A genome-wide analysis of biomineralization-related proteins in the sea urchin Strongylocentrotus purpuratus.

Biomineralization, the biologically controlled formation of mineral deposits, is of widespread importance in biology, medicine, and engineering. Mineralized structures are found in most metazoan phyla and often have supportive, protective, or feeding functions. Among deuterostomes, only echinoderms and vertebrates produce extensive biomineralized structures. Although skeletons appeared independently in these two groups, ancestors of the vertebrates and echinoderms may have utilized similar components of a shared genetic "toolkit" to carry out biomineralization. The present study had two goals. First, we sought to expand our understanding of the proteins involved in biomineralization in the sea urchin, a powerful model system for analyzing the basic cellular and molecular mechanisms that underlie this process. Second, we sought to shed light on the possible evolutionary relationships between biomineralization in echinoderms and vertebrates. We used several computational methods to survey the genome of the purple sea urchin Strongylocentrotus purpuratus for gene products involved in biomineralization. Our analysis has greatly expanded the collection of biomineralization-related proteins. We have found that these proteins are often members of small families encoded by genes that are clustered in the genome. Most of the proteins are sea urchin-specific; that is, they have no apparent homologues in other invertebrate deuterostomes or vertebrates. Similarly, many of the vertebrate proteins that mediate mineral deposition do not have counterparts in the S. purpuratus genome. Our findings therefore reveal substantial differences in the primary sequences of proteins that mediate biomineral formation in echinoderms and vertebrates, possibly reflecting loose constraints on the primary structures of the proteins involved. On the other hand, certain cellular and molecular processes associated with earlier events in skeletogenesis appear similar in echinoderms and vertebrates, leaving open the possibility of deeper evolutionary relationships.

Proteins and saccharides of the sea urchin organic matrix of mineralization: characterization and localization in the spine skeleton.

Properties of the echinoderm skeleton are under biological control, which is exerted in part by the organic matrix embedded in the mineralized part of the skeleton. This organic matrix consists of proteins and glycoproteins whose carbohydrate component is specifically involved in the control mechanisms. The saccharide moiety of the organic matrix of the spines of the echinoid Paracentrotus lividus was characterized using enzyme-linked lectin assays (ELLAs). O-glycoproteins, different types of complex N-glycoproteins, and terminal sialic acids were detected. Sialic acids are known to interact with Ca ions and could play an important role in the mineralization process. Some of the carbohydrate components detected by ELLAs as well as two organic matrix proteins (SM30 and SM50) were localized within different subregions of the spine skeleton using field-emission scanning electron microscopy. The mappings show that some of these components are not homogeneously distributed in the different skeletal subregions. For example, some N-glycoproteins were preferentially located in the putative amorphous subregion of the skeleton, whereas some O-glycoproteins were localized in the subregion where skeletal growth is inhibited. These results suggest that the biological control exerted on the skeletal properties can be partly modulated by local differences in the organic matrix composition.

Roles of two homotetrameric kinesins in sea urchin embryonic cell division.

To improve our understanding of the roles of microtubule cross-linking motors in mitosis, we analyzed two sea urchin embryonic kinesin-related proteins. It is striking to note that both of these proteins behave as homotetramers, but one behaves as a more compact molecule than the other. These observations suggest that these two presumptive motors could cross-link microtubules into bundles with different spacing. Both motors localize to mitotic spindles, and antibody microinjection experiments suggest that they have mitotic functions. Thus, one of these kinesin-related proteins may cross-link spindle microtubules into loose bundles that are "tightened" by the other.

Expression of spicule matrix proteins in the sea urchin embryo during normal and experimentally altered spiculogenesis.

