Syndecan-3 in Limb Skeletal DevelopmentROBERT A. KOSHER*Department of Anatomy, University of Connecticut Health Center, Farmington, CT 06030

KEY WORDS syndecan-3; chondrogenesis; osteogenesis; joint formation; ossification

ABSTRACT Syndecan-3 is a member of a family of heparan sulfate proteoglycans that functionas extracellular matrix receptors and as co-receptors for growth factors and signalling molecules. Avariety of studies indicate that syndecan-3 is involved in several aspects of limb morphogenesis andskeletal development. Syndecan-3 participates in limb outgrowth and proliferation in response tothe apical ectodermal ridge; mediates cell-matrix and/or cell-cell interactions involved in regulatingthe onset of chondrogenesis; may be involved in regulating the onset of osteogenesis and jointformation and, plays a role in regulating the proliferation of epiphyseal chondrocytes duringendochondral ossification. Microsc. Res. Tech. 43:123–130, 1998. r 1998 Wiley-Liss, Inc.

INTRODUCTIONSyndecan-3 is a member of a family of four integral

membrane heparan sulfate proteoglycans that containhighly conserved cytoplasmic and transmembrane do-mains, and share other structural characteristics (seeBernfield et al., 1992; David, 1993; Carey, 1995 forreviews). Members of the syndecan family includingsyndecan-3 function as extracellular matrix receptors,and as co-receptors for growth factors (Bernfield et al.,1992; David, 1993; Carey, 1995). Thus, syndecan-3, andother syndecan family members, may mediate theinteraction of cells with extracellular components andsignalling molecules that control cell shape, adhesion,proliferation, and differentiation (Bernfield et al., 1992;David, 1993; Carey, 1995). As summarized in this briefreview, a variety of studies indicate that syndecan-3 isinvolved in several aspects of limb morphogenesis andskeletal development. Syndecan-3 participates in limboutgrowth and proliferation in response to the apicalectodermal ridge; mediates cell-matrix and/or cell-cellinteractions involved in regulating the onset of chondro-genesis; may be involved in regulating the onset ofosteogenesis and joint formation and, plays a role inregulating the proliferation of epiphyseal chondrocytesduring endochondral ossification.

STRUCTURE OF SYNDECAN-3A cDNA has been isolated from a chicken limb bud

cDNA library that appears to encompass the entireprotein coding sequence of syndecan-3 mRNA (Gould etal., 1992, 1995). This syndecan-3 cDNA encodes a 405amino acid core protein with a predicted molecularweight of 43,002 Da, making syndecan-3 the largestmember of the syndecan family described. Syndecan-3core protein possesses a 33 amino acid cytoplasmicdomain potentially associated with the cytoskeletonand a 25 amino acid hydrophobic transmembrane do-main (Fig. 1), and these domains are highly similar tothose of the other members of the syndecan family(Gould et al., 1992, 1995). The 347 amino acid extracel-lular domain of syndecan-3 has a secretory signalsequence and eight putative glycosaminoglycan (GAG)attachment sites arranged in N-terminal and C-terminalclusters (Fig. 1) (Gould et al., 1995). These GAG chains

are potential sites of interaction with other extracellu-lar matrix or cell surface molecules, and also arepotential sites of interaction with heparin-bindinggrowth factors and signalling molecules (see below). Inaddition, syndecan-3 contains a threonine, serine, pro-line (T,S,P)-rich domain located between its N-terminaland C-terminal GAG attachment clusters that closelyresembles similar domains in mucin-like proteins inwhich O-linked oligosaccharides are bound to the T andS residues (Fig. 1) (Gould et al., 1992, 1995). Thispotentially highly glycosylated mucin-like domain rep-resents a potential functional domain involved in cell-cell or cell-matrix interactions that is not present inother members of the syndecan family. The ectodomainof syndecan-3 also has a single dibasic protease-cleavage site adjacent to the transmembrane domain,which might be involved in the regulated shedding ofthe ectodomain (Fig. 1) (Gould et al., 1992, 1995).

