MICROSCOPY RESEARCH AND TECHNIQUE 43:102–110 (1998) The Role of Tenascin-C and Related Glycoproteins in Early Chondrogenesis ELEANOR J. MACKIE1* AND LYN I. MURPHY2 1School of Veterinary Science, University of Melbourne, Parkville, Victoria 3052, Australia of Veterinary Basic Sciences, The Royal Veterinary College, London, United Kingdom 2Department KEY WORDS fibronectin; thrombospondin; extracellular matrix; chondrocyte ABSTRACT A number of large multidomain extracellular matrix glycoproteins, including fibronectin and members of the tenascin and thrombospondin families, are expressed in locations that suggest they may be involved in the process of chondrogenesis. During early limb morphogenesis, tenascin-C is selectively associated with condensing chondrogenic mesenchyme. With progressive development of endochondral bones, tenascin-C is absent from the matrix surrounding proliferating and hypertrophic chondrocytes, but remains in a restricted distribution in peripheral epiphyseal cartilage. During long bone development, patterns of expression of tenascin-C splice variants differ between chondrogenic and osteogenic regions, suggesting that different isoforms may have different functional roles. Tenascin-C presented as a substratum for chick wing bud mesenchymal cells induces chondrogenic differentiation. In early studies, fibronectin was found to inhibit chondrogenesis, despite being abundant in early chondrogenic mesenchyme. Recent studies showing differential effects of fibronectin splice variants on prechondrogenic mesenchymal condensation may explain this paradox. Members of the thrombospondin gene family are expressed in chondrogenic tissues at different stages, suggesting that they each play a unique role in cartilage development. Microsc. Res. Tech. 43:102–110, 1998. r 1998 Wiley-Liss, Inc. INTRODUCTION Glycoproteins of the extracellular matrix (ECM) are important in regulating cell behaviour in many tissues during development (Adams and Watt, 1993). Aspects of cell function that may be influenced by ECM glycoproteins include proliferation, migration, differentiation, and apoptosis. Cell surface receptors that mediate many of these responses have been identified, and include members of the integrin family of ECM receptors. Chondrogenesis is one of many biological processes where ECM glycoproteins appear to make important contributions. A common feature of many ECM glycoproteins is their large multidomain structure, which confers on them a multiplicity of binding sites for cells and other ECM components. In some cases structural motifs are shared by products of different genes, but the proteins are still sufficiently diverse to interact with distinct (although related) receptors and, therefore, potentially to play a number of different roles. The repertoire of ECM effects is further expanded by alternative splicing of individal gene products. In this review, the role of ECM glycoproteins in early events in the establishment of the chondrocyte phenotype will be discussed, with particular emphasis on tenascin-C, which has been extensively studied in this context. TENASCIN-C Structure A consideration of the role of tenascin-C in chondrogenesis requires an understanding of its structure. Tenascin-C is the first described and best characterized member of an expanding family of glycoproteins that is r 1998 WILEY-LISS, INC. currently known to be comprised of tenascins-C, -R, -W, -X, and -Y (Bristow et al., 1993; Chiquet-Ehrismann et al., 1994; Hagios et al., 1996; Rathjen et al., 1991; Weber et al., 1998). The four structural features common to members of this family are an NH2-terminal region comprised of heptad repeats, a series of epidermal growth factor (EGF)-like repeats, a series of fibronectin type III-like (FNIII) repeats, and a COOHterminal fibrinogen-like domain. In the case of tenascin-C, the heptad repeats take part in oligomerization to form hexamers, and the region of FNIII repeats is subject to alternative splicing (Fig. 1). The numbered FNIII repeats (1–6) are present in all isoforms, and exclusion of varying combinations of the lettered repeats (A–D) results in the existence of a number of different isoforms. Distribution of Tenascin-C in Chondrogenic Tissues The association of tenascin-C with cartilage development was first noted by Chiquet and Fambrough (1984), who described the protein as being present in the perichondrium of the formed cartilage models of chicken embryo long bones. These observations led to more detailed studies of the expression patterns of tenascin-C during cartilage development, together with in vitro studies investigating its possible functions. Tenascin-C appears in the mesenchymal cell condensation *Correspondence to: Dr E.J. Mackie, School of Veterinary Science, University of Melbourne, Cnr Flemington Road and Park Drive, Parkville, Victoria 3052, Australia. E-mail: firstname.lastname@example.org Received 00; accepted in revised form MATRIX PROTEINS AND CHONDROGENESIS 103 Fig. 1. Schematic diagram of the structure of chick tenascin-C, showing the largest possible splice variant, and the variants most highly expressed in connective tissues. The names of variants refer to their apparent relative molecular mass when run on polyacrylamide gels under reducing conditions. Tn 260: the largest possible splice variant including all known chick FNIII repeats. Tn 230: two possible combinations of FNIII repeats giving rise to subunits migrating at 230 kD. Tn 200, Tn 190: the two smallest known subunits of chick tenascin-C. that precedes overt chondrocyte differentiation in developing cartilage, but is absent from the surrounding non-chondrogenic mesenchyme. Initial immunohistochemical studies were carried out in rodent embryos where the above observation was made not only in mesenchymal condensations preceding cartilage models of endochondral