Concanavalin A and wheat germ agglutinin binding to sea urchin embryo basal laminae

Summary The basal laminae and inner extracellular matrices of Lytechinus pictus and Arbacia punctulata embryos were characterized on the basis of lectin binding. Developmental stage specific patterns of lectin binding were observed after microinjection of Con A-FITC and WGA-FITC. Lectin-specific patterns differed between control, sulfate free sea water (SFSW) and tunicamycin treated embryos. Con A injection resulted in the rounding-up of cells in the epithelium and was most pronounced in embryos cultured in the presence of tunicamycin. Basal laminae were isolated by Triton X-100 extraction of whole embryos. Proteins were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose and incubated in biotinylated lectins. Lectin-binding glycoproteins were detected with avidin peroxidase. The electrophoretic pattern of Con A-binding proteins in early developmental stages of Arbacia was similar with several low molecular weight species appearing at gastrulation in control and SFSW embryos. WGA-binding in Arbacia and Lytechinus control embryos was limited to a 125,000 Mr glycoprotein (gp125). In addition to gp125, several high molecular weight WGA-binding glycoproteins were also detected in SFSW embryos. The evidence suggests that mesenchyme migration and gastrulation are correlated with changes in the molecular composition of the ECM.     

Elongated microvilli support the sea urchin embryo concentrically within the perivitelline space until hatching

Summary The early sea urchin embryo is supported in a concentric position within the perivitelline space by elongated microvilli which are attached to the fertilization envelope by extracellular matrix fibers. This “attachment complex,” of microvillus tip: extracellular matrix fibers: fertilization envelope, was revealed by two methods: the use of pronase or calcium-free sea water to dissolve the extracellular matrix fibers, thus causing the eggs to lose their concentric location, and the visualization of the “attachment complex” using video-enhanced differential interference contrast microscopy and transmission electron microscope images. The presence of the “attachment complex” helps in understanding two types of early developmental events: (1) the apparently continual change in microvillus length during cleavage stages which retains the embryos in their concentric position and (2) the hatching process.     

The dynamics of secretion during sea urchin embryonic skeleton formation

Skeleton formation involves secretion of massive amounts of mineral precursor, usually a calcium salt, and matrix proteins, many of which are deposited on, or even occluded within, the mineral. The cell biological underpinnings of this secretion and subsequent assembly of the biomineralized skeletal element is not well understood. We ask here what is the relationship of the trafficking and secretion of the mineral and matrix within the primary mesenchyme cells of the sea urchin embryo, cells that deposit the endoskeletal spicule. Fluorescent labeling of intracellular calcium deposits show mineral precursors are present in granules visible by light microscopy, from whence they are deposited in the endoskeletal spicule, especially at its tip. In contrast, two different matrix proteins tagged with GFP are present in smaller post-Golgi vesicles only seen by electron microscopy, and the secreted protein are only incorporated into the spicule in the vicinity of the cell of origin. The matrix protein, SpSM30B, is post-translationally modified during secretion, and this processing continues after its incorporation into the spicule. Our findings also indicate that the mineral precursor and two well characterized matrix proteins are trafficked by different cellular routes.     

Expression of univin, a TGF-beta growth factor, requires ectoderm-ECM interaction and promotes skeletal growth in the sea urchin embryo

Pl-nectin is an ECM protein located on the apical surface of ectoderm cells of Paracentrotus lividus sea urchin embryo. Inhibition of ECM-ectoderm cell interaction by the addition of McAb to Pl-nectin to the culture causes a dramatic impairment of skeletogenesis, offering a good model for the study of factor(s) involved in skeleton elongation and patterning. We showed that skeleton deficiency was not due to a reduction in the number of PMCs ingressing the blastocoel, but it was correlated with a reduction in the number of Pl-SM30-expressing PMCs. Here, we provide evidence on the involvement of growth factor(s) in skeleton morphogenesis. Skeleton-defective embryos showed a strong reduction in the levels of expression of Pl-univin, a growth factor of the TGF-beta superfamily, which was correlated with an equivalent strong reduction in the levels of Pl-SM30. In contrast, expression levels of Pl-BMP5-7 remained low and constant in both skeleton-defective and normal embryos. Microinjection of horse serum in the blastocoelic cavity of embryos cultured in the presence of the antibody rescued skeleton development. Finally, we found that misexpression of univin is also sufficient to rescue defects in skeleton elongation and SM30 expression caused by McAb to Pl-nectin, suggesting a key role for univin or closely related factor in sea urchin skeleton morphogenesis.     