Syndecan-3 antibodies recognize a proteoglycan iso-lated from embryonic chicken brain with an averagemolecular weight of 170 kDa on SDS-PAGE (Gould etal., 1995). In contrast, syndecan-3 isolated from day 6–7chick limb buds migrates as a broad band at about 250kD (Gould et al., 1995), suggesting that syndecan-3may be polymorphic from tissue to tissue with respectto the number and/or size of GAG chains, as has beendemonstrated for syndecan-1 (Bernfield et al., 1992).Syndecan-3 is a heparan sulfate (HS) proteoglycan,since its electrophoretic mobility is reduced to 120 kDaafter treatment with nitrous acid or heparitinase tospecifically remove HS GAG chains (Gould et al., 1995).ChondroitinaseABC has no effect on its mobility indicat-ing the absence of chondroitin sulfate GAG chains(Gould et al., 1995). Total chemical deglycosylation withTFMS results in a further reduction in its mobilitysuggesting the presence of additional carbohydrate onsyndecan-3 (Gould et al., 1995). This carbohydrate maybe associated with oligosaccharide chains in the T,S,P-

Contract grant sponsor: NIH; Contract grant numbers: HD 22896, HD 22610.*Correspondence to: Robert A. Kosher, Ph.D., Department of Anatomy, Univer-

sity of Connecticut Health Center, Farmington, CT 06030. E-mail:kosher@sun.uchc.edu

Received 3 August 1998; accepted in revised form 6 August 1998.

MICROSCOPY RESEARCH AND TECHNIQUE 43:123–130 (1998)

r 1998 WILEY-LISS, INC.

rich, mucin-like domain of syndecan-3 core protein. Themultiple functional domains of syndecan-3, as well asits temporal and spatial pattern of expression (seebelow), make it an ideal candidate for regulating manyof the cell-cell and cell-matrix interactions involved inlimb morphogenesis and skeletal differentiation.

OVERVIEW OF SYNDECAN-3 EXPRESSIONDURING LIMB MORPHOGENESIS AND

SKELETAL DEVELOPMENTIn situ, Northern, and dot blot hybridization analy-

ses, as well as immunohistochemistry, have been usedto examine the spatial and temporal pattern of expres-sion of syndecan-3 transcripts and protein during thedevelopment of the embryonic chick limb bud (Gould etal., 1995; Koyama et al., 1995, 1996) and during theprogression of limb cartilage differentiation in vitro(Gould et al., 1992). Syndecan-3 exhibits several dis-crete spatially and temporally regulated domains ofexpression that are consistent with its involvement inseveral aspects of limb morphogenesis and skeletaldevelopment, as follows: 1) At early stages of limbdevelopment, syndecan-3 is highly expressed by thedistal mesenchymal cells of the developing limb budthat are undergoing outgrowth and proliferation inresponse to the apical ectodermal ridge (AER) (Gould etal., 1995; Dealy et al., 1997), suggesting it may play animportant role in the reciprocal interactions betweenthe AER and subridge mesoderm that are required forthe outgrowth and patterning of the developing limbbud (Dealy et al., 1997); 2) Syndecan-3 is transientlyexpressed in high amounts during the formation of theprecartilage condensations of the skeletal elements ofthe limb in vitro (Gould et al., 1992) and in vivo (Gouldet al., 1995), suggesting it is involved in mediating thecell-cell and cell-matrix interactions involved in regulat-ing the onset of chondrogenesis (Seghatoleslami et al.,1996); 3) After the formation of the cartilage models ofthe bones of the limb, syndecan-3 is highly expressed inthe perichondrium (Gould et al., 1995; Koyama et al.,

1995, 1996), which contributes to appositional growthof the cartilage models by giving rise to newly differen-tiating chondrocytes, and which interacts with matur-ing chondrocytes in the cartilage models to regulate therate of differentiation of chondrocytes to hypertrophy;4) Syndecan-3 is highly expressed at sites of incipientjoint formation in the developing limb, suggesting itmay play a role in regulating the onset of joint develop-ment (Gould et al., 1995; Koyama et al., 1995); 5)Syndecan-3 is highly expressed in the inner layers ofthe periosteum along the diaphyses of the cartilagemodels of the limb where osteoblasts are differentiatingand depositing the periosteal bony collar, suggesting itmay be involved in regulating the onset of bone differen-tiation (Gould et al., 1995); 6) During endochondralossification, syndecan-3 is expressed by immature pro-liferating chondrocytes in the top zone of the growthplate, suggesting it may be involved in the maturationof chondrocytes during endochondral ossification byparticipating in regulation of the proliferative phase ofthe process (Shimazu et al., 1996).

OVERVIEW OF THE ONSET OF LIMBCARTILAGE DIFFERENTIATION

The apical ectodermal ridge (AER), a thickened cap ofectoderm along the distal tip of the developing limbbud, is required for the outgrowth and formation of theskeletal elements of the limb in their appropriateposition and sequence along the proximodistal axis(Saunders et al., 1948). A major function of the AER isto maintain the mesenchymal cells directly subjacent toit in an actively outgrowing, labile, undifferentiatedcondition (Kosher et al., 1979a; Solursh et al., 1981).When the mesenchymal cells in the central core of thelimb bud leave the AER’s influence, they initiate chon-drogenesis, the onset of which is characterized by atransient cellular condensation process in which thecells become closely juxtaposed prior to depositing acartilage matrix. During this condensation processintimate cell-cell and cell-matrix interactions occur

Fig. 1. Diagrammatic representation of the structure of chicken syndecan-3 and its multiple potentialfunctional domains.