bones, but also in examples of condensations preceding permanent hyaline cartilage (tracheal cartilage) and fibrocartilage (intervertebral disc; Mackie et al., 1987). The observations have also been confirmed in avian embryos, both in limb bud and Meckel’s cartilage of the mandible (Gluhak et al., 1996; Pacifici et al., 1993). An example of tenascin-C expression in precartilage mesenchymal condensation is seen in Figure 2A, which shows a section through the forming Meckel’s cartilage from an embryonic day 13 (E13) rat embryo. It is also of interest to note that tenascin-C expression precedes chondrogenesis at sites of formation of experimentally induced ectopic digits (Hurle and Colombatti, 1996). Tenascin-C expression is gradually lost from the differentiating chondroblasts that become separated from each other by the deposition of cartilage-specific ECM components (including type II collagen and aggrecan), but remains throughout the periphery of the developing cartilage (Fig. 2B; Gluhak et al., 1996; Mackie et al., 1987; Pacifici et al., 1993; Thesleff et al., 1988). Thus in developing endochondral bones tenascin-C expression surrounds the cartilage model adjacent to the prospective diaphysis and metaphysis, as well as in the region of the elongating epiphysis. In the latter region, tenascin-C expression extends some distance from the end of the cartilage model at the time of onset of ossification, but recedes towards the forming articular surfaces with increasing organisation of the cartilage model into specialized layers, and the concomitant expansion of the ossification front. At any particular stage of this process, tenascin-C is found in association with flattened perichondrial cells as well as rounded, densely packed epiphyseal chondrocytes. It is, however, absent from the zones of proliferation and hypertrophy of the forming avian and rodent growth plate at all stages so far investigated (Glumoff et al., 1994; Mackie et al., 1987; Mackie and Ramsey, 1996; Pacifici et al., 1993). In order to analyse the nature of these tenascin-Cexpressing cells, it is necessary to consider the diverse functions of chondrocytes in different regions of the cartilage model during the dynamic process of long bone development. Once the primary centre of ossification is established, the major contribution to longitudinal growth occurs as a result of interstitial growth within the physeal (growth plate) zone of proliferating chondrocytes. Having proliferated, these cells undergo hypertrophy before being replaced by the bone of the expanding primary centre of ossification. Simultaneously, proliferative chondrocytes in the periphery of the epiphysis are taking part in appositional growth, which results in growth in girth of the model, as well as some additional longitudinal growth and modelling of the ends of the bone to give rise to the characteristic irregular shape of individual bones (Hinchliffe and Johnson, 1983). The proliferative chondrocytes taking part in appositional growth are round, but smaller and more closely packed than those of the physis, with the result that they appear less differentiated. An overview of all the studies on tenascin-C distribution in the cartilage models of growing endochondral bones leads to the conclusion that expression is restricted to cells with the capacity to differentiate into chondrocytes (perichondrial cells), and chondrocytes in regions taking part in appositional growth (Koyama et al., 1993; Mackie et al., 1987; Pacifici et al., 1993; Thesleff et al., 1988). Indeed, Pacifici et al. (1993) have demonstrated that some of the most peripheral tenascin-C-stained 104 E.J. MACKIE AND L.I. MURPHY Fig. 2. Expression of tenascin-C in developing cartilage. Indirect immunofluorescence of cryosections stained with anti-tenascin C. A: Section of head from E13 rat, showing positive staining throughout the condensing mesenchyme of Meckel’s cartilage (c) and the adjacent osteogenic mesenchyme (m), which will give rise to the membranous part of the mandible. B: Section of head from E15 rat, showing restriction of tenascin-C expression to the perichondrium of Meckel’s cartilage once chondrocyte differentiation has taken place. Bar ⫽ 100 µm. cells in the prospective articular cartilage of E18 chick femur are incorporating thymidine. Tenascin-C expression is, however, absent from the proliferating and hypertrophic chondrocytes of the growth plate. In Meckel’s cartilage of the chick, which does not undergo ossification, tenascin-C expression is also associated with regions of appositional growth (Gluhak et al., 1996). The distance that the tenascin-C expression extends from the articular surface into the epiphysis varies not only with age, but also with species. Just after the initiation of ossification, tenascin-C expression extends much further towards the primary centre of ossification in chick bones than it does in rodent bones (Mackie et al., 1987; Mackie and Tucker, 1992). This difference can probably be attributed to species-related differences in growth rates. It should also be mentioned that in studies comparing protein and transcript expression patterns in cartilages undergoing appositional growth, tenascin-C protein is detectable in the matrix surrounding cells that no longer express its mRNA (Gluhak et al., 1996; Mackie and Tucker, 1992). These observations suggest that tenascin-C remains stable in cartilage tissue for some time after incorporation into the matrix. Removal of tenascin-C with time is presumably executed by members of the matrix metalloproteinase family, some of which are known both to cleave tenascin-C and to be present in growth cartilage (Brown et al., 1989; Imai et al., 1994; Siri et al., 1995). By the time the secondary centre of ossification develops (in mammals), tenascin-C expression within the epiphysis is restricted to articular