Exogenous hyalin and sea urchin gastrulation. Part III: biological activity of hyalin isolated from Lytechinus pictus embryos

Hyalin is a large glycoprotein, consisting of the hyalin repeat domain and non-repeated regions, and is the major component of the hyaline layer in the early sea urchin embryo of Strongylocentrotus purpuratus. The hyalin repeat domain has been identified in proteins from organisms as diverse as bacteria, sea urchins, worms, flies, mice and humans. While the specific function of hyalin and the hyalin repeat domain is incompletely understood, many studies suggest that it has a functional role in adhesive interactions. In part I of this series, we showed that hyalin isolated from the sea urchin S. purpuratus blocked archenteron elongation and attachment to the blastocoel roof occurring during gastrulation in S. purpuratus embryos, (Razinia et al., 2007). The cellular interactions that occur in the sea urchin, recognized by the U.S. National Institutes of Health as a model system, may provide insights into adhesive interactions that occur in human health and disease. In part II of this series, we showed that S. purpuratus hyalin heterospecifically blocked archenteron-ectoderm interaction in Lytechinus pictus embryos (Alvarez et al., 2007). In the current study, we have isolated hyalin from the sea urchin L. pictus and demonstrated that L. pictus hyalin homospecifically blocks archenteron-ectoderm interaction, suggesting a general role for this glycoprotein in mediating a specific set of adhesive interactions. We also found one major difference in hyalin activity in the two sea urchin species involving hyalin influence on gastrulation invagination.     

Microplate assay for quantifying developmental morphologies: effects of exogenous hyalin on sea urchin gastrulation

It is often difficult to determine the effects of various substances on the development of the sea urchin embryo due to the lack of appropriate quantitative microassays. Here, a microplate assay has been developed for quantitatively evaluating the effects of substances, such as hyalin, on living sea urchin embryos. Hyalin (330 kDa) is a major constituent of the sea urchin hyaline layer, an extracellular matrix that develops 20 min postinsemination. Function of the hyaline layer and its major constituent, is the adhesion of cells during morphogenesis. Using wide-mouthed pipette tips, 25 microl of 24-h Strongylocentrotus purpuratus embryos were transferred to each well of a 96-well polystyrene flat-bottom microplate yielding about 12 embryos per well. Specific concentrations of purified hyalin diluted in low calcium seawater were added to the wells containing the embryos, which were then incubated for 24 h at 15 degree C. The hyalin-treated and control samples were observed live and after fixation with 10% formaldehyde using a Zeiss Axiolab photomicroscope. The small number of embryos in each well allowed quantification of the developmental effects of the added media. Specific archenteron morphologies-attached, unattached, no invagination and exogastrula-were scored and a dose-dependent response curve was generated. Hyalin at high concentrations blocked invagination. At low concentrations, it inhibited archenteron elongation/attachment to the blastocoel roof. While many studies have implicated hyalin in a variety of interactions during morphogenesis, we are not aware of any past studies that have quantitatively examined the effects of exogenous hyalin on specific gastrulation events in whole embryos.     

Sea urchin morphogenesis and cell-hyalin adhesion are perturbed by a monoclonal antibody specific for hyalin

We have generated and characterized a monoclonal antibody (McA Tg-HYL) that recognizes sea urchin hyalin as evidenced by immunofluorescence staining of the hyaline layer (HL) and immunoblot staining of the hyalin protein band. On immunoblots of HL extracts only the hyalin protein reacted with McA Tg-HYL. Immunoprecipitates of radioactive proteins from embryos incubated with [35S]methionine yielded radioactive hyalin and 190, 140 and 105 x 10(3) Mr proteins associated with hyalin. McA Tg-HYL was generated against Tripneustes gratilla embryos but reacts with hyalin from the distantly related sea urchin species, Colobocentrotus atratus, Strongylocentrotus purpuratus, Arbacia punctulata, Lytechinus variegatus and Lytechinus pictus. Developing embryos of the above-mentioned six species were treated with McA Tg-HYL and did not gastrulate or form arms. Observations of treated embryos revealed areas of separation of the hyaline layer from the underlying embryonic cells, suggesting that McA Tg-HYL was interfering with binding of the cells to the HL. Using the centrifugation-based adhesion assay of McClay et al. (Proc. natn. Acad. Sci. U.S.A. 78, 4975-4979, 1981), Fab' fragments of McA Tg-HYL were found to inhibit cell-hyalin binding. McA Tg-HYL did not inhibit hyalin gelation in vitro or the reaggregation of dissociated blastula cells. We postulate that McA Tg-HYL recognizes an evolutionarily conserved hyalin domain involved in cell-hyalin binding and required for normal epithelial folding.     