During its embryonic development, the sea urchin embryo forms an endoskeletal calcitic spicule. This instance of biomineralization is experimentally accessible and also offers the advantage of occurring within a developmental context. Here we investigate the time course of appearance and localization of two proteins among the four dozen that constitute the protein matrix of the skeletal spicule. SM50 and SM30 have been studied in some detail, and polyclonal antisera have been prepared against them (C. E. Killian and F. H. Wilt, 1996, J. Biol. Chem. 271, 9150-9159). Using these antibodies we describe here the localization and time course of accumulation of these two proteins in Strongylocentrotus purpuratus, both in the intact embryo and in micromere cultures. We also investigate the disposition of the matrix proteins, SM50, SM30, and PM27, in the three-dimensional spicule by studying changes in protein localization during experimental manipulation of isolated skeletal spicules. We conclude that SM50, PM27, and SM30 probably play different roles in biomineralization, based on their localization and patterns of expression. It is unlikely that these proteins are solely structural elements within the mineral. SM50 and PM27 may play a role in defining the extracellular space in which spicule deposition occurs, while SM30 may play a role in secretion of spicule components. Finally, we report on the effects of serum on expression of some primary mesenchyme-specific proteins in micromere cultures; withholding serum severely depresses accumulation of SM30 but has only modest effects on the accumulation of other proteins.

Matrix and mineral in the sea urchin larval skeleton.

The endoskeletal spicules of sea urchin larvae are composed of calcite, a surrounding extracellular matrix, and small amounts of occluded matrix proteins. The spicules are formed by primary mesenchyme cells (PMCs) in the blastocoel of the embryo, where they adopt stereotypical locations, thereby specifying where spicules will form. PMCs also fuse to form cytoplasmic cords connecting the cell bodies, and it is within the cords that spicules arise. The mineral phase contains 5% Mg as well as Ca, and about 0.1% of the mass is protein. The matrix and mineral form concentric plies, and the composite has different physical properties than those of pure calcite. The calcite diffracts as a single crystal and is composed of well-ordered, but not perfectly ordered, microdomains. There is evidence for adsorption of matrix proteins to specific crystal faces at domain boundaries, which may help regulate crystal growth and texture. Immature spicules contain considerable precipitated amorphous CaCO3, and PMCs also have vesicles that contain amorphous CaCO3. This suggests the hypothesis that the cellular precursor to the spicules is actually amorphous CaCO3 stabilized in the cell by protein. The spicule s enveloped by the PMC cord, but is topologically exterior to the cell. The PMC plasmalemma is tightly applied to the developing spicules, except perhaps at the elongating tip. The characteristics, localization, and possible function of the four identified matrix proteins are discussed. SM50, SM37, and PM27 all primarily enclose the mineral, though small amounts are occluded. SM30 is found in cellular vesicles and is probably the principal occluded protein of the spicule.

Ultrastructural localization of proteins involved in sea urchin biomineralization.

Three skeletal tissues of the adult echinoid Paracentrotus lividus (the pedicellaria primordium, the test, and the tooth) were immunolabeled with three sera raised against the total mineralization organic matrix and two specific matrix proteins (SM30 and SM50) from the embryo of the echinoid Strongylocentrotus purpuratus. Two conventional chemical fixation protocols and two high-pressure freezing/freeze-substitution protocols were tested. One conventional protocol is recommended for its good preservation of the ultrastructure, and one high-pressure freezing/freeze-substitution protocol is recommended for its good retention of antigenicity. Immunolabeling was obtained in the three adult tissues. It was confined to the active skeleton-forming cells and to the structured organic matrix. The results indicate that the matrix proteins follow the classical routes of secretory protein assembly and export and suggest that SM30 and SM50 are a part of the tridimensional network formed by the organic matrix before the onset of mineralization. They show that the genetic program of part of skeletogenesis is conserved among different calcification models and developmental stages.

Functional organization of DNA elements regulating SM30alpha, a spicule matrix gene of sea urchin embryos.

