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Cell Biology International (2005) 29, 952958 (Printed in Great Britain)
Mesogleal cells of the jellyfish Aurelia aurita are involved in the formation of mesogleal fibres
Tatiana Shaposhnikovaa*, Ivan Matveevb, Tatiana Naparac and Olga Podgornayab
aDepartment of Cytology and Histology, Faculty of Biology and Soil Sciences, St-Petersburg State University, Universitetskaja nab., 7/9, St-Petersburg 199034, Russia
bInstitute of Cytology RAS, Tikhoretsky pr, 4, St-Petersburg 194064, Russia
c3Fiona LTD, Podolskaja ul, 40, St-Petersburg 198013, Russia
The extracellular matrix of the jellyfish Aurelia aurita (Scyphozoa, Cnidaria), known as the mesoglea, is populated by numerous mesogleal cells (Mc). We determined the pattern of the Mc and the mesoglea, raised polyclonal antibodies (RA47) against the major mesogleal protein pA47 (47
Keywords: Extracellular matrix, Cnidaria, Aurelia aurita, Mesogleal cells, Mesoglea.
Comparative analysis of extracellular matrix (ECM) structure and synthesis gives information about the formation, function and evolution of ECM. The Cnidaria phylum represents one of the most interesting models for this kind of research due to its phylogenetic position. An extracellular substance, the mesoglea, is situated between epidermal and gastrodermal layers. The mesoglea is equivalent to the ECM of vertebrates and, as in vertebrates, it performs important functions (Chapman, 1974; Sarras and Deutzmann, 2001). Essentially it serves as a skeleton that maintains the specific animal body structure, and takes part in the transport and storage of nutrients (Chapman, 1966; Bouillon and Coppois, 1977; Weber and Schmid, 1985). The ECM is also involved in buoyancy regulation (Denton, 1963; Mackay, 1969). The forces generated by the contraction of the umbrella muscle system cause propulsive swimming of the animal at the medusa stage (Chapman, 1966; Gladfelter, 1972; Weber and Schmid, 1985; Demont and Gosline, 1988), while the elasticity of the ECM counteracts the muscle contraction (Bouillon and Coppois, 1977). The mesoglea plays a key role in the control of cell migration and morphogenetic processes (for review, see Schmid, 1992; Sarras et al., 1993; Frank and Rinkevich, 1999; Shimizu et al., 2002; Kleinman et al., 2003). Factors affecting the structural integrity of the ECM and interfering with cell–substrate interactions alter the state of cellular differentiation (for review, see Schmid, 1992; Schmid et al., 1993).
Two types of fibres in the jelly-like ground substance have been described in cnidarian ECM (Bouillon and Coppois, 1977). Evidence for the existence of collagen fibrils was reported in the late 1970s (Chapman, 1974; Bouillon and Coppois, 1977; Adams, 1978). A collagen fibre system lends resilience to the mesoglea (Adams, 1978; Franc et al., 1984; Miura and Kimura, 1985; Napara et al., 1996a,b). Another type of fibril with a ramified and undulating appearance was revealed by electron microscopy (Chapman, 1959). It was found that these fibrils consisted of a central core encircled by a fine fibrillary meshwork. Because of their external similarities, these fibrils were designated “elastic” fibrils and were initially thought to consist of elastin (Bouillon and Vandermeerssche, 1956; Bouillon, 1959; Demont and Gosline, 1988). However, phylogenetic analysis showed that elastin was present only in vertebrates (for review, see Rosenbloom et al., 1993).
Some cnidarian ECM proteins have been characterized, e.g. several different types of fibrillary collagens (Franc et al., 1984; Miura and Kimura, 1985; Sarras et al., 1991; Tillet et al., 1996; Deutzmann et al., 2000). In Hydra sp., immunoreactive non-fibrillary collagen type IV, fibronectin, heparan sulfate and metalloproteinases have been reported (Sarras et al., 1991; Fowler et al., 2000; Leontovich et al., 2000). Laminin was identified by immunohistology and rotary shadowing in Podocoryne carnea (Beck et al., 1989) and cloned from Hydra sp. (Sarras et al., 1994; Zhang et al., 2002). Another protein involved in tightening the ECM network is fibrillin (for review, see Har-El and Tanzer, 1993). Fibrillin is a 330–340
The mesoglea of some species of the Scyphozoa and Anthozoa classes contains free motile cells – mesogleal cells (Chapman, 1966, 1974). The origin of these cells has not been elucidated and little is known about their functions. Their capacity for amoeboid movement and active phagocytosis has been described (Metchnikoff, 1880; Napara et al., 1994).
