ARTICLE |
Correspondence to: Laurent Ameye, Laboratoire de Biologie Marine, CP 160 / 15, Université Libre de Bruxelles, 50 Av. F.D. Roosevelt, 1050 Bruxelles, Belgium.
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Summary |
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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/freezesubstitution protocols were tested. One conventional protocol is recommended for its good preservation of the ultrastructure, and one high-pressure freezing/freezesubstitution 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. (J Histochem Cytochem 47:11891200, 1999)
Key Words: biomineralization, sea urchin, immunocytolabeling, organic matrix, tooth, skeleton, high-pressure freezing, freezesubstitution
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Introduction |
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Sea urchin (echinoid) larvae have a simple skeleton consisting of a few spicules. These spicules develop anastomosing trabeculae and support the food capture device, the larval arms (
The larval spicules and adult skeleton share common characteristics. Both are composed of magnesium calcite with a 0.1% (w/w) mineralization organic matrix and both are formed in a vacuole, the calcification site, enclosed in a syncytial pseudopodium generated by the skeleton-forming cells (for review see
Two genes controlling the production of intraspicular proteins, i.e., SM30 and SM50, are known from larvae of several echinoid species. SM30 is a 30-kD acidic N-glycoprotein (
Accordingly, the goal of the present work was to investigate the ultrastructural localization of SM30 and SM50, using immunogold labeling, in three adult mineralized tissues, i.e., the primordium of an outer appendage (the pedicellaria), the test, and the tooth.
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Materials and Methods |
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Sampling
Pedicellaria primordia and test plates were dissected from individuals of Paracentrotus lividus ranging from 0.8 to 1.2 cm in ambital diameter (i.e., the equatorial diameter of the hemispherical test); teeth were dissected from P. lividus with a minimal ambital diameter of 3 cm. The former echinoids were obtained from the aquaculture in the Centre de Recherches et d'Etudes Côtières (University of Caen, France), whereas the latter were collected intertidally in Morgat (Brittany, France). All the echinoids were kept in a closed-circuit aquarium (35, 13C) and fed dried seaweed (Lessonia sp.) once a week. The primordia were processed by conventional chemical fixation (CF) and by high-pressure freezing/freezesubstitution (HPF/FS), the teeth were processed by HPF/FS, and the test plates by CF. All the samples were dissected under a dissecting microscope. The sampled primordia ranged from 100 to 200 µm in size. For CF, the primordia were collected with the underlying test plates using scissors. For HPF/FS, the primordia were cut away from the underlying test plates with a scalpel. (For practical reasons, the test plates could not be processed by HPF; the small size of the holders used in the high-pressure freezer prevents their handling). The teeth were collected with scissors.
Conventional Chemical Fixation (CF)
Two protocols were tested on the pedicellaria primordia and on the test plates. The first protocol (CF1) had been demonstrated to be optimal for the preservation of the spatial organization of the organic matrix within the calcification sites (
The test plates were decalcified, to allow sectioning by the double-embedding method of
High-pressure Freezing (HPF)
HPF was used on the pedicellaria primordia and on the plumulae of the teeth. The samples were sandwiched between two aluminum plates and cryoimmobilized with a high-pressure freezer (HPM 010; Balzers Union, Balzers, Liechtenstein) (
FreezeSubstitution (FS)
The frozen sandwiches were opened under liquid nitrogen and the samples (still attached to one of the plates) were freeze-substituted using a freeze-substitution unit (Balzers FSU 010). Two protocols were tested. The first one (HPF/FS1) attempted to preserve the spatial organization of the organic matrix, whereas the second (HPF/FS2) attempted to better preserve the antigenicity.
In the HPF/FS1 protocol, the samples were freeze-substituted in anhydrous acetone with 0.2% gallic acid (low molecular weight tannic acid; C7H6O5; Fluka, Buchs, Switzerland) and 6% glutaraldehyde (Polysciences) for 48 hr at 183K and 12 hr at 213K, washed three times at 213K with anhydrous acetone, transferred at 213K to anhydrous acetone with 2% osmium tetroxide (Johnson Matthey; Herts, UK), and kept for 30 hr at 213K, 8 hr at 243K, and 1 hr at 273K. The samples were then washed three times in anhydrous acetone and embedded stepwise in Epon/Araldite (30%, 70%, 100%) (Fluka). The infiltration times were 3 hr at each resin concentration. The final polymerization was completed at 333K.