cartilage, where it is found in pericellular and territorial matrix (Mackie and Ramsey, 1996; Pacifici et al., 1993; Salter et al., 1995). The articular-epiphyseal growth cartilage, which arises with establishment of the secondary centre of ossification, is situated deep to the permanent articular cartilage and consists of chondrocytes arranged in layers similar to those of the physis, although much thinner. With the cessation of growth, the articularepiphyseal growth cartilage is replaced by bone of the secondary centre of ossification. Articular-epiphyseal growth cartilage lacks tenascin-C expression, as does its physeal counterpart both before and after formation of the secondary centre of ossification (Mackie and Ramsey, 1996). The expression of tenascin-C in adult articular cartilage varies between species. In 2-monthold chickens, strong tenascin-C staining is observed in the superficial layers of articular cartilage of the proximal tibia (Pacifici et al., 1993). Similarly, in normal adult human articular cartilage, tenascin-C is present in the most superficial cell layers (Chevalier et al., 1994; Salter, 1993). In contrast, tenascin-C is absent from most of the articular surface of the distal femur in 10-week-old rats (Mackie and Ramsey, 1996). It is possible that these species differences in tenascin-C expression are related to differences in articular cartilage thickness, which are in turn related to the size of the adult animal. In addition to articular cartilage, permanent hyaline cartilages in other anatomical locations have also been investigated for tenascin-C expression. Nasal cartilages in growing rats (20 days old) show only a thin line of tenascin-C expression in the perichondrium (Thesleff et al., 1988). Tracheal cartilage from adult rats retains tenascin-C expression only in the perichondrium (Mackie et al., 1987), whereas in avian tracheal cartilage some weak tenascin-C expression remains in the centre of the cartilage in addition to the perichondrial expression (Pacifici et al., 1993). In contrast to the situation in hyaline cartilage, tenascin-C expression is not lost with progressive chondrocyte differentiation in fibrocartilage. Tenascin-C expression remains throughout development in avian articular fibrocartilage (Pacifici et al., 1993). Tenascin-C staining is strong in the developing meniscus of the embryonic human knee at a stage when only superficial articular staining is present in the adjacent femur (Salter et al., 1995). Similarly, in E18 rat knees, tenascin-C staining of the meniscus is intense at a stage when staining of adjacent articular cartilage is very much reduced (Mackie and Ramsey, 1996). The fibrocartilaginous anterior cruciate ligament in the rat expresses abundant pericellular tenascin-C throughout development, with a slight weakening of expression in the adult (Mackie and Ramsey, 1996). MATRIX PROTEINS AND CHONDROGENESIS It is clearly difficult to explain in simple terms the observed patterns of expression of tenascin-C in developing cartilage. It has been suggested that the explanation may be that it is expressed by permanent cartilages (for example articular cartilage), but not by cartilage that will be replaced by bone (physeal growth cartilage; Pacifici et al., 1993). This generalization does not, however, appear to hold even for avian species, since Meckel’s cartilage, which forms the first skeletal element of the developing mandible and is retained as a permanent cartilage in birds, shows restriction of tenascin-C expression to the perichondrium from an early stage of development (Gluhak et al., 1996). There are also examples of permanent cartilages in mammalian species that do not express tenascin-C except in the perichondrium. The rat trachea is mentioned above, and an additional example is provided by the rodent physeal growth plate, which is retained throughout life. A more appropriate explanation for tenascin-C8s distribution may be that it is expressed by chondrogenic mesenchymal cells and committed chondrocytes that have not proceeded beyond a certain stage of differentiation. The morphological differences between tenascin-Cexpressing and non-expressing chondrocytes tend to support this suggestion. The tenascin-negative resting and proliferative chondrocytes of the physis are large and surrounded by abundant cartilage matrix, in contrast to the small, densely packed peripheral chondrocytes that express tenascin-C during endochondral bone development. Amongst the latter cell population are the proliferative cells that participate in appositional growth. The association of such cells with tenascin-C expression, in contrast to the lack of expression by cells contributing to interstitial growth, was first noted by Thesleff et al. (1988). Differential Expression of Tenascin-C Splice Variants The observations described above relate to studies using antibodies or cDNA probes that recognise regions of tenascin-C present in all splice variants. It is now known, however, that there are considerable variations between tissues in their expression of tenascin-C isoforms. Some studies have included a consideration of the isoforms expressed in developing endochondral bones. Most of these studies have been carried out in chick, and since there are species variations in the nature of the tenascin-C isoforms, only studies related to the chick will be considered here. The isoform identified as Tn 260 in Figure 1 includes all FNIII repeats so far identified for chick tenascin-C (Tucker et al., 1994). This isoform appears to be expressed very rarely, and apparently not at all in chondrogenic and osteogenic mesenchyme, since probes only recognising the ‘‘C’’ or ‘‘AD2’’ repeats do not hybridize in such regions (Derr et al., 1997; Tucker et al., 1994). The most abundant isoforms expressed by fibroblasts are those labelled Tn 230, Tn 200, and Tn 190 in Figure 1. The names of isoforms refer to their apparent relative molecular mass when run on polyacrylamide gels under reducing conditions. There are multiple possible isoforms that migrate at 230 kD, but the two illustrated in Figure 1 appear to be the only ones expressed by chondrogenic and osteogenic tissues. 