Extracellular matrix in development of the early embryo

PERGAMON

Micron 32 (2001) 427±438

www.elsevier.com/locate/micron

Review

Extracellular matrix in development of the early embryo N. Zagris* Division of Genetics and Cell and Developmental Biology, Department of Biology, University of Patras, Patras, Greece Received 7 October 1999; received in revised form 14 February 2000; accepted 15 February 2000

Abstract The extracellular matrix interacts with cells and promotes and regulates cellular functions such as adhesion, migration, proliferation, differentiation, and morphogenesis. Extracellular molecules are linked to one another by multiple binding domains and form a stable, multifunctional matrix. Cells respond to the extracellular matrix through plasma membrane receptors, which include integrin and nonintegrin receptors. The regulation of these interactions requires the coordination of a multiplicity of signals both spatially and temporally. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Extracellular matrix; Transmembrane signaling; Proteolytic remodeling; Adhesion; Migration; Morphogenesis; Laminin; Embryo

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Extracellular matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Glycosaminoglycans and proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Collagens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Basement membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Extracellular matrix receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Extracellular matrix remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction During development, the individual embryonic cells become integrated to form an increasingly complex system of cell±cell and cell±matrix interactions. The interactions between cells and the extracellular matrix initiate a ¯ow of information that acts to regulate many fundamental processes of development which include cell migration in the early embryo, morphogenesis during organ formation, and the modulation of growth and differentiation programs of cells. Cells from different tissues display selective af®nities, in ways that are region-speci®c and stage-dependent, which helps to establish and maintain the spatial order of * Tel.: 1 30-61-997-650; fax: 1 30-61-997-650. E-mail address: [email protected] (N. Zagris).

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different tissues in the embryo. The acquisition by cells of the transient ability to migrate is indeed one of the most complex phenomena during development and is the focus of robust activity. The organized making and breaking of cell contacts is an integral part of morphogenesis. The different classes of molecules that play a major regulatory role in morphogenesis are the extracellular matrix molecules, cell adhesion molecules, cell surface proteoglycans, and integrins. The extracellular matrix, including the basement membrane (basal lamina), interacts with cells (ªdynamic reciprocityº) and promotes and regulates cellular functions such as migration, adhesion, proliferation, differentiation, and morphogenesis (Hay, 1991; Adams and Watt, 1993; Chung, 1993, 1995; DeSimone, 1994; Timpl and Brown, 1994; Roskelley et al., 1995). Cellular functions of extracellular matrix are mediated by a large diversity of

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Fig. 1. Extracellular matrix accumulation and the ®rst major cellular migrations in early chick embryo. Transverse sections of chick embryos at stage X (late morula; A, a), stage XIII (blastula; B, b), and stage 2±3 (mid-gastrula; C); photomicrographs of paraf®n sections (10 mm) stained with PAS (PAS staining as in Humason, 1972). a and b show higher magni®cations of A and B, respectively. In D, d and E, e immunogold labeling photomicrographs showing migrating cells that contact and follow gold-labeled laminin-containing extracellular material during gastrulation; transverse sections (7 mm) of chick embryo at mid-gastrula (st. 2±3). D is focusing on the cells and E is the same section focusing on the extracellular matrix; d and e are frames from D and E, respectively, but the migrating cells and extracellular matrix are shown against a darker background. Immunogold labeling as in Zagris and Chung (1990). b, blastoderm; e, epiblast; ex, extracellular matrix; h, hypoblast; ps, primitive streak; and v, vitelline membrane. Bar ˆ 5 mm (A, B); 20 mm (a, b); 20 mm (C); 20 mm (D, d; E, e).