Immunolocalization of hyalin in sea urchin eggs and embryos using an antihyalin-specific monoclonal antibody.

Monoclonal antibodies were raised against purified cortical secretory vesicles (CVs) from the eggs of Strongylocentrotus purpuratus. One of the monoclonal antibodies (MAb 69-10, an IgA) was shown by immunofluorescence labeling of intact and detergent-lysed CVs to be directed against a CV content antigen. Immunoblot analysis of CVs revealed that MAb 69-10 bound to a major CV polypeptide with an Mr similar to that of hyalin (i.e., 300,000). MAb 69-10 was subsequently shown to bind to purified hyalin prepared from S. purpuratus and to cross react with hyalin prepared from Lytechinus pictus. Immunogold labeling on thin sections of unfertilized S. purpuratus eggs showed that hyalin was localized to the electron-lucent portion of CVs. This result is in agreement with the labeling pattern obtained by Hylander and Summers (Dev Biol 93:368-380, 1982) using polyclonal antihyalin antibodies. In fertilized eggs and later-stage embryos, hyalin was observed to be located on the external surface of the embryo. MAb 69-10 should be useful in studies of the structure of hyalin and its function in morphogenesis.     

Inhibitors of procollagen C-terminal proteinase block gastrulation and spicule elongation in the sea urchin embryo

In the sea urchin embryo, inhibition of collagen processing and deposition affects both gastrulation and embryonic skeleton (spicule) formation. It has been found that cell-free extracts of gastrula-stage embryos of Strongylocentrotus purpuratus contain a procollagen C-terminal proteinase (PCP) activity. A rationally designed non-peptidic organic hydroxamate, which is a potent and specific inhibitor of human recombinant PCP (FG-HL1), inhibited both the sea urchin PCP as well as purified chick embryo tendon PCP. In the sea urchin embryo, FG-HL1 inhibited gastrulation and blocked spicule elongation, but not spicule nucleation. A related compound with a terminal carboxylate rather than a hydroxamate (FG-HL2) did not inhibit either chick PCP or sea urchin PCP activity in a procollagen-cleavage assay. However, FG-HL2 did block spicule elongation without affecting spicule nucleation or gastrulation. Neither compound was toxic, because their effects were reversible on removal. It was shown that the inhibition of gastrulation and spicule elongation were independent of tissue specification events, because both the endoderm specific marker Endo1 and the primary mesenchyme cell specific marker SM50 were expressed in embryos treated with FG-HL1 and FG-HL2. These results suggest that disruption of the fibrillar collagen deposition in the blastocoele blocks the cell movements of gastrulation and may disrupt the positional information contained within the extracellular matrix, which is necessary for spicule formation.     

Gastrulation in the Sea Urchin,Strongylocentrotus purpuratus,Is Disrupted by the Small Laminin Peptides YIGSR and IKVAV

Laminin is present on the apical and basolateral sides of epithelial cells of very early sea urchin blastulae. We investigated whether small laminin-peptides, known to have cell binding activities, alter the development of sea urchin embryos. The peptide YIGSR-NH₂ (850 μM) and the peptide PA22-2 (5 μM), which contains the peptide sequence IKVAV (Tashiro et al., J. Biol. Chem. 264, 16174, 1989), typically blocked archenteron formation when added to the sea water soon after fertilization. At lower doses, the YIGSR peptide allowed invagination of the archenteron but blocked archenteron extension and differentiation and evagination of the feeding arms. The effect of YIGSR and PA22-2 peptides declined when added to progressively older stages until no effect was seen when added at the mesenchyme blastula stage (24 hours after fertilization). Control peptides GRGDS, YIGSE, and SHA22, a dodeca-peptide with a scrambled IKVAV sequence, had no effect on development. The YIGSK peptide containing a conserved amino acid modification had only a small effect on gastrulation. The results suggest that YIGSR and IKVAV peptides specifically disrupt cell/extracellular matrix interactions required for normal development of the archenteron and feeding arms. Our recent finding that YTGIR is at the cell binding site of the B1 chain of S. purpuratus laminin supports this conclusion. Evidently, laminin or other laminin-like molecules are among the many extracellular matrix components needed for the invagination and extension of the archenteron during the gastrulation movements of these embryos.     