105 Studies have not been carried out that would allow a definitive description of the pattern of expression of splice variants during chondrogenesis. If some assumptions are made, however, a scheme describing the likely expression pattern of tenascin-C isoforms in chondrogenic tissues can be proposed on the basis of a number of observations in different systems. In the cartilage models of chick bones soon after the onset of osteogenesis, cDNA probes that recognise all tenascin-C isoforms hybridize with a continuous band of peripheral tissue surrounding the entire bone rudiment and extending a small distance into the cartilaginous epiphysis (Fig. 3A,E; Mackie and Tucker, 1992; Prieto et al., 1990). In contrast, cDNA probes corresponding to the FNIII repeats A and B or B and D hybridize only to the outermost region of the model and most intensely in the region of the periosteal bone collar (Fig. 3B,G; Mackie and Tucker, 1992; Prieto et al., 1990). These observations have led to the conclusion that whereas Tn 190, Tn 200, and Tn 230 are expressed by mesenchymal cells with the capacity to differentiate into chondrocytes or osteoblasts, Tn 230 is not expressed by the more differentiated chondrocytes that still express tenascin-C (illustrated schematically in Fig. 4). This conclusion is supported by the observation that tenascin-C extracted from E17 chick sternal cartilage consists of Tn 190 and Tn 200 (Vaughan et al., 1987). The hybridization pattern of cDNAs corresponding to FNIII AD1 in cartilage models undergoing early osteogenesis is similar to that of AB, indicating that both forms of Tn 230 shown in Figure 1 are colocalized in the periphery of the bone model (Derr et al., 1997). Primary osteoblasts cultured from embryonic chick calvarial bones express only Tn 230, whereas periosteal fibroblasts from the same bones express Tn 190, Tn 200, and Tn 230, as detected on Western blots of conditioned medium (Mackie and Tucker, 1992). It is likely that osteoblasts in endochondral bones express only Tn 230, like their membrane bone counterparts (Fig. 4). Results obtained in older chick long bones (E19 tibia) using antibodies that specifically recognise different isoforms of tenascin C support the scheme proposed in Figure 4, but suggest that there is an additional layer of specialization in the maturing articular cartilage; the use of an antibody specific for FNIII repeat D has allowed the distinction between the outer articular cartilage, which expresses Tn 200 and probably Tn 190, and the bulk of the articular cartilage, which expresses exclusively Tn 190 (Pacifici et al., 1993). By this stage, Tn 230 is no longer expressed at the joint surface. It is of interest to note that in contrast to the developing hyaline articular cartilage, the fibrocartilaginous meniscus in the E19 chick femorotibial joint expresses FNIII repeats B and D, which suggests that Tn230 is expressed in this type of cartilage (Pacifici et al., 1993). No studies have yet addressed the question of which tenascin-C isoforms are expressed in the limb bud when tenascin-C is first detectable. If it is assumed that the spatial progression from peripheral to more central chondrogenic cells in the cartilage model (illustrated in Fig. 4) is representative of the temporal sequence of chondrogenic differentiation in the early limb bud, it can be concluded that the earliest tenascin-C expressed consists of a mixture of Tn 190, Tn 200, and Tn 230, and 106 E.J. MACKIE AND L.I. MURPHY Fig. 3. Differential expression of tenascin-C splice variants in E14 chick phalanges. Film overlay images (A–C) and darkfield photomicrographs (E–G) of adjacent cryosections hybridized with a probe corresponding to the region of EGF-like repeats (cTn8; A,E), a probe detecting FNIII repeats A and B (cTn230; B,G), or the control probe (pUC; C,F). D: Adjacent section stained with hematoxylin and eosin. The box in A shows the region that is enlarged (in the opposite orientation) in D–G. Both probes label the periosteal bone collar (double arrowheads in D–G), and the most superficial chondrocytes at the developing joint surface (small arrows in D–G). cTn230 is absent from a region of rounded, more differentiated chondrocytes that are, however, labelled by cTn8 (large arrows in D–G). Bars: A–C , 1 mm; D–G, 200 µm. that the number of isoforms expressed is restricted with progressive differentiation. a pattern of temporal regulation of tenascin-C expression that reflects that seen in vivo (Gluhak et al., 1996; Mackie et al., 1987). Tenascin-C presented as a culture substratum for E4 chick limb bud cells was found to cause an increase in the number of chondrogenic nodules as compared with tissue culture plastic or fibronectin (Mackie et al., 1987). These results were later confirmed by Chuong et al. (1993), who also demonstrated that antibodies to tenascin-C added to the culture medium inhibited the formation of chondro- Tenascin-C and Chondrogenesis In Vitro The finding that tenascin-C is expressed selectively in chondrogenic mesenchyme led to the hypothesis that tenascin-C stimulates chondrogenesis, and in vitro experiments were carried out accordingly. Cultures of prechondrogenic mesenchyme from chick embryos have been used for such experiments, and appear to undergo MATRIX PROTEINS AND CHONDROGENESIS 107 Fig. 4. Schematic diagram showing probable distribution of tenascin-C splice variants during endochondral bone development (based on observations made by Derr et al., 1997; Mackie and Tucker, 1992; Prieto et al., 1990; Vaughan et al., 