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cellular receptors such as the integrins that bind speci®cally to some of its components (Hynes, 1992; Haas and Plow, 1994). In addition, some extracellular matrix components such as the proteoglycan decorin act as repositories for inductive signaling molecules and growth factors such as TGF-b that bind to matching receptors in cell plasma membranes (Hildebrand et al., 1994). The occupancy of cell receptors to extracellular matrix components or extracellular matrix-bound signals feeds back on the behavior and gene activity of a cell. Cell migration is a major feature of animal morphogenesis with cells moving over relatively long distances from one site to another. In several morphogenetic events, cells that begin as part of an epithelium break away from their neighbors and migrate individually. In sea urchins, mesenchyme cells separate from the vegetal plate and ingress during gastrulation. In the embryo of higher vertebrates during gastrulation (Vakaet, 1970; Vakaet and Bortier, 1995), individual cells from the epithelial epiblast break away from their neighbors, become mesenchymal, ingress through the primitive streak, and migrate as individual cells before they band together to form epithelia such as somites and the lateral plates of the mesoderm. As development progresses, remodeling these epithelia to form the dermis, muscle, and sclerotome involves local migration of cells before they acquire their de®nitive position in the embryo. Stimulatory and inhibitory signals are important for cell adhesion and/or migration during development. The path®nding and the avoidance of inappropriate tracks by cells, and the development of organized layers and establishment of boundaries are crucial events. A variety of anti-adhesion molecules participate in establishing cellular boundaries and restricting cell migration. Many established extracellular matrix proteins have been shown to be anti-adhesive, at least under certain experimental conditions and for certain types of cells. For example, laminins are generally known to provide substrates to cells and to promote cellular migration and neurite outgrowth (reviewed by Sanes, 1989; Chung, 1995). However, the tripeptide sequence LRE in S-laminin (laminin-3) inhibits neurite extension of motorneurons at the neuromuscular junctions (Porter et al., 1995; Patton et al., 1998). Anti-adhesive mechanisms are prominent in the nervous system, where they seem to be important for guiding neuronal growth cones. Several chondroitin sulfate proteoglycans present in brain, spinal cord and along neural crest cell pathways are implicated in anti-adhesive functions and inhibition of neurite outgrowth (Margolis and Margolis, 1993; Landolt et al., 1995). The interactive nature of the extracellular matrix is of particular interest in embryos, where the extracellular matrix molecules help to direct cellular and epithelial movements and to localize inductive signals. 2. Extracellular matrix The extracellular matrix is a complex cell product

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consisting of glycoproteins, collagens, glycosaminoglycans, and proteoglycans as major structural and functional components. The extracellular matrix molecules secreted and self-assembled into the immediate cellular environment are linked to one another by multiple binding regions and with speci®c receptor molecules in the plasma membrane of cells into an organized multifunctional network resident in extracellular spaces. The extracellular matrix consists of a meshwork of ®bers embedded in a gel-like ground substance. The ground substance consists of glycosaminoglycans and proteoglycans, which form extended random coils that hold water by osmotic pressure. The ®brous extracellular matrix components are glycoproteins that reinforce the ground substance and resist its expansive forces. The matrix provides attachment sites that could guide migrating cells into de®ned pathways and in¯uence the extent and direction of their movements, and matrix±cell interactions affect differentiation of cells and are required for the maintenance of the proper tissue architecture of the developing embryo (England, 1982, 1984; Harrisson, 1989; Adams and Watt, 1993; DeSimone, 1994; Timpl and Brown, 1994; Chung, 1995; Zagris and Stavridis, 1995). Among the most puzzling questions concerning gastrulation have been what triggers the initiation of cellular migrations and how cells reach their appropriate destinations in the embryo. Integrated movements of cells permit new interactions, which in turn promote the formation of the three germ layers, ectoderm, mesoderm, and endoderm. Whatever the mechanism of control of triggering the initiation of cellular migration, initiation of morphogenetic movements of gastrulation correlates with extracellular matrix deposition. Cells deposit extracellular matrix molecules at very early stages of embryonic development. The chick embryo at stage X (blastoderm, Eyal-Giladi and Kochav, 1976) is a ¯at compacted disc (Fig. 1A, a) and is homologous to late morula (Eyal-Giladi, 1995). Deposition of extracellular matrix was ®rst detected in the space between the epiblast and the hypoblast (homologous to blastocoele in amphibia) in embryos at the blastula stage (stage XIII)(Fig. 1B, b). A great number of doughnut-like granules were accumulating in the blastocoele and these granules seemed to originate from both the epiblast and the hypoblast. The extracellular matrix was assembled as a ®brillar network in the blastocoele preceding the extensive cellular migrations of gastrulation (Fig. 1C). During gastrulation, cells move as a stream of loosely packed cells away from the primitive streak (homologous to the blastopore in amphibia) in the blastocoele between the epiblast and the hypoblast to form the de®nitive endoderm and mesoderm; migrating cells interact directly with the matrix through their ®lopodia that contact and follow gold labeled laminin-containing ®brils (Fig. 1D, d; E, e). Gold particles are bound at regular intervals along laminin-containing ®brils in extracellular matrix; laminin is also detected on the cell surface of the migrating cells and in the epiblast and hypoblast (Fig. 1D, d; E, e). These results indicated a possible direct interaction between cell