The SM30a gene encodes a protein in the embryonic endoskeleton of the sea urchin Strongylocentrotus purpuratus, and is specifically expressed in the skeletogenic primary mesenchyme cell lineage. To clarify the mechanism for the differentiation of this cell lineage, which proceeds rather autonomously in the embryo, regulation of the SM30alpha gene was investigated previously and it was shown that the distal DNA region upstream of this gene from - 1.6 to - 1.0 kb contained numerous negative regulatory elements that suppressed the ectopic expression of the gene in the gut. Here we study the influence of the proximal region from - 303 to + 104 bp. Analysis of the expression of reporter constructs indicated that a strong positive enhancer element existed in the region from -142 to -105bp. This element worked both in forward and reverse orientations and additively when placed tandemly upstream to the reporter gene. In addition, other weaker positive and negative regulatory sites were also detected throughout the proximal region. Electrophoretic gel mobility shift analyses showed that multiple nuclear proteins were bound to the putative strong enhancer region. One of the proteins binding to this region was present in ear y blastulae, a time when the SM30 gene was still silent, but it was not in prism embryos actively expressing the gene. The binding region for this blastula-specific protein was narrowed down to the region from - 132 to -122 bp, which included the consensus binding site for the mammalian proto-oncogene product, Ets. Two possible SpGCF1 binding sites were identified in the vicinity of the enhancer region. This information was used to make a comparison of the general regulatory architecture of genes that contribute to the formation of the skeletal spicule.

Matrix metalloproteinase inhibitors disrupt spicule formation by primary mesenchyme cells in the sea urchin embryo.

The primary mesenchyme cells of the sea urchin embryo construct an elaborate calcareous endoskeletal spicule beginning at gastrulation. This process begins by ingression of prospective primary mesenchyme cells into the blastocoel, after which they migrate and then fuse to form a syncytium. Skeleton deposition occurs in spaces enclosed by the cytoplasmic cables between the cell bodies. Experiments are described which probe the role of proteases in these early events of spicule formation and their role in the continued elaboration of the spicule during later stages of embryogenesis. We find that several inhibitors of metalloproteinases inhibit the continuation of spiculogenesis, an effect first reported by Roe et al. (Exp. Cell Res. 181, 542-550, 1989). A detailed study of one of these inhibitors, BB-94, shows that fusion of primary mesenchyme cells still occurs in the presence of the inhibitor and the formation of the first calcite granule is not impeded. Continued elaboration of the spicule, however, is completely stopped; addition of the inhibitor during the active elongation of the spicule stops further elongation immediately. Removal of the inhibitor allows resumption of spicule growth. The inhibition is accompanied by almost complete cessation of massive Ca ion transport via the primary mesenchyme cells to the spicule. The inhibitor does not prevent the continued synthesis of several spicule matrix proteins. Electron microscopic examination of inhibited primary mesenchyme cells shows an accumulation of characteristic vesicles in the cytoplasm. Gel zymography demonstrates that although most proteases in homogenates of primary mesenchyme cells are not sensitive to the inhibitor in vitro, a protease of low abundance detectable in the medium of cultured primary mesenchyme cells is inhibited by BB-94. We propose that the inhibitor is interfering with the delivery of precipitated calcium carbonate and matrix proteins to the site(s) of spicule growth.

Looking into the sea urchin embryo you can see local cell interactions regulate morphogenesis.

The transparent sea urchin embryo provides a laboratory for study of morphogenesis. The calcareous endoskeleton is formed by a syncytium of mesenchyme cells in the blastocoel. The locations of mesenchyme in the blastocoel, the size of the skeleton, and even the branching pattern of the skeletal rods, are governed by interactions with the blastula wall. Now Guss and Ettensohn show that the rate of deposition of CaCO3 in the skeleton is locally controlled in the mesenchymal syncytium, as is the pattern of expression of three genes involved in skeleton formation. They propose that short range signals emanating from the blastula wall regulate many aspects of the biomineralization process.

Oral-aboral ectoderm differentiation of sea urchin embryos is disrupted in response to calcium ionophore.

Intracellular signaling mediated by calcium ions has been implicated as important in controlling cell activity. The ability of calcium ionophore (A23187), which causes an increase in calcium ion concentration in the cytoplasm, to alter the pattern of differentiation of cells during sea urchin development was examined. The addition of A23187 to embryos for 3 h during early cleavage causes dramatic changes in their development during gastrulation. Using tissue-specific cDNA probes and antibodies, it was shown that A23187 causes the disruption of oral-aboral ectoderm differentiation of sea urchin embryos. The critical period for A23187 to disturb the oral-aboral ectoderm differentiation is during the cleavage stage, and treatment of embryos with A23187 after that time has little effect. The A23187 does not affect the formation of the three germ layers. These results indicate that intracellular signals mediated by calcium ions may play a key role in establishment of the oral-aboral axis during sea urchin development.