1987). Scanned darkfield image of E14 chick phalanx (and adjacent tissues), with the regions expressing different combinations of splice variants indicated by patterned shad- ing. See Figure 1 for splice variant structures. All of the major variants are expressed by peripheral prechondrogenic and preosteogenic cells, Tn 190 and Tn 200 are expressed by differentiating chondrocytes, and two forms of Tn 230 are expressed in the periosteal bone collar and centre of ossification. Fig. 5. Induction of chondrogenesis by tenascin-C. E4 chick wingbud cells were cultured on plastic coated with recombinant Tn 190 (A) or Tn 190 lacking the fibrinogen-like domain (B; recombinants kindly provided by D. Fischer and R. Chiquet-Ehrismann), then stained with Alcian blue and photographed by brightfield microscopy. Chondrogen- esis assays were carried out as described in Mackie et al. (1987). Alcian blue-stained nodules are almost completely absent from cultures plated on tenascin-C lacking the fibrinogen-like domain. Bar ⫽ 200 µm. genic nodules. Recently we have attempted to identify the region of tenascin-C responsible for stimulation of chondrogenesis using hexameric tenascins lacking different structural domains. Preliminary results indicate that the fibrinogen-like terminal domain is necessary for chondrogenesis to occur (Fig. 5). Receptors so far identified for this region of tenascin-C include cell surface heparan sulfate proteoglycans, an unspecified integrin, and receptor tyrosine phosphatase-␤ (Aukhil et al., 1993; Joshi et al, 1993; Milev et al., 1997). It remains to be seen which, if any, of these receptors mediates tenascin-C-induced chondrogenesis. The differential distribution of tenascin-C splice variants described above leads to the speculation that different isoforms vary in their effects on chondrogenesis and osteogenesis. The progressive loss of the larger splice variants with chondrocyte differentiation suggests that the additional FNIII repeats may be inhibitory for chondrogenesis. Further studies will be needed to investigate this possibility. Any discussion of the function of tenascin-C is incomplete without mention of results obtained with mice genetically incapable of expressing tenascin- C (Forsberg et al., 1996; Saga et al., 1992). These mice are 108 E.J. MACKIE AND L.I. MURPHY described as developing normally, but detailed morphological analysis of the skeleton and other cartilagecontaining tissues has not been carried out. Ultrastructural investigations have been required for the detection of effects of lack of other ECM proteins such as type X collagen and osteopontin (Kwan et al., 1997; Liaw et al., 1998), and it seems likely that similar investigations may reveal abnormalities in chondrogenesis in tenascinC-null mice. It is also possible that other proteins with similar functions, either members of the tenascin gene family or other ECM glycoproteins, may substitute for tenascin-C. Whether any of these have appropriate expression patterns for such a task is discussed below. OTHER MEMBERS OF THE TENASCIN GENE FAMILY The tenascin family is currently known to consist of tenascins-C, -R, -W, -X, and -Y. Tenascin-R (previously named ‘‘restrictin’’ and ‘‘janusin’’) is restricted in its expression to the nervous system (Rathjen et al. 1991). Tenascin-Y is described as being predominantly expressed in heart and skeletal muscle, but expression in chondrogenic tissues has not been mentioned (Hagios et al., 1996). Tenascin-X is expressed during early limb development, but in muscle-associated connective tissues rather than developing skeletal elements (Burch et al., 1995). The newest tenascin to be identified is tenascin-W, which has so far only been described in zebrafish (Weber et al., 1998). In embryonic zebrafish, tenascin-W is expressed by cells that contribute to the formation of the cartilaginous skeleton, and is likely to play a role in chondrogenesis. Tenascin-W is, therefore, the only tenascin other than tenascin-C that is known to appear in the developing skeleton. If tenascin-W exists in mammals it may substitute for tenascin-C during skeletal development in tenascin-C-null mice. FIBRONECTIN Expression of fibronectin is enhanced in chondrogenic mesenchyme from the onset of condensation, but unlike that of tenascin-C is not absent from the surrounding non-chondrogenic mesenchyme (Dessau et al., 1980; Melnick et al., 1981). In further contrast to tenascin-C, fibronectin is retained in mature cartilage (Melnick et al., 1981). Early functional studies indicated that fibronectin inhibits chondrogenesis (Pennypacker et al., 1979; Swalla and Solursh, 1984; West et al., 1979). More recent studies have, however, implicated fibronectin in the process of pre-cartilage condensation (Frenz et al., 1989; Tavella et al., 1997). Fibronectin, like tenascin-C, is subjected to alternative splicing in the region of its type III repeats, and plasma fibronectin (used in the early functional studies) lacks type III repeats A and B (Schwarzbauer et al., 1987), which are now known to be present in condensing mesenchyme undergoing chondrogenesis (Gehris et al., 1996). These findings, together with the observation that antibodies to repeat IIIA disrupt chondrogenesis both in vitro and in vivo, may help to explain the failure of fibronectin to stimulate chondrogenesis in the early studies mentioned above (Gehris et al, 1997). Once again, these recent experiments suggest that fibronectin’s role is in the initiation of cellular condensation. Changes in the expression of fibronectin splice variants with progressive chondrocyte differentiation have been described for both chick and human, with loss of repeat IIIA in both species, and loss of repeat IIIB in humans (ffrench-Constant et al., 1989; Gehris et al., 1996; Salter et al., 1995). These observations are reminiscent of the loss of the related