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Fig. 2. Laminin distribution in the early chick embryo. Immunogold labeling photomicrographs showing laminin immunoreactivity. Transverse sections (7 mm) of chick embryos at blastula (A), mid-gastrula (B, b), late gastrula (st. 4; C), early neurula (st. 5±6; D) and at seven somites (st. 9; E, F, G) incubated in the presence of laminin antibodies; b shows magni®ed area of B, and F and G show magni®ed areas of E. Methods as in Zagris and Chung (1990). e, epiblast; ec, ectoderm; en, endoderm; ex, extracellular matrix; h, hypoblast; m, mesoderm; n, neural plate; nc, neural crest; nt, neural tube; and v, vitelline membrane. Bar ˆ 20 mm (A, B, C, D, F, G), 50 mm (b), and 5 mm (E).

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Fig. 3. Antibodies to laminin perturb morphogenetic movements of early chick embryo. Chick embryos at the morula stage (st. X) cultured in plain Ringer solution (A) or in Ringer solution containing laminin antibodies (B) for 4 h, then cultured for 22 h in plain egg albumen (methods as in Zagris and Chung, 1990). Photomicrographs of embryos at the end of culture. a, an atypical primitive streak; ps, primitive streak. Bar ˆ 500 mm.

processes and laminin during active migration and that laminin was involved in providing the substrate and cues for the earliest morphogenetic movements. The extracellular matrix is a dynamic entity undergoing relatively rapid qualitative and quantitative changes of the constituent molecules, which seem to dictate the properties of the embryonic cellular environments. The development and differentiation of extracellular matrices may re¯ect the acquisition of differentiated functions of the cells that produce them and, in turn, may in¯uence the phenotypic expression of the differentiated state. Matrix may also play a role, direct or indirect, in events of tissue and organ morphogenesis. 2.1. Glycoproteins Extracellular matrices contain a variety of glycoproteins such as ®bronectin, tenascin, entactin, and laminin, which are involved in cell and tissue adhesion processes at speci®c developmental times. These are multi-domain molecules that interact with one another, with other extracellular matrix molecules, and with cell surfaces and are probably responsible for organizing the collagen, proteoglycan, and cells into an ordered structure. Laminin is the ®rst glycoprotein to appear in the extracellular matrix in the developing embryo (Leivo et al., 1980; Cooper and MacQueen, 1983; Dziadek and Timpl, 1985; Martin and Timpl, 1987; reviewed by Zagris and Stavridis, 1995). The early appearance and the highly conserved basic structure of laminin in diverse species from anthomedusa to human points to an important role in the regulation of tissue development (reviewed by Zagris and Stavridis, 1995). In chick embryos at stage X (late morula), cells may synthesize laminin but do not deposit it on their surfaces (Zagris and Chung, 1990). Deposition of laminin to the extracellular

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space was ®rst detected on cell surfaces on the ventral surface of the epiblast and in the hypoblast in embryos at stage XIII (blastula) (Fig. 2A). The detectable localization of laminin on the ventral surface of epiblast is coincident with completion of cell polyingression from the epiblast and formation of the hypoblast and the assembly of the ®rst basement membrane below the epiblast in the early chick embryo. The basement membrane could serve as barrier to continued polyingression of cells, could provide structural support and may be crucial to the epithelization of the epiblast. When the ®rst migration movements start, it may also serve as a substrate for migration of mesodermal cells. The extracellular matrix seems to have a dual origin resulting from interaction of epiblast-derived materials and noncollagenous glycoprotein synthesized by the hypoblast (Figs. 1b, 2b and 2C, and Harrisson, 1993). As gastrulation proceeds, the extracellular matrix was organized in the space between the epiblast and the hypoblast and showed strong laminin immunoreactivity (Fig. 2B). When examined at higher magni®cation, the extracellular matrix displayed a ®ne ®brillar and granular structure (Fig. 2b). The migrating cells utilize the extracellular ®bers as a contact guidance system in the gastrulating chick embryo. The chick early neurula (stage 5±6; Hamburger and Hamilton, 1951) contains an elaborate laminin-rich extracellular matrix in which laminin appeared in punctate deposits, resembling strung beads, below the neural plate (Fig. 2D). Extracellular matrix may be playing an active part in neural plate bending to form the neural tube during primary neurulation. In embryo at stage 9 (seven somites), extracellular matrix was organized in the neural tube and in the other embryonic cavities (Fig. 2E, F, G); the laminin-containing network of extracellular matrix present between the migrating neural crest cells and the lateral margins of the neural tube may provide the proper substrate and cues for guiding these cells during their migration (Fig. 2E, G). A much more direct demonstration of the involvement of laminin in cell migration comes from the inhibition of the function of this molecule. Fig. 3B shows an embryo treated with antibodies to laminin at the morula stage. In the treated embryo, migrating cells followed atypical trails, dispersed and formed an atypical primitive streak not in the center of the area pellucida but at its margin. A control embryo in a parallel culture formed a primitive streak and is shown for comparison in Fig. 3A. The primitive streak is formed as the direct result of speci®c movements taking place in the epiblast, and these movements are induced by the underlying hypoblast and mark initiation of the gastrula stage. Our results on the inhibition of laminin function indicated that laminin does not interfere with triggering of cell movements but in¯uences the directionality of cell migration. The laminins are large hetero-trimeric glycoproteins that are major components of the extracellular matrix, including the basement membrane, and of embryonic tissues (reviewed by Engel, 1992; Chung, 1993; Timpl and Brown, 1994). The prototype of laminin (PYS/EHS

