of the yolk platelets in vivo was equal to the pH optimum of the enzyme measured in vitro, indicating that this acidification is sufficient to activate the enzyme. For both S. purpuratus and Lytechinus pictus, the observed decrease in the pH was approximately 0.8 U, from 7.0 to 6.2. The trypsin inhibitor benzamidine was found to inhibit the yolk proteinase in vivo. By virtue of the fact that this inhibitor was reversible we established that the activity of the yolk proteinase is developmentally regulated even though the enzyme is present throughout the course of development. These findings indicate that acidification of yolk platelets is a developmentally regulated process that is a prerequisite to initiation of the catabolism of the major yolk glycoprotein.     

Calcium-vesicles perform active diffusion in the sea urchin embryo during larval biomineralization

Biomineralization is the process by which organisms use minerals to harden their tissues and provide them with physical support. Biomineralizing cells concentrate the mineral in vesicles that they secret into a dedicated compartment where crystallization occurs. The dynamics of vesicle motion and the molecular mechanisms that control it, are not well understood. Sea urchin larval skeletogenesis provides an excellent platform for investigating the kinetics of mineral-bearing vesicles. Here we used lattice light-sheet microscopy to study the three-dimensional (3D) dynamics of calcium-bearing vesicles in the cells of normal sea urchin embryos and of embryos where skeletogenesis is blocked through the inhibition of Vascular Endothelial Growth Factor Receptor (VEGFR). We developed computational tools for displaying 3D-volumetric movies and for automatically quantifying vesicle dynamics. Our findings imply that calcium vesicles perform an active diffusion motion in both, calcifying (skeletogenic) and non-calcifying (ectodermal) cells of the embryo. The diffusion coefficient and vesicle speed are larger in the mesenchymal skeletogenic cells compared to the epithelial ectodermal cells. These differences are possibly due to the distinct mechanical properties of the two tissues, demonstrated by the enhanced f-actin accumulation and myosinII activity in the ectodermal cells compared to the skeletogenic cells. Vesicle motion is not directed toward the biomineralization compartment, but the vesicles slow down when they approach it, and probably bind for mineral deposition. VEGFR inhibition leads to an increase of vesicle volume but hardly changes vesicle kinetics and doesn’t affect f-actin accumulation and myosinII activity. Thus, calcium vesicles perform an active diffusion motion in the cells of the sea urchin embryo, with diffusion length and speed that inversely correlate with the strength of the actomyosin network. Overall, our studies provide an unprecedented view of calcium vesicle 3D-dynamics and point toward cytoskeleton remodeling as an important effector of the motion of mineral-bearing vesicles.     

Micromere-derived signal regulates larval left-right polarity during sea urchin development

The micromeres (Mics) lineage functions as a morphogenetic signaling center in early embryos of sea urchins. The Mics lineage releases signals that regulate the specification of cell fates along the animal-vegetal and oral-aboral axes. We tested whether the Mics lineage might also be responsible for differentiation of the left-right (LR) axis by observing of the placement of the adult rudiment, which normally forms only on the left side of the larvae, after removal of the Mics lineage. When all of the Mics lineage were removed from embryos of the regular sea urchin Hemicentrotus pulcherrimus between the 16- and 64-cell stages, the LR placement of the rudiment became randomized. However, the immediate retransplantation of the Mics rescued the normal LR placement of the rudiment, indicating that the Mics lineage releases a signal that specifies LR polarity. Additionally, we investigated whether the specification of LR polarity of whole embryos in the indirect-developing sea urchin H. pulcherrimus is affected by LiCl exposure, which disturbs the establishment of LR asymmetry in a direct-developing sea urchin. Larvae derived from normal animal caps combined with LiCl-exposed Mics descendants were defective in normal LR placement of the rudiment, suggesting that LiCl interferes with the Mics-derived signal. In contrast, embryos of two sand dollar species (Scaphechinus mirabilis and Astriclypeus manni) were resistant to alteration of LR placement of the rudiment by either removal of the Mics lineage or LiCl exposure. These results indicate that the Mics lineage is involved in specification of LR polarity in the regular sea urchin H. pulcherrimus, and suggest that LiCl impairs the normal LR patterning by affecting Mics-derived signaling.     

Membrane formation and membrane contacts during the development of the sea urchin embryo

Summary Sea urchin embryos, 8-cell stage to pluteus stage, fixed in osmium tetroxide and embedded in Epon 812 were observed by electron microscopy. At no point in the development were syncytial junctions between the embryonic cells found. During the cleavage stages the membrane contact was closer than in later stages. In early blastula stages intercellular clefts appeared which in the gastrula stage demarcate every cell. At the same time a ringshaped desmosome structure develops at the outer cell surface. In the pluteus stage a closer cell contact is re-established. With proceeding embryogenesis endoplasmic membranes will attach to the cell membrane. These membrane structures may even be of nuclear origin. Gradually, long protrusions, vesicles and lamellae begin to be formed from the nuclear membrane. The commencement of this nuclear activity coincides in time with the formation of nucleoli. At cell division the new cell membrane seemed to arise partly independently of the cleavage furrow from a system of cytoplasmic vesicles.The investigation was facilitated by grants from the Nordic Insulin Foundation.I am indebted to Dr. Torsten Olsson and Miss Brita Nilsson for procuring the material and to Mrs. Mariann Carleson for technical aid.     