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.     

Evolutionary dissociation between cleavage, cell lineage and embryonic axes in sea urchin embryos.

Using vital dye staining and the microinjection of fluorescent cell lineage-autonomous tracers, the relationship between the first cleavage plane and the prospective larval dorsoventral axis was examined in several sea urchin species, including: Strongylocentrotus purpuratus, S. droebachiensis, Lytechinus pictus, Clypeaster rosaceus, Heliocidaris tuberculata and H. erythrogramma. The results indicate that there is no single relationship between the early cleavage pattern and the dorsoventral axis for all sea urchins; however, specific relationships exist for individual species. In S. purpuratus the first cleavage plane occurs at an angle 45 degrees clockwise with respect to the prospective dorsoventral axis in most cases, as viewed from the animal pole. On the other hand, in S. droebachiensis, L. pictus and H. tuberculata, the first cleavage plane generally corresponds with the plane of bilateral symmetry. There does not appear to be a predominant relationship between the first cleavage plane and the dorsoventral axis in C. rosaceus. In the direct-developing sea urchin H. erythrogramma the first cleavage plane bisects the dorsoventral axis through the frontal plane. Clearly, evolutionary differences have arisen in the relationship between cleavage pattern and developmental axes. Therefore, the mechanism of cell determination is not necessarily tied to any particular pattern of cell cleavage, but to an underlying framework of axial systems resident within sea urchin eggs and embryos.     

The role of secondary mesenchyme cells during sea urchin gastrulation studied by laser ablation

It has long been thought that traction exerted by filopodia of secondary mesenchyme cells (SMCs) is a sufficient mechanism to account for elongation of the archenteron during sea urchin gastrulation. The filopodial traction hypothesis has been directly tested here by laser ablation of SMCs in gastrulae of the sea urchin, Lytechinus pictus. When SMCs are ablated at the onset of secondary invagination, the archenteron doubles in length at the normal rate of elongation, but advance of the tip of the archenteron stops at the 2/3 gastrula stage. In contrast, when all SMCs are ablated at or following the 2/3 gastrula stage, further elongation does not occur. However, if a few SMCs are allowed to remain in 2/3-3/4 gastrulae, elongation continues, although more slowly than in controls. The final length of archenterons in embryos ablated at the 1/3-1/2 gastrula stage is virtually identical to the final length of everted archenterons in LiCl-induced exogastrulae; since filopodial traction is not exerted in either case, an alternate, common mechanism of elongation probably operates in both cases. These results suggest that archenteron elongation involves two processes: (1) active, filopodia-independent elongation, which depends on active cell rearrangement and (2) filopodia-dependent elongation, which depends on mechanical tension exerted by the filopodia.     

Tandem duplication and divergence of a sea urchin protein belonging to the troponin C superfamily

The Spec1 and Spec2 proteins of the sea urchin Strongylocentrotus purpuratus are related to calmodulin, troponin C, and myosin light chains by sequence similarity in their four calcium binding domains. These domains, the EF-hands, are distinct helix-loop-helix structures of about 40 amino acids. The Spec1 and Spec2 genes are expressed specifically in aboral ectoderm cells of the developing embryo; however, the function of the Spec proteins in these cells is unknown. To find conserved regions of the proteins that might be important for structure and function, Spec homologues from Lytechinus pictus, a distantly related sea urchin, were sought. L. pictus embryos do not synthesize detectable amounts of the 14,000-17,000-Da Spec proteins as determined by two-dimensional gel electro-phoresis, but do synthesize three 34,000-Da proteins that cross-react with Spec1 antibodies and display a similar ontogenetic pattern of expression. cDNA clones were isolated by hybridization to a synthetic oligonucleotide corresponding to the EF-hand. One clone, LpS1, encodes an mRNA with developmental properties like those of the S. purpuratus Spec mRNAs. However, LpS1 contains an open reading frame for a protein of 34,000 Da rather than 17,000 Da, and antibodies raised against part of the LpS1 reading frame demonstrate that LpS1 encodes a 34,000-Da protein in L. pictus embryos. The sequence of LpS1 reveals the presence of eight EF-hand domains, which share structural homology with the Spec1 or Spec2 EF-hands; however, little else in the protein sequence is conserved. The results support the hypothesis that the LpS1 gene arose from a duplication of an ancestral Spec gene and that the overall structural features of the Spec family of proteins are more conserved than the amino acid sequences.     