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tumor; Chung et al., 1979 and Timpl et al., 1979) consists of three distinct polypeptide chains designated as a 1 (400 kDa), b 1 (210 kDa), and g 1 (210 kDa). Eleven distinct heterotrimeric laminin isoforms assembled from ®ve a , three b , and two g genetically distinct subunit chains have been identi®ed to date with distinct tissue distributions and co-distributions, unique properties and developmentally regulated expression (Hunter et al., 1989; Sanes et al., 1990; reviewed by Timpl and Brown, 1994; Champliaud et al., 1996; Patton et al., 1998). Laminin has a variety of functions which can be assigned to different clearly de®ned domains of the molecule. Cell binding to laminin occurs by a large diversity of cellular integrin receptors and several less well characterized non-integrin receptors and may trigger various intracellular signal transduction pathways (Mecham, 1991; Hynes, 1992; Haas and Plow, 1994; Giancotti, 1997). Cell interaction with laminin matrices plays an important role in cell attachment, directional migration, mitogenetic modulation, neurite outgrowth, axon guidance, survival of cells, maintenance of differentiated cell phenotypes and the induction of new expression patterns (reviewed by Engel, 1992; Adams and Watt, 1993; Timpl and Brown, 1994; Yurchenco and O'Rear, 1994; Chung, 1995; Streuli et al., 1995; Zagris and Stavridis, 1995; Luckenbill-Edds, 1997). Entactin/nidogen forms a particularly stable complex with laminin and it also binds to collagen IV, the protein core of perlecan, and to ®bronectin (Martin and Timpl, 1987; Yurchenco and O'Rear, 1994; Chung, 1995). Entactin appears at the early stages of chick embryogenesis and its presence appears to be correlated with directional migration of cells (Zagris et al., 1993). The recognition of entactin by more than one plasma membrane receptor is reminiscent of other extracellular matrix glycoproteins such as laminin and ®bronectin (reviewed by Chung, 1995). Fibronectin is a large (460 kDa), homodimeric, multidomain glycoprotein that can bind to collagen, entactin, glycosaminoglycans, and cells (Ruoslahti and Obrink, 1996). It is essential for mesodermal cell migration during gastrulation. Tenascin has been shown to have both adhesive and anti-adhesive properties on isolated cells and its expression was shown to be tightly regulated during development (Chiquet-Ehrismann et al., 1986; Chiquet-Ehrismann, 1995). Fibronectin and tenascin collaborate in regulating collagenase gene expression in ®broblasts in culture and this may be an important feature for regulation of cell adhesion, cell migration and morphogenesis (Tremble et al., 1994). 2.2. Glycosaminoglycans and proteoglycans Glycosaminoglycans (e.g. hyaluronate, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate) are a major class of components of embryonic extracellular matrix which have been implicated as important factors in the control of cell proliferation, migration, differentiation, and maintenance of morphogenetic structures. Glycosamino-