Expression of spicule matrix protein gene SM30 in embryonic and adult mineralized tissues of sea urchin Hemicentrotus pulcherrimus.

We have isolated a cDNA clone for spicule matrix protein, SM30, from sea urchin Hemicentrotus pulcherrimus and have studied the expression of this gene in comparison with that of another spicule matrix protein gene, SM50. In cultured micromeres as well as in intact embryos transcripts of SM30 were first detectable around the onset of spicule formation and rapidly increased with the growth of spicules, which accompanied accumulation of glycosylated SM30 protein(s). When micromeres were cultured in the presence of Zn2+, spicule formation and SM30 expression were suppressed, while both events resumed concurrently after the removal of Zn2+ from the culture medium. Expression of SM50, in contrast, started before the appearance of spicules and was not sensitive to Zn2+. Differences were also observed in adult tissues; SM30 mRNA was detected in spines and tube feet but not in the test, while SM50 mRNA was apparent in all of these mineralized tissues at similar levels. These results strongly suggest that the SM30 gene is regulated by a different mechanism to that of the SM50 gene and that the products of these two genes are differently involved in sea urchin biomineralization. A possible role of SM30 protein in skeleton formation is discussed.

Characterization of the proteins comprising the integral matrix of Strongylocentrotus purpuratus embryonic spicules.

In the present study, we enumerate and characterize the proteins that comprise the integral spicule matrix of the Strongylocentrotus purpuratus embryo. Two-dimensional gel electrophoresis of [35S]methionine radiolabeled spicule matrix proteins reveals that there are 12 strongly radiolabeled spicule matrix proteins and approximately three dozen less strongly radiolabeled spicule matrix proteins. The majority of the proteins have acidic isoelectric points; however, there are several spicule matrix proteins that have more alkaline isoelectric points. Western blotting analysis indicates that SM50 is the spicule matrix protein with the most alkaline isoelectric point. In addition, two distinct SM30 proteins are identified in embryonic spicules, and they have apparent molecular masses of approximately 43 and 46 kDa. Comparisons between embryonic spicule matrix proteins and adult spine integral matrix proteins suggest that the embryonic 43-kDa SM30 protein is an embryonic isoform of SM30. An adult 49-kDa spine matrix protein is also identified as a possible adult isoform of SM30. Analysis of the SM30 amino acid sequences indicates that a portion of SM30 proteins is very similar to the carbohydrate recognition domain of C-type lectin proteins.

Two cis elements collaborate to spatially repress transcription from a sea urchin promoter.

The expression pattern of many territory-specific genes in metazoan embryos is maintained by an active process of negative spatial regulation. However, the mechanism of this strategy of gene regulation is not well understood in any system. Here we show that reporter constructs containing regulatory sequence for the SM30-alpha gene of Stronglyocentrotus purpuratus are expressed in a pattern congruent with that of the endogenous SM30 gene(s), largely as a result of active transcriptional repression in cell lineages in which the gene is not normally expressed. Chloramphenicol acetyl transferase assays of deletion constructs from the 2600-bp upstream region showed that repressive elements were present in the region from -1628 to -300. In situ hybridization analysis showed that the spatial fidelity of expression was severely compromised when the region from -1628 to -300 was deleted. Two highly repetitive sequence motifs, (G/A/C)CCCCT and (T/C)(T/A/C)CTTTT(T/A/C), are present in the -1628 to -300 region. Representatives of these elements were analyzed by gel mobility shift experiments and were found to interact specifically with protein in crude nuclear extracts. When oligonucleotides containing either sequence element were co-injected with a correctly regulated reporter as potential competitors, the reporter was expressed in inappropriate cells. When composite oligonucleotides, containing both sequence elements, were fused to a misregulated reporter, the expression of the reporter in inappropriate cells was suppressed. Comparison of composite oligonucleotides with oligonucleotides containing single constituent elements show that both sequence elements are required for effective spatial regulation. Thus, both individual elements are required, but only a composite element containing both elements is sufficient to function as a tissue-specific repressive element.

Genomic organization of a gene encoding the spicule matrix protein SM30 in the sea urchin Strongylocentrotus purpuratus.