Fibronectin from the ovary of the sea urchin,Pseudocentrotus depressus

Summary Fibronectin, with a subunit molecular weight of 220,000 daltons, was isolated from the ovary of the sea urchin,Pseudocentrotus depressus, using affinity chromatography on heat-denatured mammalian collagen coupled to Sepharose 4B. The distribution of fibronectin in the sea urchin ovary was examined by indirect immunofluorescence using antifibronectin serum. The basement membrane and the connective tissues exhibited strong fluorescence. The fibronectin was localized closely together with collagen bundles in the sea urchin ovary. Biochemical and immunological examinations indicate that sea urchin fibronectin has similar properties as those of mammalian fibronectin.     

Cell-to-substratum adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus

Summary Some plant lectins, Concanavalin agglutinin (Con A), succinyl Con A and wheat germ agglutinin (WGA) increased the adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus, to the substratum (plastic and glass surface) in vitro. Other plant lectins,Ulex europeus agglutinin (UEA) andDolichos biflorus agglutinin (DBA) had no effect on the cell-to-substratum interaction. A specific monocarbohydrate inhibitor of lectins, -methyl-d-mannoside, inhibited the Con A-induced cell-to-substratum adhesion of dissociated embryonic cells. This observation suggests that the Con A-induced cell-to-substratum adhesion may be attributed to the Con A-carbohydrate interaction. In Millipore-filtered sea water (MPFSW) containing Con A (0.1 mg/ml), dissociated embryonic cells adhered to the substratum for more than 6 h at 18°C, while in MPFSW as control, almost all the dissociated cells were released from the substratum after 1 h. A scanning electron microscopic study showed that dissociated embryonic cells adhered to the substratum were surrounded by an extracellular fibrous material, when the cells were cultured in MPFSW containing Con A. The induction of the extracellular fibrous material by Con A was inhibited by -methyl-d-mannoside. The appearance of this material may be related to the cell-to-substratum adhesion of dissociated cells. Sequential extractions of Con A-treated dissociated cells with Triton X 100 and urea solubilized most of the cellular components, leaving the fibrous material on the surface. Biochemical conponents of the isolated fibrous material included sea urchin fibronectin, Con A and minor components (88 and 140 kilodalton proteins). Fibronectin preformed in the cells was excreted after the dissociation, while the 88 and 140 kilodalton proteins were synthesized and released to the extracellular space.     

The origin of skeleton forming cells in the sea urchin embryo

Summary In embryos of the modern sea urchin species, subclass Euechinoidea, primary mesenchyme cells are derived from the progeny of micromeres formed at the sixteen cell stage of embryogenesis. The micromeres reside within the vegetal plate epithelium and later ingress into the blastocoel as primary mesenchyme cells which form the larval skeleton. Embryos of Eucidaris tribuloides, a member of the primitive subclass Perischoechinoidea, exhibit several noteworthy differences from euechinoid primary mesenchyme cell lineage including variable numbers and sizes of micromeres, the absence of mesenchyme ingression, and the lack of any detectable primary mesenchyme although a larval skeleton forms. In the present study, the cell lineage of the spiculogenic mesenchyme has been studied in Eucidaris tribuloides and in the euechinoid Lytechinus pictus by microinjecting the fluorescent tracer, Lucifer Yellow, into individual blastomeres of the embryo. In addition, wheat germ agglutinin, a lectin which binds only to primary mesenchyme cells of the early euechinoid embryo, was injected into the blastocoel of embryos of both species in order to examine the distribution of cells which possess primary mesenchyme-specific cell surface markers. The results of these experiments demonstrate that the spiculogenic mesenchyme of both Lytechinus and Eucidaris arise from descendants of micromeres formed at the sixteen cell stage, although the temporal and spatial distribution of these mesenchyme cells varies considerably between species. Furthermore, the evidence obtained suggests that the information necessary for spicule formation is already segregated to the vegetal pole by the eight cell stage. The results also suggest that there are no gap junctions present between the blastomeres of the early sea urchin embryo.     