glycans occur as large polymers of repeating disaccharides and, with the possible exception of hyaluronate, are covalently linked to protein to form the proteoglycans. Hyaluronate consists of thousands of repeating disaccharides (d-glucuronic acid and N-acetyl-d-glucosamine) which create an enormous molecule of several million daltons and carries negatively charged carboxyl groups. In glycosaminoglycans with sulfate groups, the density of negative charges is even higher. Their polyanionic properties due to sulfate ester and/or carboxylic acid groups and hydrophilic nature suggest that glycosaminoglycans are fundamental in determining the properties of the extracellular matrix environment during development. Hyaluronate, a predominant glycosaminoglycan in the early embryo (Solursh, 1976; Fisher and Solrush, 1977; Derby, 1978; Vanroelen et al., 1980; Brown and Papaioannou, 1993; Fenderson et al., 1993; Sherman et al., 1994) interacts with sulfated proteoglycans and collagen to form gel±®ber networks. It is known that dilute solutions of hyaluronate can have a hydrated volume of 10 3 times larger than that of the unhydrated state (Ogston and Stanier, 1951; Laurent and Fraser, 1992). Toole et al. (1972) originally postulated that expansion of the entangled meshwork of extracellular matrix during hydration of the hyaluronate domain would produce an expansion of the extracellular space, which would allow cells to migrate into previously inaccessible areas. It is known that cells bind and respond to hyaluronate through cell-surface receptor proteins CD44 and RHAMM (reviewed by Sherman et al., 1994), and we can expect in the years to come progress on the regulation and role of these and/or similar proteins in the developing embryo. The sequential increase and decrease in relative concentrations of hyaluronate have now been correlated with cell adhesion and de-adhesion, the regulation of cell migration and the creation of cell-free spaces in several developmental systems. The proteoglycan superfamily now contains more than 30 molecules formally de®ned as proteoglycans. Matrix proteoglycans can be separated into three groups: the basement membrane proteoglycans (perlecan, agrin, bamacan), the hyalectans (versican, aggrecan, neurocan, brevican) which are proteoglycans interacting with hyaluronan and lectins, and small leucine-rich proteoglycans (e.g. decorin, biglycan). Proteoglycans act as tissue organizers, in¯uence the cell proliferation and maturation of specialized tissues, play a role as biological ®lters and modulate growth factor activities, regulate collagen ®brillogenesis and skin tensile strength, and in¯uence neurite outgrowth (reviewed by Iozzo, 1998). Current work focuses at understanding the mechanisms that regulate the generation of proteoglycan isoforms, their tissue distributions and generation of biologically active growth factor/proteoglycan complexes. The following examples illustrate functions of representative proteoglycan molecules. Perlecan was detected in preimplantation mouse embryo and may be fundamental in cell differentiation and tissue morphogenesis (Dziadek et

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al., 1985; Carson et al., 1993). Perlecan has been reported to play a key role as a regulator of FGF-2 signaling and as a gatekeeper to limit access of growth factors to subjacent target cells (reviewed by Iozzo, 1998). It is interesting that distribution of FGF-2 in various basement membranes (Friedlander et al., 1994) parallels that observed for perlecan. A perlecan splice variant is known to control the amount of FGFs during early neuronal development (Joseph et al., 1996). The hyalectants can bind complex carbohydrates such as hyaluronate at their N-termini, less complex sugars at their C-termini and a varied number (2±100) glycosaminoglycan chains at their central non-homologous regions. It is interesting that the different splice variants of hyalectants are often expressed in distinct spatial and temporal patterns (Dours-Zimmermann and Zimmermann, 1994). It has been discussed in the literature that the major functional role of hyalectants would be to provide a means to introduce glycosaminoglycans into various extracellular matrices. For example, each aggrecan monomer presents a high concentration of chondroitin sulfate side chains and the formation of large supramolecular hyaluronate aggregates which occupy a large hydrodynamic volume in cartilage. Chondroitin sulfate proteoglycans have been implicated in the regulation of cell migration, axonal outgrowth, and pattern formation in the developing peripheral nervous system (Margolis and Margolis, 1993; Meyer-Puttlitz et al., 1995). For example, versican is selectively expressed in embryonic tissues that act as barriers to neural crest cell migration and motor and sensory axonal outgrowth (Landolt et al., 1995) and neurocan binds to the neural cell adhesion molecules Ng-CAM and N-CAM, inhibits their homophilic interactions and blocks neurite outgrowth (Grumet et al., 1993). The mechanisms for the anti-adhesive activity of proteoglycans remain uncertain. It has been discussed that molecules bearing chondroitin sulfate epitopes may prevent access to substrate molecules by literally covering them up thus providing an anti-adhesive function through steric hindrance at the cell surface (Oakley et al., 1994). Another type of anti-adhesive signaling cascade that acts through the binding of a chondroitin sulfate proteoglycan core protein (most probably neurocan) to a cell surface N-acetylgalactosaminylphosphotransferase points to a transmembrane signal transduction event (Balsamo et al., 1995). Decorin, the small leucine-rich proetoglycan, binds TGFb and its isoforms with high af®nity and functions as a reservoir for these growth factors in the extracellular matrix (Hildebrand et al., 1994). Recent studies indicate that decorin causes rapid phosphorylation of the EGF receptor which results in mobilization of intracellular Ca 21 and activation of the mitogen-activated protein (MAP) kinase signal pathway (Moscatello et al., 1998; Patel et al., 1998). 2.3. Collagens The most abundant structural components of extracellular