The mesoglea, with its mesogleal cells, has been shown to be similar to the connective tissues of more advanced animals (Zawarzin, 1945; Chapman, 1974). Its development has been studied by several methods, including autoradiography after 3H-proline incorporation (Hausman and Burnett, 1971; Singer, 1974; Young, 1974; Franc et al., 1984). Even so, none of these methods has shed light on the possible role of mesogleal cells in mesoglea formation.
The “elastic” fibres of the Scyphozoan jellyfish Aurelia aurita contain lysine-rich proteins that bare heavily positive charge. “Elastic” fibre proteins are also enriched in cysteine and, consequently, in S–S bonds (Schmid et al., 1991; Napara et al., 1996a). A. aurita possesses numerous mesogleal cells at the medusa stage of the life cycle (Napara et al., 1994). Therefore, this species was chosen to determine whether mesogleal cells are involved in mesoglea development. We determined the protein pattern of mesogleal cells and the mesoglea, and raised antibodies against one of the major mesogleal proteins. Immunoblots and data from light and electron microscopic immunostaining demonstrate that mesogleal cells are involved in the formation of mesogleal “elastic” fibres.
2 Materials and methods
Jellyfish A. aurita were collected in the vicinity of the White Sea Biological Station of the Zoological Institute RAS “Kartesh” (Chupa Inlet, Kandalaksha Bay in the White Sea) during the summers of 1996–2003. To obtain mesogleal cells (Mc) from the growing medusae, pieces of about 0.5
Hydra vulgaris animals were kindly donated by Dr. M. Samoilovich (Central Roentgeno-Radiological Research Institute, Russia) and stored alive in water or in a freezer at −20
2.2 Gels and antibodies
The protein composition of the preparations was determined using SDS-PAGE (Laemmli, 1970). Western blotting was performed according to Towbin et al. (1979) with modifications. Methanol-activated polyvinylidene difluoride (PVDF) membranes (0.45
Polyclonal antibodies (Ab) against the major mesoglea protein pA47 were raised as previously described (Podgornaya and Shaposhnikova, 1998). The 47
Polypeptide composition (I) and immunoblot with RA47 (II). (I) Coomassie-stained 10% SDS-PAGE. 1 – gastrodermal cells (G); 2 – epidermal cells (E); 3 – mesogleal cells (Mc); 4 – mesoglea (Mes). In parentheses – 3 collagen lines on Mes indicated. Molecular masses of marker proteins (in kDa) are indicated on the left. (II) 1 – E; 2 – Mc; 3 – Mes. 1–3 correspond to lanes 2–4 in (I). Additionally loaded: 4 – Hydra ECM; 5 – the whole Hydra. The molecular masses of the stained polypeptides in addition to p47 are indicated on the right. Serum dilution 1:3000.
Pieces from a growing medusa body were fixed in Bouin solution for 24
For electron microscopy the pieces of mesoglea were fixed in 2.5% glutaraldehyde in phosphate-buffered saline (PBS) with 10% sucrose for 2
For immunostaining, the materials were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in PBS at 10
2.4 Spectrometry and sequencing
Several attempts were made to perform mass-spectrometry (MALDI; Pappin et al., 1993) of pA47 transferred to a PVDF membrane at the mass-spectrometry facility of Laval University (Quebec, Canada). The MALDI procedure for comparing pA80 with pA47 was carried out by the firm “PYNNY”, CJSC, Moscow. The MALDI results were processed using the program MASCOT (http://www.matrixscience.com). N-terminal sequencing and internal fragment sequencing were done in the Protein Resource Center of The Rockefeller University, USA.