124 R.A. KOSHER

that are necessary to trigger chondrogenic differentia-tion (Kosher, 1983; Solursh et al., 1983).

The onset of the critical condensation phase of chon-drogenesis may be initiated, at least in part, by aprogressive decline in the accumulation of extracellularhyaluronate (Kosher et al., 1981; Knudson and Toole,1985; Kulyk and Kosher, 1987). Precartilage condensa-tion is thought to be mediated by interactions amongseveral cell surface and extracellular matrix moleculesthat are transiently expressed during the process,including fibronectin (Tomasek et al., 1982; Kulyk etal., 1989b); type I collagen (Dessau et al., 1980); hepa-ran sulfate proteoglycans (HSPG) (Frenz et al., 1989);tenascin (Mackie et al., 1987); the chondroitin sulfateproteoglycan PG-M (versican) (Kimata et al., 1986);N-CAM (Widelitz et al., 1993), and N-cadherin (Ober-lender and Tuan, 1994).

The onset of condensation and chondrogenesis in thedeveloping limb may be regulated at least in part bymembers of the TGF-b family. TGF-bs 1, 2, and 3 arepotent promoters of the chondrogenic differentiation oflimb mesenchymal cells in vitro (Kulyk et al., 1989a;Leonard et al., 1991) and in vivo (Ganan et al., 1996),and can promote chondrogenesis even under cultureconditions in which chondrogenic differentiation doesnot normally occur (Kulyk et al., 1989a). Furthermore,TGF-bs are expressed just before and during condensa-tion in vitro (Leonard et al., 1991; Roark and Greer,1994), and thus may regulate chondrogenesis by promot-ing the expression of matrix molecules such as FN,HSPGs, and tenascin that are involved in mediatingthe formation of prechondrogenic condensations (Leo-nard et al., 1991). The homeobox-containing gene MHox,mutations in which impair skeletal development, hasbeen suggested to regulate the expression of cell adhe-sion molecules that mediate condensation formation(Martin et al., 1995).

Regulatory events occurring during condensationresult in the initiation of cartilage-specific gene expres-sion. Concurrent initiation of the expression of mRNAsfor the core protein of the cartilage proteoglycan aggre-can (Kosher et al., 1986a; Mallein-Gerin et al., 1988;Kulyk et al., 1991) and cartilage-specific type IX colla-gen (Kulyk et al., 1991), and a dramatic increase incartilage-characteristic type II collagen mRNA expres-sion (Kosher et al., 1986b; Nah et al., 1988) occurduring condensation in vitro and in vivo. Several regu-latory factors have been implicated in controlling carti-lage-specific gene expression during condensation in-cluding cAMP (Kosher et al., 1979b; Kosher and Savage,1980; Leonard and Newman, 1987; Rodgers et al., 1989;Kosher and Gay, 1985), gap junctional communication(Coelho and Kosher, 1991), and a cytoskeletal-mediatedchange in the shape of the cells from a flattenedmorphology to a rounded configuration (Zanetti andSolursh, 1984). The onset of cartilage-specific geneexpression is accompanied by initiation of the expres-sion of Sox9, a transcription factor that is a keyregulator of cartilage differentiation (Zhao et al., 1997;Ng et al., 1997; Lefebvre et al., 1997; Bell et al., 1997).Human Sox9 mutations result in severe skeletal malfor-mations (Foster et al., 1994; Kwok et al., 1995; Wagneret al., 1994). Sox9 appears to regulate chondrogenesisat least in part by controlling the expression of the typeII collagen gene by binding to a cartilage-specific en-

hancer element in the middle of the first nitron of thegene (Lefebvre et al., 1997; Bell et al., 1997). The HLHtranscription factor scleraxis (Cserjesi et al., 1995; Liuet al., 1997) and the homeobox-containing gene Cart-l(Zhao et al., 1993) have also been implicated in regulat-ing cartilage-specific gene expression at the onset ofchondrogenesis.