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matrices in all tissues are the collagens. Detailed information about the structure and expression of the more than 30 different genes that encode members of this large family of at least 19 proteins has been assembled (reviewed by Olsen, 1995; Prockop and Kivirikko, 1995). A vast literature is also available on the structure and functions of collagens, and on mutations in collagen genes and their consequences (reviewed by Yurchenco and O'Rear, 1994; Olsen, 1995; Prockop and Kivirikko, 1995). The collagen family is a highly diverse group of proteins which includes the classical ®brillar collagens (types I, II, III, V and XI), collagens that form a variety of sheet or lattice structures in basement membranes (the type IV family, and types VIII and X), collagens that are found on the surface of collagen ®brils and are known as ®bril-associated collagens with interrupted triple helices (FACITs that include types IX, XII, XIV, XVI, and XIX), the collagen that forms beaded ®laments (type VI), the collagen that forms anchoring ®brils for basement membranes (type VII), collagens with a transmembrane domain (types XIII and XVII), the newly discovered collagen types XV and XVIII; an additional group consists of proteins containing triple-helical domains that have not been formally de®ned as collagens (reviewed by Prockop and Kivirikko, 1995). Collagens are distributed in a tissue-speci®c way. The most abundant collagens form extracellular ®brils or network-like structures, but the others ful®l a variety of biological functions. Type IV collagen is the ®rst collagen to appear in the early embryo (Adamson and Ayers, 1979; Leivo et al., 1980). Six different collagen IV genes have been identi®ed in mammals and they show tissue-speci®c expression patterns (reviewed by Yurchenco and O'Rear, 1994). The chains most widely distributed and present in largest amount are the a1(IV) and a2(IV) chains [a1(IV)2 a2(IV)]. Some basement membranes contain smaller amounts of the a3(IV) and a4(IV) or of a5(IV) and a6(IV) chains that are similar but not identical (Hudson et al., 1994). The exon±intron patterns of most of the collagen genes are widely distributed in the genome, but the genes of type IV collagens are located in a unique head-to-head arrangement on different chromosomes so that the promoter regions overlap. The COL4A1/COL4A2 genes have a bi-directional (head-to-head) promoter arrangement on chromosome 13, the COL4A3/ COL4A4 genes are head-to-head on chromosome 2, and the COL4A5/COL4A6 genes are head-to-head on the X chromosome (reviewed by Yurchenco and O'Rear, 1994; Olsen, 1995; Prockop and Kivirikko, 1995). The two other network-forming collagens, types VIII and X, are very different in structure from type IV but similar to each other (Muragaki et al., 1991; reviewed by Van der Rest and Garrone, 1991; Hulmes, 1992 and Prockop and Kivirikko, 1995). Collagen type VIII is in the form of stacks of hexagonal lattices in Descemet's membrane which separates the corneal endothelial cells from the stroma. Type X collagen is among the most specialized of the collagens and is synthesized primarily by hypertrophic chondrocytes in the