We report the characterization of a genomic clone containing portions of two tandemly arranged genes that encode a spicule matrix protein, SM30, of the sea urchin Strongylocentrotus purpuratus. The isolated 18.4-kilo-base genomic clone contains the complete genomic sequence of one SM30 gene, designated SM30-alpha, and a portion of another SM30 gene, designated SM30-beta. Southern blot analysis shows that SM30 protein is encoded by a small gene family of two to four members. RNase protection assays indicate that the SM30-alpha gene is expressed at the time of spicule formation in the sea urchin embryo. In addition, mapping of SM30-alpha shows that a large single intron interrupts the coding sequence. Comparison of the nucleic acid and amino acid sequences of the SM30-alpha genomic sequence and the previously isolated SM30 cDNA reveals them to be very similar, but not identical. We also demonstrate that 2.6 kilobases of upstream sequence of SM30-alpha are sufficient to direct primary mesenchyme cell-specific expression of a reporter gene construct.

Field research on veterinary problems in group-housed sows--a survey of lameness.

Investigations on two experimental farms with group-housing revealed that lameness occurred mainly at the hind legs of sows, with a higher incidence in first parity sows. The highest incidence of lameness was seen during the first 2 months of gestation and the highest prevalence in the last 2 months of gestation. No relation could be established between lameness of a sow in the last month of gestation and reproduction results. The incidence of claw lesions increased too, during the group-housing during pregnancy and was higher compared to sows kept in crates. This increase was clearest in first parity sows. In addition the observations suggest a pattern in the course of development of lesions. Claw lesions mainly occur on the outer claws of a sow. For two types of lesions left-right symmetry has been established for both the hind and the front legs. On the level of the individual animal there was no relation between the incidence of claw lesions and lameness.

Cell interactions in invertebrate development.

By utilizing both genetics and physical manipulations, studies on invertebrate embryos have provided substantial information on how cell interactions influence cell fate during development. The picture emerging from these studies suggest that while there may be varied and intricate patterns of cell interactions during the development of different embryos, the signaling molecules that convey positional information across cell membranes have been highly conserved.

Review of health problems in group-housed sows, with special emphasis on lameness.

In the Netherlands, there is a trend towards housing gestating sows in groups. Vulva biting and lameness have been described as major health problems in group-housed sows. Besides these problems, the effects of group housing on morbidity, reproduction parameters and the occurrence of infectious disease have been investigated. The literature is reviewed, with special emphasis on lameness.

A role for regulated secretion of apical extracellular matrix during epithelial invagination in the sea urchin.

Epithelial invagination, a basic morphogenetic process reiterated throughout embryonic development, generates tubular structures such as the neural tube, or pit-like structures such as the optic cup. The 'purse-string' hypothesis, which proposes that circumferential bands of actin microfilaments at the apical end of epithelial cells constrict to yield a curved epithelial sheet, has been widely invoked to explain invaginations during embryogenesis. We have reevaluated this hypothesis in two species of sea urchin by examining both natural invagination of the vegetal plate at the beginning of gastrulation and invagination induced precociously by Ca2+ ionophore. Neither type of invagination is prevented by cytochalasin D. In one species, treatment with A23187 three hours before the initiation of invagination resulted in the deposition of apical extracellular matrix at the vegetal plate, rather than invagination. This apical matrix contains chondroitin sulfate, as does the lumen of the archenteron in normal gastrulae. When the expansion of this secreted matrix was resisted by an agarose gel, the vegetal plate buckled inward, creating an archenteron that appeared 3-4 hours prematurely. Pretreatment with monensin, which blocks secretion, inhibits both Ca2+ ionophore-stimulated folding and natural invagination, demonstrating that secretion is probably required for this morphogenetic event. These results indicate that alternatives to the purse-string hypothesis must be considered, and that the directed deposition of extracellular matrix may be a key Ca(2+)-regulated event in some embryonic invaginations. A bending bilayer model for matrix-driven epithelial invagination is proposed in which the deposition of hygroscopic material into a complex, stratified extra-cellular matrix results in the folding of an epithelial sheet in a manner analagous to thermal bending in a bimetallic strip.