SYNDECAN-3 IN LIMB OUTGROWTH ANDPATTERNING IN RESPONSE TO THE AERSyndecan-3 is highly expressed by the distal mesen-

chymal cells of the limb bud undergoing proliferationand outgrowth in response to the AER (Gould et al.,1995; Dealy et al., 1997), a process that appears to bemediated at least in part by members of the FGF familyof growth factors (Niswander et al., 1993; Fallon et al.,1994; Crossley et al., 1996; Vogel et al., 1996). Recentstudies have indicated that FGFs need to interact withHSPGs in order to bind to and activate their specific cellsurface receptors (Yayon et al., 1991; Rapraeger et al.,1991; Quarto and Amalric, 1994; Olwin and Rapraeger,1992; Ornitz et al., 1995). Syndecan-3 can bind FGFs(Chernousov and Carey, 1993), suggesting that it mayplay an essential role in limb outgrowth by serving as aco-receptor for FGFs produced by the AER (Gould et al.,1995; Dealy et al., 1997).

The maintenance of syndecan-3 expression by thesubridge mesoderm of the developing limb bud isindeed dependent on the AER (Dealy et al., 1997).Surgical extirpation of the AER results in a cessation ofsyndecan-3 expression by the subridge mesoderm con-comitant with the cessation of limb outgrowth. Further-more, syndecan-3 expression is severely impaired in thedistal mesoderm of the limb buds of the amelic chickmutants limbless and wingless, which lack morphologi-cally-distinct and functional AERs capable of directinglimb outgrowth (Dealy et al., 1997). In addition, FGFscan substitute for the AER in maintaining the distalsubridge domain of syndecan-3 expression in normallimb buds from which the AER has been removed, andcan induce syndecan-3 expression in the subridge meso-derm of amelic mutant limb buds which lack functionalAERs (Dealy et al., 1997). These studies indicate thatthe expression of syndecan-3 by the subridge mesodermof the limb bud is regulated by FGFs produced by theAER.

Moreover, syndecan-3 antibodies inhibit the ability ofFGFs to promote the outgrowth and proliferation oflimb mesoderm (Dealy et al., 1997). Control explants oflimb mesoderm lacking an AER undergo little or nooutgrowth or proliferation, whereas the subridge meso-derm of such explants undergo considerable directedoutgrowth and proliferation in response to exogenousFGF-2. However, when FGF-treated explants are cul-tured in the presence of syndecan-3 antibodies, theiroutgrowth is dramatically inhibited and they exhibitlittle proliferation. Syndecan-3 antibodies reduce FGF-stimulated outgrowth and proliferation almost to con-trol levels (Dealy et al., 1997). These results suggestthat syndecan-3 plays an essential role in limb out-growth by mediating the interaction of FGFs producedby the AER with the underlying mesoderm of the limbbud (Dealy et al., 1997).

125SYNDECAN-3 IN LIMB SKELETAL DEVELOPMENT

THE ROLE OF SYNDECAN-3 IN REGULATINGTHE ONSET OF LIMB CARTILAGE

DIFFERENTIATIONSyndecan-3 is transiently expressed in high amounts

during the cellular condensation process that character-izes the onset of limb cartilage differentiation in vitro(Gould et al., 1992) and in vivo (Gould et al., 1995) (Fig.2). During condensation, limb mesenchymal cells be-come closely juxtaposed and undergo cell-cell and cell-matrix interactions that are necessary to trigger carti-lage differentiation and cartilage-specific geneexpression (see the previous section). The onset ofcondensation appears to be mediated by several cellsurface and extracellular matrix macromolecules thatare transiently expressed during the process (see theprevious section). The functional domains of synde-can-3 including its HS GAG chains and its glycosylatedmucin-like domain provide potential sites for mediatingthe cell-matrix interactions required for precartilagecondensation and cartilage differentiation (Gould et al.,1992).

To directly test the possible involvement of synde-can-3 in regulating the onset of limb chondrogenesis,the effect of polyclonal antibodies against a syndecan-3fusion protein on the chondrogenic differentiation ofchick limb mesenchymal cells in micromass culture hasbeen examined (Seghatoleslami et al., 1996). Synde-can-3 antiserum elicits a dose dependent inhibition ofthe accumulation of Alcian blue stainable cartilagematrix by high density limb mesenchymal cell micro-mass cultures (2 3 105 cells/10 µl) and a correspondingreduction in steady state levels of mRNAs for cartilage-characteristic type II collagen and the core protein ofthe cartilage proteoglycan aggrecan (Seghatoleslami etal., 1996). Proliferating cells are limited to the periph-ery of areas of cartilage matrix deposition in preim-mune serum-treated control cultures, whereas largenumbers of proliferating cells are uniformly distributedthroughout the undifferentiated cultures supplementedwith syndecan-3 antiserum.