Functional analysis of the promoter of a sea urchin metallothionein gene.

The 5'-flanking region of the metallothionein (MT) gene LpMT1 of the sea urchin Lytechinus pictus includes three copies of a conserved sequence that includes the metal-responsive element (MRE) consensus core sequence required for heavy metal induction of other MT genes, a GC box, a G box of a putative basal level enhancer element which includes another MRE core element, and a poly(C) tract. A fragment of LpMT1 DNA from nucleotides +31 to -309 fused to a chloramphenicol acetyltransferase reporter gene was inducible with cadmium after injection into L. pictus embryos. This induced activity was greatly reduced in a deletion mutant which retained only 195 base pairs of 5'-flanking sequence, including the proximal pair of MREs and the G box, but excluding the poly(C) tract, GC box, and distal MRE. A potent human hMT-IIA gene promoter is marginally functional in L. pictus embryos. In contrast, the LpMT1 promoter is active in HeLa cells and in embryos of the sea urchin Strongylocentrotus purpuratus. The hMT-IIA gene may lack a cis-acting sequence element required for expression of MT genes in L. pictus embryos. The LpMT1 promoter is a powerful, inducible, promiscuous promoter useful for driving the expression of heterologous genes in sea urchin embryos.     

Archenteron elongation in the sea urchin embryo is a microtubule-independent process

Earlier studies using colchicine (L. G. Tilney and J. R. Gibbins, 1969, J. Cell Sci. 5, 195-210) had suggested that intact microtubules (MTs) are necessary for archenteron elongation during the second phase of sea urchin gastrulation (secondary invagination), presumably by allowing secondary mesenchyme cells (SMCs) to extend their long filopodial processes. In light of subsequently discovered effects of colchicine on other cellular processes, the role of MTs in archenteron elongation in the sea urchin, Lytechinus pictus, has been reexamined. Immunofluorescent staining of ectodermal fragments and isolated archenterons reveals a characteristic pattern of MTs in the ectoderm and endoderm during gastrulation. Ectodermal cells exhibit arrays of MTs radiating away from the region of the basal body/ciliary rootlet and extending along the periphery of the cell, whereas endodermal cells exhibit a similar array of peripheral MTs emanating from the region of the apical ciliary rootlet facing the lumen of the archenteron. MTs are found primarily at the bases of the filopodia of normal SMCs. beta-Lumicolchicine (0.1 mM), an analog of colchicine which does not bind tubulin, inhibits secondary invagination, indicating that the effects previously ascribed to the disruption of MTs are probably due to the effects of colchicine on other cellular processes. The MT inhibitor nocodazole (5-10 micrograms/ml) added prior to secondary invagination does not prevent gastrulation or spontaneous exogastrulation, even though indirect immunofluorescence indicates that cytoplasmic MTs are completely disrupted in drug-treated embryos. Transverse tissue sections indicate that a comparable amount of cell rearrangement occurs in nocodazole-treated and control embryos. Significantly, SMCs in nocodazole-treated embryos often detach prematurely from the tip of the gut rudiment and extend abnormally large broad lamellipodial protrusions but are also capable of extending long slender filopodia comparable in length to those of control embryos. These results indicate that cytoplasmic MTs are not essential for either filopodial extension by SMCs or for the active epithelial cell rearrangement which accompanies elongation during sea urchin gastrulation.     

On the ultrastructure of hyalin, a cell adhesion protein of the sea urchin embryo extracellular matrix