In the HPF/FS2 protocol, the samples were freeze-substituted in anhydrous ethanol with 0.5% uranyl acetate (Sigma; St Louis, MO) at 183K, 213K, and 243K for 8 hr at each step (
Microtomy, Immunolabeling, and Electron Microscopy
Ultrathin sections were cut on an Ultracut UCT Leica with diamond knives (Diatome; Biel, Switzerland), picked up on gold grids, and immunolabeled. Three primary sera (anti-total matrix, anti-SM30, and anti-SM50) raised in rabbit against larval organic matrix components of the echinoid Strongylocentrotus purpuratus were used. The characterization of the anti-total matrix serum was reported in
Anti-total matrix, anti-SM30, and anti-SM50 single immunolabeling was applied to sections placed on Formvar-coated or uncoated grids. To block unspecific staining, the sections were immersed in drops of 1 M NH4Cl in PBS buffer (0.01 M, 0.15 M NaCl) for 1 hr. They were then rinsed three times with PBS and incubated in primary antibodies diluted 1:500 for 1 hr at 20C. After six rinses with PBS, goat anti-rabbit IgG conjugated to 10- or 15-nm gold particles (Biocell; Cardiff, UK) diluted 1:100 with PBS (0.01 M, NaCl 0.15 M) plus 0.1% Tween-20 (Sigma) and 1% BSA (Sigma) was applied to the grids for 1 hr at 20C. The sections were then rinsed six times with PBS and three times with Milli-Q (Millipore; Bedford, MA) water.
Immunolabeling omitting the primary antibody was used as control. All the sections were stained with uranyl acetate and lead citrate and examined in a Philips EM 300 or a JEOL 2000 FX II transmission electron microscope.
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Results |
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Preservation of Antigenicity According to the Fixation Protocol
Three primary sera (anti-total matrix, anti-SM30, and anti-SM50) raised against larval organic matrix components of the echinoid S. purpuratus were used on pedicellaria primordia, test plates, and teeth of the echinoid P. lividus. The anti-total matrix serum is known to react with at least eight components of the larval organic matrix, whereas the anti-SM30 and anti-SM50 sera are specific to, respectively, the SM30 and SM50 proteins (
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Immunolabeling of the Pedicellaria Primordia
The pedicellariae are small appendages consisting of three movable jaws on a stalk. They form on the test plates within primordia, which are made up of a cluster of tightly packed dermal cells covered by the epidermis (
In the primordia, the anti-total matrix serum labeled the sclerocytes, the sclerocyte pseudopodia, and the organic matrix of the calcification sites (Figure 1). The epidermis (Figure 1) and the undifferentiated cells of the primordia (Figure 2) were not labeled. In the sclerocytes, the labeling was observed on the trans side of the Golgi stacks, in the electron-translucent Golgi vesicles, and along the plasma membranes (Figure 3 and Figure 4) but not in the medium gray vesicles (Figure 1). In the sclerocyte pseudopodia, the labeling was localized along the inner and outer pseudopodium membrane and in the vesicles (Figure 5). The anti-SM30 and anti-SM50 labeling was weaker than the anti-total matrix labeling (compare Figure 6 and Figure 8 with Figure 1). They presented the same localization and were observed in the translucent Golgi vesicles of the sclerocyte cell bodies (Figure 6 Figure 7 Figure 8), in the translucent vesicles of the sclerocyte pseudopodia (Figure 6), on the organic matrix of the calcification sites, and along the edges of the perforations within the calcification sites (Figure 9). The latter labeling did not result from a nonspecific effect due to the perforation. Indeed, no labeling was observed along the accidental perforations localized outside calcification sites. With each of the three sera separately, the basal lamina and the extracellular organic matrix of the primordia were lightly labeled (Figure 1).
Immunolabeling of the Test Plates
The test plates comprise a tridimensional mineral network of trabeculae (the stereom) which delimits an internal and complementary network filled by a connective tissue (the stroma, Figure 14) containing some sparse sclerocytes. The trabeculae are surrounded by an outer organic matrix layer; the trabecular coat. In our sections, no sclerocyte cell body was observed and the trabecular coat was the only structure to be labeled (Figure 12 Figure 13 Figure 14 Figure 15). It was labeled uniformly by the anti-total matrix serum and by patches with the anti-SM30 and anti-SM50 sera (Figure 13 and Figure 14). For the anti-SM30 labeling, the labeled patches were often bordered by electron-lucent areas of the stereom (Figure 13).