FNIII repeats from tenascin-C with chondrocyte maturation (described above). Repeat IIIA does not appear to be the only region of fibronectin involved in prechondrogenic condensation. Reagents that inhibit interactions between the aminoterminal heparin-binding domain of fibronectin and the mesenchymal cell surface inhibit precartilage condensation in wing but not leg mesenchyme (Downie and Newman, 1995; Frenz et al., 1989). Contradictory results have been obtained with respect to the role of the tenth type III repeat of fibronectin (present in all isoforms), which interacts with integrins through its RGD sequence. Frenz et al. (1989) concluded that this site was not involved in fibronectin-mediated condensation of substratum-attached cells. In contrast, experiments by Tavella et al. (1997) carried out with cells in suspension culture suggested that the RGD-containing site of fibronectin may be required for condensation. Thus, it appears that fibronectin-induced condensation of chondrogenic mesenchyme is mediated by repeat IIIA, the amino-terminal heparin-binding site, and possibly also the RGD-containing tenth type III repeat, and that these domains of fibronectin may vary in their contribution to condensation in different anatomical locations. THROMBOSPONDINS The thrombospondins, like the tenascins, belong to an expanding family of extracellular matrix proteins composed of modules of repeating structural units (Adams and Lawler, 1993). The structural features common to all thrombospondins are a series of ‘‘type II’’ (epidermal growth factor-like) repeats, a series of ‘‘type III’’ (calmodulin-like) repeats, and a globular carboxyterminal domain. Five members of the family have been described so far: thrombospondins-1, -2, -3, and -4, and cartilage oligomeric matrix protein (COMP). No studies have been carried out to investigate the ability of thrombospondins to influence early chondrogenesis, although distribution patterns of these proteins suggest that they may all play distinctive roles during this process (Tucker et al., 1997). Distribution of Thrombospondins-1, -2, -3, and -4 in Chondrogenic Tissues Thrombospondins-1 and -2 show a similar distribution to tenascin-C in chondrogenic tissues in the chick. They are both present in condensing mesenchyme, and expression is lost with progressive chondrocyte differentiation, only being retained in the periphery of the cartilage model (O’Shea and Dixit, 1988; Tucker, 1993; Tucker et al., 1995, 1997). Like tenascin-C, both thrombospondins-1 and -2 are absent from the regions of proliferative and hypertrophic chondrocytes in the avian growth plate (Tucker et al., 1995, 1997). There appear to be species differences, however, since during murine development there is additional expression of thrombospondin-1 in the zone of hypertrophic chondrocytes and thrombospondin-2 in the late proliferative zone of the growth plate (Iruela-Arispe et al., 1993). MATRIX PROTEINS AND CHONDROGENESIS Thrombospondin-3 appears later in chondrogenesis than do thrombospondins-1 and -2. The strongest expression in both developing murine and avian cartilage is in the zone of proliferating chondrocytes of the growth plate, but there is some expression in adjacent resting and hypertrophic chondrocytes (Iruela-Arispe et al., 1993; Tucker et al., 1997). Thrombospondin-4 shows the most restricted distribution in developing cartilage of all the thrombospondins, being expressed only weakly by the proliferative chondrocytes of the avian growth plate (Tucker et al., 1995). Cartilage Oligomeric Matrix Protein Cartilage oligomeric matrix protein is abundant in embryonic cartilage, but only after chondrogenesis is well advanced (Franzen et al., 1987), and remains in the territorial matrix of mature cartilage (Hedbom et al., 1992). Mutations in COMP have recently been found to be responsible for pseudoachondroplasia, a dwarfing condition in humans (Briggs et al., 1995; Hecht et al., 1995). The mutations so far detected are within the TSP type III repeats, or putative calciumbinding region of COMP. Patients with pseudoachondroplasia are not normally identified until the second year of life, suggesting that COMP’s role occurs late in cartilage development. The mechanism of the defect is not clearly understood, but in cartilage from pseudoachondroplasia patients, COMP accumulates within the rough endoplasmic reticulum (Maddox et al., 1997). The structural defect that occurs may, therefore, result from a deficiency of COMP or the presence of abnormal COMP in the cartilage matrix, or both. In either case, it seems likely that the defect is in the organisation of cartilage matrix, rather than in cell-matrix interactions. CONCLUSION During the process of early chondrogenesis, a large number of related ECM glycoproteins are expressed, with unique but overlapping spatiotemporal expression patterns. It is possible that for normal chondrogenesis to occur, the co-ordinated expression of all these proteins is required, although it also seems likely that in some cases of pathological failure of expression substitution by related proteins can occur. Functional evidence for a role for tenascin-C and fibronectin in early chondrogenesis exists. Members of the thrombospondin family are expressed in appropriate locations to be able to influence early chondrogenesis, but functional studies have not been carried out. ACKNOWLEDGMENTS The authors thank Ed Ghiocas for assistance with preparation of figures, and Richard Tucker for provision of micrographs. REFERENCES Adams, J.C. and Lawler, J. (1993) The thrombospondin family. Curr. Biol., 3:188–190. Adams, J.C., and Watt, F.M. (1993) Regulation of development and differentiation by the extracellular matrix. Development, 117:1183–– 1198. Aukhil, I., Joshi, P., Yan, Y., and Erickson, H. P. (1993) Cell- and heparin-binding domains of the hexabrachion arm identified by tenascin expression proteins. J. Biol. Chem., 268:2542–2553. 