Cholinergic regulation of the sea urchin embryonic and larval development

Choline esters of polyenoic fatty acids block cleavage divisions of sea urchins and evoke the formation of one-cell multinuclear embryos. If the fatty acids AA-Ch or DHA-Ch are added at the mid or late blastula stage, many cells are extruded, forming extra-embryonic cell clusters near the animal pole of embryos or larvae. Both effects are prevented by dimethylaminoethyl esters of polyenoic fatty acids (AA-DMAE or DHA-DMAE) or their 5-hydroxytryptamides. Nicotinic acetylcholine receptor antagonists, imechine, d-tubocurarine or QX-222 provide partial protection against AA-Ch or DHA-Ch. The organophosphate pesticide, chlorpyrifos, or a combination of (-)-nicotine + phorbol 12-myristate 13-acetate, also evoke the mass extrusion of transformed embryonic cells at the animal pole of larvae. These effects are similarly antagonized by AA-DMAE, DHA-DMAE, or fatty acids 5-hydroxytryptamides. Taking together, these results suggest that AA-Ch and DHA-Ch act on sea urchin embryos and larvae as agonists of acetylcholine receptors, whereas AA-DMAE and DHA-DMAE act as antagonists. The ability of fatty acids 5-hydroxytryptamides to prevent the effects of AA-Ch or DHA-Ch may be due to restoration of the normal dynamic balance of cholinergic and serotonergic signaling during cleavage divisions and gastrulation.     

Metabolic interconversion of dolichol and dolichyl phosphate during development of the sea urchin embryo

The in vivo and in vitro synthesis and turnover of dolichol and dolichyl phosphate have been studied over the course of early development in sea urchin embryos. Synthesis of dolichol and dolichyl phosphate was studied in vivo and in vitro using [3H]acetate and [14C] isopentenylpyrophosphate, respectively, as precursors. Both the in vivo and in vitro results indicate that the principal labeled end product of de novo synthesis is the free alcohol, and that this alcohol is subsequently phosphorylated to produce dolichyl phosphate. The presence of 30 microM compactin inhibits the de novo synthesis of dolichol from [3H]acetate by greater than 90%, but has no effect on the incorporation of 32Pi into dolichyl phosphate for more than 6 h, thus suggesting that during this time interval the major source of dolichyl phosphate is preformed dolichol. The rate of turnover of the [3H]acetate-labeled polyisoprenoid backbone of dolichyl phosphate is very slow (t1/2 = 40-70 h). In contrast, the rate of loss of the [32P]phosphate headgroup is more rapid (t1/2 = 5.7-7.7 h) and increases over the course of development. Finally, dolichyl phosphate phosphatase activity has been measured in vitro. The activity of this enzyme, which can be distinguished from phosphatidic acid phosphatase, was found to increase as a function of development, in qualitative agreement with the increased turnover of 32P from dolichyl phosphate observed in vivo. These results suggest that the phosphate moiety of dolichyl phosphate is in a dynamic state, and that dolichol kinase and dolichyl phosphate phosphatase play key roles in regulating the cellular level of dolichyl phosphate.     

Analysis of changes in a yolk glycoprotein complex in the developing sea urchin embryo

A sea urchin yolk glycoprotein complex (YGC) was isolated from several developmental stages by velocity centrifugation on sucrose gradients. The YGCs were analyzed by SDS-polyacrylamide gel electrophoresis to determine if the molecular composition of the YGC was changing during development. The mass of the YGC did not change with development. However, as development proceeded there were significant changes in the glycoprotein composition of the YGC isolated from either Lytechinus pictus or Strongylocentrotus purpuratus embryos. In both species the YGC isolated from eggs contained three major glycoproteins. The most abundant one had an apparent molecular weight of 190,000 and was designated GP-190. During development the three major egg YGC glycoproteins decreased in relative amounts as intermediate-molecular-weight glycoproteins increased. While these changes were detected in YGCs isolated from either species, the rate of change was much greater in S. purpuratus than in L. pictus. The most significant difference was observed in the rate of decrease in GP-190. In S. purpuratus, GP-190 showed a significant decrease by 8-10 hr postfertilization, while a similar decrease did not occur in L. pictus YGCs until 72 hr postfertilization. To determine how these changes were occurring, both amino acid and carbohydrate analyses were done on the YGC isolated from various stages. From examination of these data, it appeared that the molecular composition of the YGC was changing via very limited proteolysis. The intermediate and low-molecular-weight glycoproteins generated apparently remain assembled in the YGC, thus conserving its mass.     