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deep-calcifying zone of cartilage (Muragaki et al., 1991; Van der Rest and Garrone, 1991; Hulmes, 1992; Prockop and Kivirikko, 1995). Type I is the most abundant collagen and is found in a variety of tissues. Many of the other ®bril-forming collagens have a more selective tissue distribution. Among the new developments concerning ®bril-forming collagens is the discovery of alternative splicing of exons in the N-terminal propeptides of type II (Ryan and Sandell, 1990; Nah and Upholt, 1991; Sandell et al., 1991) and type XI (Oxford et al., 1995; Tsumaki and Kimura, 1995; Zhidkova et al., 1995) collagens so that even more varieties of collagen peptides are generated. Another new development concerning ®bril-forming collagens is the ®nding that many ®brils in vivo are composed of two or more different collagen types (reviewed by Van der Rest and Garrone, 1991; Hulmes, 1992; Prockop and Kivirikko, 1995). The important structural/mechanical function of collagen in extracellular matrices is well documented. Current work focuses at understanding the role of collagen in development and morphogenesis and has opened novel perspectives in extracellular matrix research. For example, the discovery that two tyrosine kinase orphan receptors, the discoidin domain receptors DDR1 and DDR2, are the receptors for ®brillar collagen I, III, and V is intriguing (Shrivastava et al., 1997; Vogel et al., 1997). Fibrillar collagen interacts with the proteoglycan decorin and this interaction would help stabilize ®brils and orient ®brillogenesis (Scott, 1996). Both ®brillar collagen and decorin converge on similar tyrosine kinase receptors, and their activation may allow a speci®c cross talk between cells and the extracellular matrix. 2.4. Basement membrane The principal embryonic tissues, mesenchyme and epithelia, have characteristic types of extracellular matrix. Beneath epithelia, the extracellular matrix is formed as a basement membrane, a dense sheet which separates the basal surface of the epithelium from the underlying mesenchyme. Mesenchymal cells are surrounded on all sides by extracellular matrix. Basement membranes create barriers to the passage of macromolecules and cells that allow embryonic cells to segregate and differentiate into speci®c tissues, serve as surfaces for cell adhesion and support, and for migration of cells during development. In the adult, basement membranes provide the scaffolding that maintains tissue form during regeneration and growth (Timpl and Dziadek, 1986; Martin and Timpl, 1987; Chung, 1993; Yurchenco and O'Rear, 1994). The basement membrane is a specialized form of the extracellular matrix. All basement membranes contain a common set of proteins that include laminin, entactin/nidogen, collagen IV and the heparan sulfate and chondroitin sulfate proteoglycans. The synthesis of laminin, collagen IV and other matrix molecules is developmentally regulated

and these molecules are distributed in a tissue speci®c way; correspondingly, the structural organization of basement membranes is not homogeneous. There is sequential assembly of components during early basement membrane formation (Dziadek and Timpl, 1985) rather than secretion of pre-formed complexes (Martin et al., 1984). In the mouse embryo, laminin and heparan sulfate proteoglycan are already present on the surface at the two-cell stage (Wu et al., 1983; Dziadek and Timpl, 1985), entactin/nidogen is ®rst detected on compacted 8±16 cell stage morulae, and collagen IV and ®bronectin are ®rst detected in the blastocyst inner cell mass (Adamson and Ayers, 1979; Wartiovaara et al., 1979; Leivo et al., 1980). The appearance of collagen IV coincides with assembly of the ®rst basement membranes in the early embryo. In vivo, these components must interact with each other to form the thin, tightly packed network of ®ne cords in a highly cross-linked network of collagen IV (Martin and Timpl, 1987). Collagen IV is unique to basement membranes and forms, by end-to-end and lateral interactions, open or planar or polygonal networks to which other glycoproteins and proteoglycans bind at speci®c sites. Collagen IV is considered the major structural component and may be instrumental in basement membrane assembly. However, the six different genes of collagen IV which show tissue-speci®c expression patterns in mammals (Yurchenco and O'Rear, 1994) points to the functional signi®cance of this molecule as well. In regions of signi®cant mechanical stress, such as skin, additional collagen types, which include collagen XVII and collagen VII, anchor basement membranes to overlying cells and the underlying stroma (reviewed by Olsen, 1995). Proteoglycans may likewise have a structural role and they are responsible for regulating the permeability of the basement membrane. Basement membranes contain polyanionic sites composed of heparan sulfate and chondroitin sulfate chains which serve as selective charge barriers to proteins (Farquhar, 1981). In an early work, Kanwar et al. (1980) reported that removal of heparan sulfate chains increases glomerular permeability to proteins and lead to proteinuria. Perlecan, agrin, bamacan and splice variants of these are the only proteoglycans associated with basement membranes. It is puzzling why only these three seemingly diverse proteoglycans are associated with the basement membrane (reviewed by Iozzo, 1998). The adhesive properties of the basement membranes are usually ascribed to the glycoproteins. Laminin, the most abundant glycoprotein in all basement membranes, is both a structural and regulatory molecule. The ability of laminin to self-associate and to interact with collagen IV, entactin/ nidogen, perlecan and other proteins is essential to the assembly of basement membrane (Martin and Timpl, 1987; Yurchenco and O'Rear, 1994). Fibronectin (Ruoslahti, 1988) and tenascin (Chiquet-Ehrismann et al., 1986) are sometimes also associated with the basement membrane. The basement membrane can be modi®ed by varying the constituent molecules, as for example insertion