Immunolabeling of the Teeth
The plumula of the tooth contains two main cell types, the odontoblasts and the preodontoblasts (
The anti-total matrix serum labeled the odontoblasts and their pseudopodia (Figure 16), the preodontoblasts (Figure 17 and Figure 18), the organic matrix of the primary plates (Figure 19), and the margins of the perforations in the calcification sites (Figure 16). No labeling was observed on the extracellular matrix or along accidental perforations outside of the calcification sites. This indicates that the labeling along the perforations of the calcification sites did not result from a nonspecific effect caused by the perforation. In the odontoblasts and their pseudopodia, labeling was observed on the plasma membranes of the odontoblasts and on the inner and outer membranes and the hyaloplasm of the pseudopodia (Figure 16). In the preodontoblasts, labeling was observed in the Golgi stacks, in the medium electron-dense Golgi vesicles, and along the plasma membranes (Figure 17 and Figure 18).
The anti-SM30 and anti-SM50 labeling was practically identical. Their intensity was lower than the intensity of the anti-total matrix labeling (compare Figure 21 and Figure 22 with Figure 16 and Figure 23 with Figure 17). The anti-SM30 and anti-SM50 labeling was confined to the Golgi apparatus of the preodontoblasts (Figure 23) and to the organic matrix of the calcification sites (Figure 21 and Figure 22). No labeling was observed in the extracellular matrix or along the plasma membrane of the odontoblasts. The only difference between the anti-SM30 and the anti-SM50 labeling was that the organic matrix was more weakly labeled with the anti-SM30 serum than with the anti-SM50 serum (compare Figure 22 and Figure 21).
A summary of the structures labeled by the three sera studied is presented in Table 1. Control experiments omitting the primary sera showed that secondary antibodies induced a background, restricted to some components of the extracellular matrix and to the epidermic basal lamina, in the primordia and the test plates (Figure 10, Figure 11, and Figure 20).
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Discussion |
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Four protocols, two conventional chemical protocols (CF1 with tannic acid and CF2 without tannic acid) and two high-pressure freezing/freeze-substitution protocols (HPF/FS1 with gallic acid and HPF/FS2 without gallic acid), were tested. The CF1 and HPF/FS1 protocols better preserved the spatial organization of the organic matrix but prevented any labeling. These protocols contain tannic or gallic acid, which are known to improve the preservation of the spatial organization of the echinoid organic matrix (
Except for the background due to labeling by the secondary antibodies of the extracellular matrix and epidermal basal lamina (in the primordia and the test plates), the anti-total matrix, anti-SM30, and anti-SM50 labeling was confined to the skeleton-forming cells and to the mineralization organic matrix (i.e., the organic material within the calcification sites) (see Table 1). The sclerocytes (in the pedicellaria primordium) and the preodontoblasts (in the tooth) had their Golgi stacks, Golgi vesicles and vesicles of the pseudopodia labeled by the three sera (Table 1). This indicates that the adult matrix glycoproteins follow the classical routes of secretory glycoprotein assembly and export, as already demonstrated in echinoid larvae (
The plasma membranes of all three skeleton-forming cells studied (sclerocytes, preodontoblasts, and odontoblasts) were labeled by the anti-total matrix serum but not by the anti-SM30- and anti-SM50-specific sera. This labeling is not related to the secretion route of the glycoproteins. It might be due to a crossreaction of the polyclonal serum with the msp 130 protein. The msp 130 is a membrane glycoprotein present in both echinoid larvae and adults (
In this study, the odontoblasts did not present well-developed Golgi apparatus and were not labeled by the anti-SM30 and anti-SM50, indicating that they were not involved at this time in the synthesis of these important matrix proteins. This is surprising because these cells closely surround still growing tooth plates (
In the tooth, anti-SM30 and anti-SM50 labeling is restricted (within the calcification site) to the structured organic matrix that is pre-formed before the onset of biomineralization (see
Unlike
The present results demonstrate the first immunolabeling of organic matrix components in the adult echinoid skeleton and tooth. The presence of SM30 and the SM50 in postmetamorphic tegumentary skeleton has previously been documented from RNA blot experiments (
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Acknowledgments |
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Supported by a FRIA grant (to LA) by FRFC grant 2.4512.95, and by NFSR grant 1.5.201.98. PhD is a Research Associate of the NFSR (Belgium). Contribution from the Centre Interuniversitaire de Biologie Marine (CIBIM).
We thank Ph. Grosjean of the Centre de Recherches et d'Etudes Côtières (University of Caen) for providing the echinoids. LA is grateful for the hospitality shown to him by Professor Muëller and colleagues while a visitor at Laboratory for Electron Microscopy 1 (ETH, Zürich, Switzerland) to carry out the cryotechnical part of this work.
Received for publication November 17, 1998; accepted April 6, 1999.
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