A protein of the basal lamina of the sea urchin embryo

The purification, biochemical characterization and functional features of a novel extracellular matrix protein are described. This protein is a component of the basal lamina found in embryos from the sea urchin species Paracentrotus lividus and Hemicentrotus pulcherrimus . The protein has been named PI-200 K or Hp-200 K, respectively, because of the species from which it was isolated and its apparent molecular weight in SDS-PAGE under reducing conditions. It has been purified from unfertilized eggs where it is found packed within cytoplasmic granules, and has different binding affinities to type I collagen and heparin, as assessed by affinity chromatography columns. By indirect immunofluorescence experiments it was shown that, upon fertilization, the protein becomes extracellular, polarized at the basal surface of ectoderm cells, and on the surface of primary mesenchyme cells at the blastula and gastrula stages. The protein serves as an adhesive substrate, as shown by an in vitro binding assay where cells dissociated from blastula embryos were settled on 200K protein-coated substrates. To examine the involvement of the protein in morphogenesis of sea urchin embryo, early blastula embryos were microinjected with anti-200K Fab fragments and further development was followed. When control embryos reached the pluteus stage, microinjected embryos showed severe abnormalities in arms and skeleton elongation and patterning. On the basis of current results, it was proposed that 200K protein is involved in the regulation of sea urchin embryo skeletogenesis.     

DNA amplification in sea urchin oocytes

Summary In situ hybridization of ribosomal RNA withParacentrotus lividus ovaries suggests that ribosomal DNA undergoes amplification in the mononucleolate oocytes of this sea urchin.     

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.     

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.     

The sea urchin histone gene complement

The only eukaryotic mRNAs that are not polyadenylated are the replication-dependent histone mRNAs in metazoans. The sea urchin genome contains two sets of histone genes that encode non-polyadenylated mRNAs. One of these sets is a tandemly repeated gene cluster with a 5.6-kb repeat unit containing one copy of each of the five alpha-histone genes and is present as a single large cluster which spans over 1 Mb. There is a second set of genes, consisting of 39 genes, containing two histone H1 genes, 34 genes encoding core histone proteins (H2a, H2b, H3 and H4) and three genes expressed only in the testis. Unlike vertebrates where these genes are clustered, the sea urchin late histone genes, expressed in embryos, larvae and adults, are dispersed throughout the genome. There are also genes encoding polyadenylated histone mRNAs, which encode histone variants, including all variants found in other metazoans, as well as a unique set of five cleavage stage histone proteins expressed in oocytes. The cleavage stage histone H1 is the orthologue of an oocyte-specific histone H1 protein found in vertebrates.     

Sea urchin arylsulfatase,an extracellular matrix component,is involved in gastrulation during embryogenesis

Arylsulfatases (Arses) have been regarded as lysosomal enzymes because of their hydrolytic activities on synthetic aromatic substrates and their lysosomal localization of their enzymatic activities. Using sea urchin embryos, we previously demonstrated that the bulk of Hemicentrotus Ars (HpArs) does not exhibit enzyme activity and is located on the apical surface of the epithelial cells co-localizing with sulfated polysaccharides. Here we show that HpArs strongly binds to sulfated polysaccharides and that repression of the synthesis by HpArs-morpholino results in retardation of gastrulation in the sea urchin embryo. Accumulation of HpArs protein and sulfated polysaccharides on the apical surface of the epithelial cells in sea urchin larvae is repressed by treatment with β-aminopropionitrile (BAPN), suggesting that deposition of HpArs and sulfated polysaccharides is dependent on the crosslinking of proteins such as collagen-like molecules. We suggest that HpArs functions by binding to components of the extracellular matrix.     

Distribution and redistribution of pigment granules in the development of sea urchin embryos

Summary Pigment granules (PGs) are embeded in the cortex of embryos of the Japanese sea urchins,Hemicentrotus pulcherrimus and Anthocidaris crassispina. PGs in the cortex actively retreated from the vegetal pole area at the 4-cell stage and then a notable PG-distribution gradient formed along the egg axis (the polar redistribution of PGs). The polar redistribution of PGs in the cortex occurred at the same time after fertilization even in solutions of microtubule disrupting reagents such as Colcemid, vinblastine sulfate or griseofulvin. Consequently, the polar redistribution of PGs was not associated with the microtubules. However, the polar redistribution of PGs was interrupted in seawater containing cytochalasin B (CB), dithiothreitol (DTT) or tetracaine, and the distribution pattern of PGs in the cortex was definitely disturbed. Moreover, CB, DTT and tetracaine altered the division pattern of vegetal blastomeres at the 4th cleavage which is normally unequal so that all the blastomeres divided equally. Microtubule disrupting reagents did not have such an effect on the cleavage pattern. Thus the cortical movement along the egg axis reflected by the polar redistribution of PGs seems to correlate with the micromere formation.     