Gastrodermal (G) and epidermal (E) cell layers were separated from adjacent mesoglea by collagenase digestion and used directly for SDS-PAGE (Fig. 1 I, G, E). The method for isolating mesogleal cells (Mc) is based on intense collagenase treatment of the mesogleal pieces followed by sedimentation and washing of the cells released from the ECM. The Mc remained viable and motile after isolation. We assume the difference in polypeptide composition of the cell types to be relevant in spite of the different methods of isolation. None of the three cell groups has an obvious major cell-type-specific protein. The major protein in each case is actin, 42
Mesoglea (Mes) contains some Mc, but the proteins of the mature medusa mesoglea preparation are not resolved by one-dimensional SDS-PAGE (Fig. 1 I, Mc, Mes). The major protein components of the mature medusa Mes are more likely to be ECM components. The three zones in the 150
Polyclonal Abs against pA47 stain pA47 itself and some minor bands in the Mes preparations (Fig. 1 II, Mes). In Mc, the pA47 stains weakly. More prominent stained bands have apparent molecular masses of 120
We suppose that pA47 is synthesized as a high molecular mass precursor and undergoes restricted proteolysis during incorporation into the ECM. RA47 stains both the high molecular bands and 45
Immunofluorescence with RA47 shows that fibrils in the Mes and Mc are stained intensely (Fig. 2). The label looks like a circle or a patch with blurred boundaries on cross-sectioned fibrils (Fig. 2 I, arrows). The cells are characterized by similar blurred boundaries. Fig. 2 II reveals granules in the Mc of the growing medusa with very pronounced staining in paraffin sections. The cell contours are blurred and the cells look as though they are surrounded by weakly stained material. The ECM material itself is not heavily stained and fibres of other types are less obvious than those revealed by immunofluorescence (Fig. 2 I). This seems to be due to secretion of granular material from the Mc. In the epidermis, the antigen is distributed throughout the cell cytoplasm but immunostaining is most prominent in the apical parts of the cells (Fig. 2 IIa). The gastrodermis is not stained by RA47, in accordance with the biochemical data (not shown). Immunostaining together with biochemical data (Fig. 1) indicate that both the epidermal and mesogleal cells of A. aurita produce the main Mes protein, which is a component of the “elastic” fibres. Mc definitely participates in pA47 production and might play a role in pA47 modification during “elastic” fibre formation.
Immunostaining of the mesoglea of a growing medusa (diameter 18
The relative sizes of Mc and “elastic” fibres in the Mes are shown in Fig. 3a. At high magnification, Mc has the typical appearance of a protein-synthesizing cell: the endoplasmic reticulum is well developed and there are several granules at different stages of maturation (Fig. 3b). The granules, which are located closer to the cell periphery, look smaller and clearer. The total number of granules in the Mc decreases as the medusa ages, at the end of its season of vegetation (data not shown). A longitudinal section of a typical-looking “elastic” fibre shows its difference from collagen fibres (Fig. 3c). The dense core previously revealed in “elastic” fibres (Bouillon, 1959) can be seen in cross-section (Fig. 3a).
Ultrastructure (a–c) and immunogold staining with RA47 in 1:300 diluted serum (d–f) of mesogleal cells and “elastic” fibres of mature medusa (diameter 26
Immunogold staining of such preparations shows that in Mc most of the label is present in granules of moderate electron density and at the ends of the endoplasmic reticulum tubes; some material on and outside the cell surface is also stained (Fig. 3d). Both the longitudinal section near the “elastic” fibre, with its characteristic turn (Fig. 3e), and the cross-section (Fig. 3f) show the label in the fibre and in material radiating from it. Collagen fibres are not stained (Fig. 3e,f).
The immunogold technique confirms that Mc synthesize and secrete the main Mes protein, which is included in the “elastic” fibres.
The life cycle of the scyphozoan A. aurita is characterized by four main successive stages: planula larva, polyp, ephyra and medusa. When a planktonic larva settles on an appropriate substrate it undergoes metamorphosis into a polyp, the scyphistoma, which can multiply by asexual reproduction. Polyps form ephyra by strobilation. Ephyra can then metamorphose into the medusa, the sexual stage. It is distinguished from the polyp not only by a different shape and anatomical organization, but also by a set of somatic cells and sense organs that are absent in the polyp. The cycle is completed by the liberation of gametes, formation of a zygote and subsequent development of the planula (Hyman, 1940). In northern latitudes, medusae exist in the plankton only in summer and autumn (Grondahl, 1988).
One of the main differences in body plans between the medusa and the polyp is the appearance of a huge amount of mesoglea (ECM) between the two layers, epidermis and gastroderm. In some species mesoglea is populated with Mc, which are capable of amoeboid movement and can uptake foreign particles by phagocytosis (Metchnikoff, 1880; Napara et al., 1994). Recent cellular and molecular data show that some cells situated between ecto- and endodermal layers at the medusa stage, e.g. muscle cells, are derived from the entocodon, a mesoderm-like third cell layer, which separate from the ectoderm (for review, see Seipel and Schmid, 2005). Mc are believed to originate from the epidermis, though it has been suggested that they might arise from both layers (Ivanova-Kazas, 1995; Tardent, 1978). The similarity between the epidermal and Mc polypeptide patterns (Fig. 1 I) argues that Mc originate from the epidermal layer or its derivate.