Hyalin is a large (ca. 350 x 10(3) kD by gel electrophoresis) molecule that contributes to the hyalin layer surrounding the sea urchin embryo. In previous work a mAb (McA Tg-HYL), specific for hyalin, was found to inhibit cell-hyalin adhesion and block morphogenesis of whole embryos (Adelson, D. L., and T. D. Humphreys. 1988. Development. 104:391-402). In this report, hyalin ultrastructure was examined via rotary shadowing. Hyalin appeared to be a filamentous molecule approximately 75-nm long with a globular "head" about 12 nm in diameter that tended to form aggregates by associating head to head. Hyalin molecules tended to associate with a distinct high molecular weight globular particle ("core"). In fractions containing the core particle often more than one hyalin molecule were seen to be associated with the core. The core particle maintained a tenacious association with hyalin throughout purification procedures. The site(s) of McA Tg-HYL binding to the hyalin molecule were visualized by decorating purified hyalin with the antibody and then rotary shadowing the complex. In these experiments, McA Tg-HYL attached to the hyalin filament near the head region in a pattern suggesting that more than one antibody binding site exists on the hyalin filament. From the ultrastructural data and from the cell adhesion data presented earlier we conclude that hyalin is a filamentous molecule that binds to other hyalin molecules and contains multiple cell binding sites. Attempts were made to demonstrate the existence of lower molecular weight hyalin precursors. Whilst no such precursors could be identified by immunoprecipitation of in vivo labeled embryo lysates, immunoprecipitation of in vitro translation products suggested such precursors (ca 40 x 10(3) kD) might exist.     

Skeletogenesis in sea urchin interordinal hybrid embryos

Reciprocal interordinal crosses were made between the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus. Previous research indicated that the expression of many L. pictus genes is reduced in the hybrid embryos. The S. purpuratus gene encoding the spicule matrix protein SM50 and the L. pictus gene encoding its orthologue LSM34 were both expressed at normal levels per gene copy in hybrid embryos, and in about 32 skeletogenic primary mesenchyme cells (PMCs) in hybrid and natural gastrulae. In many embryos of all crosses, 16 PMCs initially ingressed, while 32-64 PMCs were present in gastrulae. The skeletal spicules of most hybrid plutei were predominantly like those of S. purpuratus, consistent with the predominance of expression of S. purpuratus genes in hybrid embryos. The spicules of some hybrid plutei showed features characteristic of L. pictus, such as recurrent rods, branched body rod tips, or convergent ventral transverse rods; a few hybrid spicules were predominantly like those of L. pictus. Based on our observations and the literature, we propose the following. Cues from the ectodermal epithelium position the PMCs as they elaborate the initial triradiate spicules. Their orientation and outgrowth appears to be responsible for the convergence of the tips of body rods in most S. purpuratus and hybrid embryos, unlike in most L. pictus embryos. Variations among hybrid and natural embryos in skeletal branching pattern reflect differences in interpretation by PMCs of patterning cues produced by the ectodermal epithelium that probably have similar spatial distributions in the two species.     

Tissue-specific, temporal changes in cell adhesion to echinonectin in the sea urchin embryo.

Echinonectin is a dimeric, glycoprotein found in the hyaline layer of the developing sea urchin embryo. It was found that echinonectin supports adhesion of embryonic cells in vitro. Previous studies have shown that the protein hyalin also supports adhesion. The purpose of this study was to examine the specificity of cell-echinonectin interactions during sea urchin development. Primary mesenchyme cells (PMCs) ingress into the blastocoel during gastrulation. In the process the PMCs lose contact with the hyaline layer. It was found experimentally that differentiating PMCs decreased their adhesion to hyalin at the time of ingression. It was of interest, therefore, to determine whether there was a coordinate loss of adhesion to echinonectin at ingression as well. When cell-echinonectin interactions were quantified using a centrifugal force-based adhesion assay, it was shown that micromeres adhered well to echinonectin. At the time of ingression, PMCs displayed reduced adhesion to echinonectin just as had been found when hyalin was tested as a substrate. There was no change in adhesion of presumptive ectoderm or endoderm to echinonectin over the same time period. Early in gastrulation presumptive ectoderm and endoderm adhered to echinonectin only half as strongly as to equimolar concentrations of hyalin. After gastrulation endoderm cells were observed to retain the same relative affinity to hyalin and echinonectin, while ectoderm cells became equally adhesive for both hyalin and echinonectin. Quantitatively, this represents an overall increase in the affinity of ectodermal cells for echinonectin. Adhesion to combined substrata of echinonectin and hyalin was reduced but not abolished by monoclonal antibodies specific for echinonectin. The antibodies did not cross-react with hyalin. We conclude that both echinonectin and hyalin independently act as adhesive substrata for the developing sea urchin embryo. PMCs lose an affinity for echinonectin and ectodermal cells later increase their affinity for this substrate.