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of one of the many laminin isoforms could have marked effects on the properties of the matrix. 3. Extracellular matrix receptors Cells respond to the extracellular matrix through plasma membrane receptors which include integrin and non-integrin receptors (Mecham, 1991; Hynes, 1992). Remarkable progress has been made in identi®cation of the integrin receptors and in studies of their mechanism of action. The integrins, a family of heterodimeric (ab) transmembrane proteins, are unique cell surface receptors in that they mediate interactions of cells with extracellular matrix proteins and with other cells (Hynes, 1992; Haas and Plow, 1994). The integrin extracellular domain forms a ligand-binding site recognizing one or more extracellular ligands or counter-receptors on other cells, and the cytoplasmic domain interacts with cytoskeletal proteins (Hynes, 1992; Giancotti, 1997). The signal transduction molecules present at adhesion sites are common to the well known components of growth factor and cytokine signaling pathways, and the emerging view is that these systems overlap substantially. There is evidence for a considerable reciprocal ¯ow of signaling information between the extracellular matrix, the cytoplasm and the nucleus (reviewed by Roskelley et al., 1995). These interactions may transmit or initiate signals between and within cells, promote cell movement or cell adhesion. In addition to cell migration and adhesion, integrins mediate or regulate a wide variety of cellular processes including organization of the cytoskeleton, cell proliferation, apoptosis and cell differentiation. The speci®city of integrin binding and functions are due to the variety of combinations of associations of the 16 known a chains and 8 b chains (reviewed by Hynes, 1992); in addition, an array of variant forms of integrins with alternatively spliced extracellular and cytoplasmic domains have been identi®ed (reviewed by Collo et al., 1993; Song et al., 1993; Ziober et al., 1993; Martin et al., 1996; Ziober et al., 1997). Different tissues express one or more combinations of these different isoforms and multiple ab heterodimers are often found on the surface of the same cell. 4. Extracellular matrix remodeling Matrix degradation and turnover are also extremely important in tissue remodeling in development and in many other processes including wound healing, tumor invasion and metastasis, in¯ammation, and even the activation of cytokines from their precursors. Some of the key components regulating matrix turnover are the metalloproteases which constitute a family of 11 or more zinc-dependent endopeptidases, and their inhibitors called TIMPs (tissue inhibitors of metalloproteases). One of the newly characterized metalloproteases is GON-1, a secreted protein that controls morphogenesis of gonads by remodeling basement

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membranes in Caenorhabditis elegans (Blelloch and Kimble, 1999). Metalloproteases seem to be critical modulators of organogenesis (Lelongt et al., 1997; Hiraoka et al., 1998; Blelloch and Kimble, 1999). Metalloprotease expression is tightly controlled by growth factors and cytokines which either induce (e.g. TGF-a, EGF, FGF) or repress (e.g. TGF-b) transcription of metalloprotease genes (StetlerStevenson et al., 1993; Birkedal-Hansen, 1995). The activities of these proteases are highly regulated by a complex network of enzymes and inhibitors, resulting in an exquisitely balanced system of matrix turnover. 5. Concluding remarks There continues to be remarkable progress towards understanding the mechanisms by which the different systems of cell±cell and cell±matrix contacts establish and regulate physical adhesive interactions and mediate transmembrane signaling processes in various tissues. Current areas of vibrant research include: the elucidation of new roles for matrix proteins and the continually expanding roles for adhesion proteins in intercellular signaling and embryonic induction. The mechanisms that govern proteoglycan diversity and the tissue speci®c constrains that favor the expression of specialized variants. The identi®cation of novel important extracellular matrix molecules involved in repulsive and inhibitory interactions and identi®cation of ligands and signal transduction molecules mediating these effects. The mechanisms that regulate the generation of biologically active growth factor/proteoglycan complexes. Proteoglycan-dependent induction of transcription factors and their modulation. The mechanisms regulating expression of adhesion molecules and the role of cytokines and growth factors. The elucidation of the reciprocal feedback between genes that control cell adhesion and histogenesis and genes that regulate spatial patterning. Acknowledgements I wish to thank Ms Maria Christopoulos for technical assistance. This work was supported by grant (99 EK 352) from the General Secretariat of Research and Technology of Greece and the European Social Fund. References Adams, J.C., Watt, F.M., 1993. Regulation of development and differentiation by the extracellular matrix. Development 117, 1183±1198. Adamson, E.D., Ayers, S.E., 1979. The localization and synthesis of some collagen types in developing mouse embryos. Cell 16, 953±965. Balsamo, J., Ernst, H., Zanin, M.K.B., Hoffman, S., Lilien, J., 1995. The interaction of the retina cell surface N-acetylgalactosaminylphosphotransferase with an endogenous proteoglycan ligand results in inhibition of cadherin-mediated adhesion. J. Cell Biol. 129, 1391±1401.

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