A new extracellular matrix protein of the sea urchin embryo with properties of a substrate adhesion molecule

Summary A new embryonic extracellular matrix protein has been purified from eggs of the sea urchin Paracentrotus lividus. The molecule is a 210 kD dimer consisting of two 105 kD subunits that are held together by S-S bridges. In the unfertilized egg, the protein is found within granules uniformly distributed throughout the cytoplasm. After the egg is fertilized, the antigen is polarized to the apical surface of ectodermal and endodermal cells during all of the developmental stages examined, until the pluteus larva is formed. The protein promotes the adhesion of blastula cells to the substrate and is antigenically distinct from echinonectin, a well characterized substrate adhesion molecule. This report adds a new candidate to the list of known extracellular matrix molecules for the regulation of differentiation and morphogenesis in the sea urchin embryo. Offprint requests to: V. Matranga     

Ultrastructure of collagen in sea urchin embryos

Summary Collagen fibrils with a main period banding of 610 Å and 220 Å in width were observed in the blastocoel of 72-h embryos of the sea urchin,Strongylocentrotus purpuratus. Non-striated fibrils of 50 Å diameter were also observed. The collagen is seen in highest concentration in the vicinity of mesenchyme cells which are richly endowed with endoplasmic reticulum and secretory vesicles. A role for collagen in cell attachment, orientation and spicule formation is discussed.     

Characterization of a fasciclin I-like protein with cell attachment activity from sea urchin (Strongylocentrotus intermedius) ovaries

Fasciclin I, a neuronal cell adhesion molecule, was first identified in the grasshopper. To date, various fasciclin I-like proteins have been identified but their biological functions have not been well characterized. Here, we have purified a fasciclin I-like protein with a molecular weight of 33kDa from sea urchin (Strongylocentrotus intermedius) ovaries using hydrophobic chromatography and gel filtration. The protein was not N-glycosylated. Partial amino acid sequences of cyanogen bromide (CNBr)-cleaved fragments were highly conserved to other sea urchin fasciclin I-like proteins identified previously. The circular dichroism (CD) spectrum analysis demonstrated that the 33kDa protein contained high content of alpha-helical structure. These results suggest that the 33kDa protein is a fasciclin I-like family. Additionally, the fasciclin I-like protein promoted HT1080 human fibrosarcoma cell attachment. Further, a synthetic peptide (P1: GLREAANIAEQVDLRQVLRDVDL) of the protein corresponding to a highly conserved region of the fasciclin I-like family promoted heparin-dependent HT1080 cell attachment. Moreover, the peptide inhibited HT1080 cell attachment to the fasciclin I-like protein. These results suggest that the 33kDa protein from sea urchin ovaries isolated here is a member of the fasciclin I family and that the N-terminal region of the protein is important for cell attachment activity. The protein has a potential to be involved in biological functions in sea urchin as a cell adhesive molecule.     

The contractility of elongated microvilli in early sea urchin embryos

Summary Elongated microvilli attach the early sea urchin embryo to the fertilization envelope and support it in a concentric position within the perivitelline space. The contractility of the elongated microvilli was demonstrated in several ways. (1) During normal cleavage, these microvilli change their length to adapt to the change in shape and numbers of blastomeres. (2) When treated with calcium-free sea water, embryos become eccentrically located and the microvilli extend further than normal on one side; when returned to normal sea water, the embryos become centered again. (3) Several agents cause the fertilization envelope to become higher and thinner than normal and the elongated microvilli to extend correspondingly if treated within ten min after fertilization. In some cases, both elongated microvilli and fertilization envelope return to normal size when returned to normal sea water. (4) Fertilization in a papain solution causes the elongated microvilli and the fertilization envelope to contract to the surface of the embryo. (5) Refertilization after the papain-induced contraction can bring about the elongation of these microvilli and the elevation of the fertilization envelope a second time. It was also shown that elongated microvilli are extended immediately upon fertilization, at the same time as the short microvilli. The firm adherence of the tips of elongated microvilli to the fertilization envelope by means of extracellular matrix fibers is shown in a high voltage electron microscope stereoimage. This allows us to understand why it is that when the elongated microvilli extend or contract, the fertilization envelope also extends and contracts accordingly.