109 Briggs, M.D., Hoffman, S.M.G., King, L.M., Olsen, A.S., Mohrenweiser, H., Leroy, J.G., Mortier, G.R., Rimoin, D.L., Lachman, R.S., Gaines, E.S., Cekleniak, J.A., Knowlton, R.G., and Cohn, D.H. (1995) Pseudoachondroplasia and multiple epiphyseal dysplasia due to mutations in the cartilage oligomeric matrix gene. Nat. Genet., 10:330–336. Bristow, J., Tee, M.K., Gitelman, S.E., Mellon, S.H., and Miller, W.L. (1993) Tenascin-X: A novel extracellular matrix protein encoded by the human XB gene overlapping P450c21B. J. Cell Biol., 122:265– 278. Brown, C.C, Hembry, R.M., and Reynolds, J.J. (1989) Immunolocalization of metalloproteinases and their inhibitor in the rabbit growth plate. J. Bone Joint Surg., 71:580–593. Burch, G.H., Bedolli, M. A., McDonough, S., Rosenthal, S.M., and Bristow, J. (1995) Embryonic expression of tenascin-X suggests a role in limb, muscle and heart development. Dev. Dyn. 203:491–504. Chevalier, X., Groult, N., Larget-Piet, B., Zardi, L., and Hornebeck, W. (1994) Tenascin distribution in articular cartilage from normal subjects and from patients with osteoarthritis and rheumatoid arthritis. Arthritis Rheum., 37:1013–1022. Chiquet, M., and Fambrough, D.M. (1984) Chick myotendinous antigen. I. A monoclonal antibody marker for tendon and muscle morphogenesis. J. Cell. Biol., 98:1926–1936. Chiquet-Ehrismann, R., Hagios, C., and Matsumoto, K.I. (1994) The tenascin gene family. Perspect. Dev. Neurobiol., 2:3–7. Chuong, C.-M., Widelitz, R.B., Jiang, T.-X., Abbott, U.K., Lee, Y.-S., and Chen, H.-M. (1993) Roles of adhesion molecules NCAM and tenascin in limb skeletogenesis: Analysis with antibody perturbation, exogenous gene expression, talpid mutants and activin stimulation. In: Limb Development and Regeneration. J.F. Fallon, P.F. Goetinck, R.O. Kelley, and D.L. Stocum, eds. Wiley-Liss, New York, pp. 465–474. Derr, L.B., Chiquet-Ehrismann, R., Gandour-Edwards, R., Spence, J., and Tucker, R.P. (1997) The expression of tenascin-C with the AD1 variable repeat in embryonic tissues, cell lines and tumors in various vertebrate species. Differentiation, 62:71–82. Dessau, W.H., von der Mark, H., von der Mark, K., and Fischer, S. (1980) Changes in the pattern of collagens and fibronectin during limb-bud chondrogenesis. J. Embryol. Exp. Morphol., 57:51–60. Downie, S.A., and Newman, S.A. (1995) Different roles for fibronectin in the generation of fore and hind limb precartilage condensations. Dev. Biol., 172:519–530. ffrench-Constant, C., and Hynes, R.O. (1989) Alternative splicing of fibronectin is temporally and spatially regulated in the chicken embryo. Development, 106:375–388. Forsberg, E., Hirsch, E., Fröhlich, L., Meyer, M., Ekblom, P., Aszodi, A., Werner, S., and Fässler, R. (1996) Skin wounds and severed nerves heal normally in mice lacking tenascin-C. Proc. Natl. Acad. Sci. U.S.A., 93:6594–6599. Franzen, A., Heinegard, D., and Solursh, M. (1987) Evidence for sequential appearance of cartilage matrix proteins in developing mouse limbs and in cultures of mouse mesenchymal cells. Differentiation, 36:199–210. Frenz, D.A., Jaikaria, N.S., and Newman, S.A. (1989) The mechanism of precartilage mesenchymal condensation: A major role for interaction of the cell surface with the amino-terminal heparin-binding domain of fibronectin. Dev. Biol., 136:97–103. Gehris, A.L., Oberlender, S.A., Shepley, K.J., Tuan, R.S., and Bennett, V.D. (1996) Fibronectin mRNA alternative splicing is temporally and spatially regulated during chondrogenesis in vivo and in vitro. Dev. Dyn., 206:219–230. Gehris, A.L., Stringa, E., Spina, J., Desmond, M.E., Tuan, R.S., and Bennett, V.D. (1997) The region encoded by the alternatively spliced exon IIIA in mesenchymal fibronectin appears essential for chondrogenesis at the level of cellular condensation. Dev. Biol., 190:191–205. Gluhak, J., Mais, A., and Mina, M. (1996) Tenascin-C is associated with early stages of chondrogenesis by chick mandibular ectomesenchymal cells in vivo and in vitro. Dev. Dyn., 205:24–40. Glumoff, V., Savontaus, M., Vehanen, J., and Vuorio, E. (1994) Analysis of aggrecan and tenascin gene expression in mouse skeletal tissues by Northern and in situ hybridization using species specific cDNA probes. Biochim. Biophys. Acta, 1219:613–622. Hagios, C., Koch, M., Spring, J., Chiquet, M., and Chiquet-Ehrismann, R. (1996) Tenascin-Y: A protein of novel domain structure is secreted by differentiated fibroblasts of muscle connective tissue. J.Cell. Biol., 134:1499–1512. Hedbom, E., Antonsson, P., Hjerpe, A., Aeschlimann, D., Paulsson, M., Rosa-Pimentel, E., Sommarin, Y., Wendel, M., Oldberg, Å., and Heinegård, D. (1992) Cartilage matrix proteins: An acidic oligomeric protein (COMP) detected only in cartilage. J. Biol. Chem., 267:6132– 6136. 110 E.J. MACKIE AND L.I. MURPHY Hecht, J.T., Nelson, L.D., Crowder, E., Wang, Y., Elder, F.F.B., Harrison, W.R., Francomano, C.A., Prange, C.K., Lennon, G.G., Deere, M., and Lawler, J. (1995) Mutations in exon 17B of cartilage oligomeric matrix protein (COMP) cause pseudoachondroplasia. Nat. Genet., 10:325–329. Hinchliffe, J.R., and Johnson, D.R. (1983) Growth of cartilage. In: Cartilage Vol. 2: Development, Differentiaion, and Growth. B.K. Hall, ed. Academic Press, New York, pp. 255–295. Hurle, J.M., and Colombatti, A. (1996) Extracellular matrix modifications in the interdigital spaces of the chick embryo leg bud during the formation of ectopic digits. Anat. Embryol., 193:355–364. Imai, K., Kusakabe, M., Sakakura, T., Nakanishi, I., and Okada, Y. (1994) Susceptibility of tenascin to degradation by matrix metalloproteinases and serine proteinases. FEBS Lett., 352:216–218. Iruela-Arispe, M.L., Liska, D.J., Sage, E.H., and Bornstein, P. (1993) Differential expression of thrombospondin 1, 2, and 3 during murine development. Dev. Dyn., 197:40–56. Joshi, P., Chung, C.