The Mc of scyphistoma have a low level of proliferation with mitotic index (MI) ≈0.1% (Napara and Chaga, 1992b). This situation changes dramatically during strobilation. The Mc in the upper part of the polyp, which transforms into the ephyra, become mitotically active (MI 2.2%) and look more like medusa Mc. In the young medusa, Mc remain mitotically active (MI 1.5%) and continue to phagocytose. Such proliferative activity makes the whole population self-supporting during medusa growth without the need for incorporation of cells from the established layers (Napara and Chaga, 1992a,b). Eviction of Mc from the epidermis probably occurs once during the first steps of ontogenesis.
Mc display synthetic activity in some species. For example, a labelled 3H-proline precursor became incorporated into Mc and the label was subsequently translocated into the ECM in the actinia Calliactis parasitica; and the Mc of A. aurita actively incorporated 3H-lysine (Young, 1974; Napara et al., 1996b). A. aurita medusa Mc are also typical protein-synthesizing cells (Fig. 3b). The material from the Mc granules is released by exocytosis. The granular material and ECM fibres share common cytochemical characteristics (Napara et al., 1996a). Thus, the Mc granules contain material for forming the ECM fibres. At least 3 types of granules could be identified morphologically in the Mc (Napara et al., 1996a). The Ab against pA47 stains granules inside the Mc as well as the “elastic” fibres of the mesoglea (Figs. 2, 3d–f). The staining of epidermis with RA47 and the absence of staining in the gastroderm also suggest that Mc originate from the epidermal layer.
In the mesoglea, pA47 is involved in the formation of undulating non-collagenous fibres. We did not observe any significant ramification, but they are definitely wavy (Figs. 2, 3). The undulated and ramified fibres in some cnidarian ECMs proved to be digestible with elastase only after oxidation with peracetic acid, and then they could be stained specifically for elastin (Bouillon and Coppois, 1977). This led to the conclusion that oxylatan fibrils were present and they were found to be morphologically similar to the elastic fibres. In invertebrates, proteins unrelated to elastin but with elastic properties have been reported: abductin in bivalvia, elastomer in the octopus, and resilin in arthropods (Rosenbloom et al., 1993). Despite the differences in apparent molecular mass, pA47 could be an ortholog of such a protein.
pA47 was subjected to the MALDI procedure and no analogue of it was found in the world databases. Maximal, but not reliable, similarity scores were obtained for the hypothetical Caenorhabditis elegans protein CBG 13184. Since pA47 could not be identified in the mass-spectra of its polypeptide profile, MALDI failed to answer the question of whether the 80
An attempt to determine the N-terminal sequence showed that the N-terminus was blocked, which also suggested post-translational protein modification during fibre formation (Figs. 1 II, 3d–f). The internal pA47 fragment was sequenced. A 7-residue sequence (YTFIENR) did not correspond to any protein or peptide sequence in the protein databases. A primer designed on the basis of this peptide, together with an arbitrary primer, was the foundation for cDNA library screening.
Our data indicate that pA47, which is involved in forming the “elastic” fibres of the mesoglea, is synthesized as high molecular mass precursor(s) by Mc and, to a lesser extent, the epidermis. Biochemical and morphological data suggest that Mc originate from the epidermis but not the gastroderm. Neither pA47 nor its precursors exist in the world protein databases; hence, gene cloning is necessary.
This work was supported by the DOE Human Genome Program (USA) and the Russian Foundation for Basic Research (grants 02-04-49420-a, 05-04-49578-a and 04-04-48734-a). The authors wish to express gratitude to Dr. O Yu Chaga, who inspired this work. We would like to thank Dr. R. Hancock (Laval University, Quebec, Canada) for help with MALDI; we are grateful to Dr. V.E. Tretiakov for the MALDI done in Moscow and to Dr. J. Fernandez (Protein Resource Centre, The Rockefeller University, NY, USA) for the protein sequencing. We are deeply appreciative of the help that we have always received at the White Sea Biological Station of the Zoological Institute RAS “Kartesh”.
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Received 27 April 2005/9 June 2005; accepted 25 August 2005doi:10.1016/j.cellbi.2005.08.008