Mathematical model for early development of the sea urchin embryo

In Xenopus and Drosophila, the nucleocytoplasmic ratio controls many aspects of cell-cycle remodeling during the transitory period that leads from fast and synchronous cell divisions of early development to the slow, carefully regulated growth and divisions of somatic cells. After the fifth cleavage in sea urchin embryos, there are four populations of differently sized blastomeres, whose interdivision times are inversely related to size. The inverse relation suggests nucleocytoplasmic control of cell division during sea urchin development as well. To investigate this possibility, we developed a mathematical model based on molecular interactions underlying early embryonic cell-cycle control. Introducing the nucleocytoplasmic ratio explicitly into the molecular mechanism, we are able to reproduce many physiological features of sea urchin development.     

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.     

A large calcium-binding protein associated with the larval spicules of the sea urchin embryo

A tissue-specific, high molecular weight, calcium-binding protein from the sea urchin embryo is described. This protein, designated as CBP 180, has a molecular weight of 180,000 under reducing conditions, and is extractable with 1% Triton X-100. It accumulates rapidly during development, starting roughly at the onset of spiculogenesis. When embryos are cultured in the presence of inhibitors of spicule formation, such as tunicamycin and zinc ions, accumulation of CBP180 is depressed or stopped. By immunofluorescence technique and by using an antibody specifically generated against this protein, CBP180 is mainly localized in primary mesenchyme cells and spicular syncytium of the pluteus larva. Little or none is detectable in ectoderm, endoderm or blastocoelar extracellular matrix. These results suggest that the protein is involved in calcium sequestration in the differentiation of larval spicules.     

Cell junctions during the early development of the sea urchin embryo (Paracentrotus lividus)

Thin sections, lanthanum tracer and the freeze-fracture technique revealed the presence of different types of cell junctions in early sea urchin (Paracentrotus lividus) embryos. During the first four cleavage cycles, which are characterized by synchrony of cell division, sister blastomeres were connected only by intercellular bridges, formed as a result of incomplete cytokinesis; no trace of other junctions was found at these stages. From the 16-cell stage onwards, septate junctions and gap junctions began to appear between blastomeres. It is postulated that cell-cell interactions may provide a mechanism for the propagation of signals necessary for the coordination of cell proliferation and differentiation.     

Inhibition of glycoprotein processing blocks assembly of spicules during development of the sea urchin embryo

Previous studies have implicated an 130-kD glycoprotein containing complex, N-linked oligosaccharide chain(s) in the process of spicule formation in sea urchin embryos. To ascertain whether the processing of high mannose oligosaccharides to complex oligosaccharides is necessary for spiculogenesis, intact embryos and cultures of spicule-forming primary mesenchyme cells were treated with glycoprotein processing inhibitors. In both the embryonic and cell culture systems 1-deoxymannojirimycin (1-MMN) and, to a lesser extent, 1-deoxynojirimycin (1-DNJ) inhibited spicule formation. These inhibitors did not affect gastrulation in whole embryos or filopodial network formation in cell cultures. Swainsonine (SWSN) and castanospermine (CSTP) had no effect in either system. Further analysis revealed the following: (a) 1-MMN entered the embryos and blocked glycoprotein processing in the 24-h period before spicule formation as assessed by a twofold increase in endoglycosidase H sensitivity among newly synthesized glycoproteins upon addition of 1-MMN; (b) 1-MMN did not affect general protein synthesis until after its effects on spicule formation were observed; (c) Immunoblot analysis with an antibody directed towards the polypeptide chain of the 130-kD protein (mAb A3) demonstrated that 1-MMN did not affect the level of the polypeptide that is known to be synthesized just before spicule formation; (d) 1-MMN and 1-DNJ almost completely abolished (greater than 95%) the appearance of mAb 1223 reactive complex oligosaccharide moiety associated with the 130-kD glycoprotein; CSTP and SWSN had much less of an effect on expression of this epitope. These results indicate that the conversion of high mannose oligosaccharides to complex oligosaccharides is required for spiculogenesis in sea urchin embryos and they suggest that the 130-kD protein is one of these essential complex glycoproteins.     

Histone phosphorylation during sea urchin development

Studies on histone phosphorylation during transitions in chromatin structure occurringin vivoduring spermatogenesis and early embryogenesis in sea urchins are reviewed and evaluated in the light of recent studies on histone phosphorylation occurring during chromatin synthesis in frog egg extractsin vitroand evidence that protein kinases and phosphatases play direct roles in the regulation of cellular structure. Sperm-specific histone variants Sp H1 and Sp H2B are maintained as phosphorylated derivatives N and O/P throughout spermatogenesis and early embryogenesis and egg specific histone variants CS H1 and CS H2A are phosphorylated during early embryogenesis. These developmental correlations provide clues about the roles of histone phosphorylation in control of chromatin structurein vivoand provide a basis for the interpretation of data obtained from in-vitro sperm chromatin remodeling in egg extracts and from biochemical studies on the effects of histone phosphorylation on DNA binding. The potential consequences for chromatin structure of the various histone phosphorylation events observed in sea urchins and frog egg extracts are discussed.