-Y., Aukhil, I., and Erickson, H. P. (1993) Endothelial cells adhere to the RGD domain and the fibrinogen-like terminal knob of tenascin. J. Cell Sci., 106:389–400. Koyama, E., Leatherman, J.L., Shimazu, A., Nah, H.-D., and Pacifici, M. (1995) Syndecan-3, tenascin-C, and the development of cartilaginous skeletal elements and joints in chick limbs. Dev. Dyn., 203:152– 162. Kwan, K.M., Pang, M.K.M., Zhou, S., Cowan, S.K., Kong, R.Y.C., Pfordte, T., Olsen, B.R., Sillence, D.O., Tam, P.P.L., and Cheah, K.S.E. (1997) Abnormal compartmentalization of cartilage matrix components in mice lacking collagen X: Implications for function. J. Cell Biol., 136:459–471. Liaw, L., Birk, D.E., Ballas, C.B., Whitsitt, J.S., Davidson, J.M., and Hogan, B.L.M. (1998) Altered wound healing in mice lacking a functional osteopontin gene (spp1). J. Clin. Invest., 101:1468–1478. Mackie, E.J., and Ramsey, S. (1996) Expression of tenascin in jointassociated tissues during development and postnatal growth. J. Anat., 188:157–165. Mackie, E.J., and Tucker, R.P. (1992) Tenascin in bone morphogenesis: Expression by osteoblasts and cell type-specific expression of splice variants. J. Cell Sci., 103:765–771. Mackie, E.J., Thesleff, I., and Chiquet-Ehrismann, R. (1987) Tenascin is associated with chondrogenic and osteogenic differentiation in vivo and promotes chondrogenesis in vitro. J. Cell Biol., 105:2569– 2579. Maddox, B.K., Keene, D.R., Sakai, L.Y., Charbonneau, N.L., Morris, N.P., Ridgway, C.C., Boswell, B.A., Sussman, M.D., Horton, W.A., Bächinger, H.P., and Hecht, J.T. (1997) The fate of cartilage oligomeric matrix protein is determined by the cell type in the case of a novel mutation in pseudoachondroplasia. J. Biol. Chem., 272:30993– 30997. Melnick, M., Jaskoll, T., Brownell, A.G., Macdougall, M., Bessem, C., and Slavkin, H.C. (1981) Spatiotemporal patterns of fibronectin distribution during embryonic development. I. Chick limbs. J. Embryol. Exp. Morphol., 63:193–206. Milev, P., Fischer, D., Häring, M., Schulthess, T., and Margolis, R.K. (1997) The fibrinogen-like globe of tenascin-C mediates its interactions with neurocan and phosphacan/protein-tyrosine phosphatase␤. J. Biol. Chem., 272:15501–15509. O’Shea, K.S., and Dixit, V.M. (1988) Unique distribution of the extracellular matrix component thrombospondin in the developing mouse embryo. J. Cell Biol., 107:2737–2748. Pacifici, M., Iwamoto, M. Golden, E.B., Leatherman, J.L., Lee, Y.-S., and Chuong, C.-M. (1993) Tenascin is associated with articular cartilage development. Dev. Dyn., 198:123–134. Prieto, A.L., Jones, F.S., Cunningham, B.A., Crossin, K.L., and Edelman, G.M. (1990) Localization during development of alternatively spliced forms of cytotactin mRNA by in situ hybridization. J. Cell Biol., 111:685–698. Pennypacker, J.P., Hassell, J.R., Yamada, K.M., and Pratt, R.M. (1979) The influence of an adhesive cell surface protein on chondrogenic expression in vitro. Exp. Cell Res., 121:411–415. Rathjen, F.G., Wolff, J.M., and Chiquet-Ehrismann, R. (1991) Restrictin: A chick neural extracellular matrix protein involved in cell attachment co-purifies with the cell recognition molecule F11. Development, 113:151–164. Saga, Y., Yagi, T., Ikawa, Y., Sakakura, T., and Aizawa, S. (1992) Mice develop normally without tenascin. Genes Dev., 6:1821–1831. Salter, D.M. (1993) Tenascin is increased in cartilage and synovium from arthritic knees. Br. J. Rheumatol., 32:780–786. Salter, D.M., Godolphin, J.L., and Gourlay, M.S. (1995) Chondrocyte heterogeneity: Immunohistologically defined variation of integrin expression at different sites in human fetal knees. J. Histochem. Cytochem., 43:447–457. Schwarzbauer, J.E., Patel, R.S., Fonda, D., and Hynes, R.O. (1987) Multiple sites of alternative splicing of the rat fibronectin gene transcript. EMBO J., 6:2573–2580. Siri A., Knäuper V., Veirana N., Caocci F., Murphy G., and Zardi L. (1995) Different susceptibility of small and large human tenascin-C isoforms to degradation by matrix metalloproteinases. J. Biol. Chem., 270:8650–8654. Swalla, B.J., and Solursh, M. (1984) Inhibition of limb chondrogenesis by fibronectin. Differentiation, 26:42–48. Tavella, S., Bellese, G., Castagnola, P., Martin, I., Piccini, D., Doliana, R., Colombatti, A., Cancedda, R., and Tacchetti, C. (1997) Regulated expression of fibronectin, laminin and related integrin receptors during the early chondrocyte differentiation. J. Cell Sci., 110:2261– 2270. Thesleff, I., Kantomaa, T., Mackie, E.J., and Chiquet- Ehrismann, R. (1988) Immunohistochemical localization of the matrix glycoprotein tenascin in the skull of the growing rodent. Arch. Oral Biol., 33:383–390. Tucker, R.P. (1993) The in situ localization of tenascin splice variants and thrombospondin 2 mRNA in the avian embryo. Development, 117:347–358. Tucker, R.P., Spring J., Baumgartner S., Martin D., Hagios C., Poss P.M., and Chiquet-Ehrismann, R. (1994) Novel tenascin variants with a distinctive pattern of expression in the avian embryo. Development, 120:637–647. Tucker, R.P., Adams, J.C., and Lawler, J. (1995) Thrombospondin-4 is expressed by early osteogenic tissues in the chick embryo. Dev. Dyn., 203:477–490, Tucker, R.P., Hagios, C., Chiquet-Ehrismann, R., and Lawler, J. (1997) In situ localization of thrombospondin-1 and thrombospondin-3 transcripts in the avian embryo. Dev. Dyn., 208:326–337. Vaughan, L, Huber, S., Chiquet, M., and Winterhalter, K.H. (1987) A major, six-armed glycoprotein from embryonic cartilage. EMBO J., 6:349–353. Weber, P., Montag, D., Schachner, M., and Bernhardt, R.R. (1998) Zebrafish tenascin-W, a new member of the tenascin family. J. Neurobiol., 35:1–16. West, C.M., Lanza, R., Rosenbloom, M., Lowe, M., and Holtzer, H. (1979) Fibronectin alters the phenotypic properties of cultured chick embryo chondroblasts. Cell, 17:491–501.