Limb mesenchymal cells cultured at lower densities(1 3 105 cells/10 µl) in the presence of preimmuneserum form extensive precartilage condensations char-acterized by the close juxtaposition of rounded cells byday 2 of culture. In contrast, in the presence of synde-can-3 antiserum the cells fail to aggregate, but rather

remain flattened and spatially separated from oneanother, suggesting that syndecan-3 antibodies impairthe formation of precartilage condensations (Seghatole-slami et al., 1996). These studies indicate that synde-can-3 plays an important role in mediating the adhe-sive cell-matrix interactions required for the criticalcondensation phase of chondrogenesis.

Furthermore, syndecan-3 possesses a cytoplasmicdomain highly similar to that of syndecan-1 (Gould etal., 1992, 1995), which is associated with the cytoskel-eton (Bernfield et al., 1990). Thus, interaction of synde-can-3 with other ligands might be involved in the cellshape changes and cytoskeletal reorganization thatoccurs during condensation and that may facilitatechondrogenesis (Zanetti and Soulursh, 1984). Indeed,the cells of lower density micromass cultures treatedwith syndecan-3 antiserum are flattened, whereas thecondensed cells of preimmune cultures are rounded(Seghatoleslami et al., 1996).

Syndecan-3 antibodies inhibit chondrogenesis evenwhen limb mesenchymal cells are cultured at highdensities which passively allow close apposition of thecells (Seghatoleslami et al., 1996). Thus, syndecan-3may be involved not only in promoting condensation,but also in the intercellular signalling that occurs as aconsequence of condensation—perhaps by interactingwith heparin-binding growth factors such as BMPs thatregulate chondrogenesis (see the next section). Thus,syndecan-3 plays an important role in regulating theonset of limb chondrogenesis, perhaps by mediating thecell-cell and cell-matrix interactions required for conden-sation and subsequent cartilage differentiation (Segha-toleslami et al., 1996).

THE POSSIBLE ROLE OF SYNDECAN-3 INTHE PERICHONDRIUM AND IN THE ONSET

OF JOINT FORMATIONAfter the formation of precartilage condensations

and the initiation of chondrogenesis, the cartilageanlagen undergo rapid longitudinal and appositionalgrowth to form the cartilage models of the bones of thelimb (Mundlos and Olsen, 1997; Erlebacher et al.,1995). The developing cartilage models are surroundedby a longitudinally-oriented group of flattened cellscalled the perichondrium. The perichondrium may con-tribute to appositional growth of the cartilage modelsby giving rise to newly differentiating chondrocytes.Furthermore, signals between the perichondrium andthe maturing chondrocytes in the cartilage modelsregulate the rate of differentiation of chondrocytes tohypertrophy (Vortkamp et al., 1996; Long and Linsen-mayer, 1998; Lanske et al., 1996).

After its transient expression during the formation ofthe precartilage condensations of the skeletal elementsof the limb (Fig. 2), syndecan-3 expression ceases in thechondrocytes of the early cartilage models (Gould et al.,1995). However, syndecan-3 is very highly expressed inthe perichondrium (Gould et al., 1995; Koyama et al.,1995, 1996). Although the role of syndecan-3 in theperichondrium is not known, one interesting possibilityis that it may modulate or mediate the activity ofheparin-binding signalling molecules that mediate thefunctions of the perichondrium. In this regard, severalmembers of the BMP family including BMPs-2, -4, and

Fig. 2. Expression of syndecan-3 transcripts detectable by in situhybridization (A) and syndecan-3 protein detectable by immunohisto-chemistry.

126 R.A. KOSHER

-7 that are expressed in the perichondrium appear tomediate the recruitment of cells into the cartilagemodels (Duprez et al., 1996; Macias et al., 1997; Brunetet al., 1998), and BMPs also appear to be involved in aregulatory loop with Indian hedgehog and PTHrP toregulate signalling between the perichondrium andmaturing chondrocytes that controls the rate of chondro-cyte maturation and hypertrophy (Vortkamp et al.,1996; Zou et al., 1997). It has recently been demon-strated that BMP-2 contains a heparin-binding site atits N-terminal end which plays a role in modulating itsbiological activity (Ruppert et al., 1996). Similar do-mains are present in many other members of the BMPfamily (Ruppert et al., 1996). Thus, the activity ofBMPs during skeletal development can be potentiallymodulated by HSPGs at the cell surface or in theextracellular matrix. Therefore, it is quite possible thatsyndecan-3 expressed in the perichondrium mightmodulate the activity of BMPs or perhaps other heparin-binding signalling molecules that regulate the growthand differentiation of the cartilage models.

In addition to being expressed in the perichondrium,syndecan-3 is highly expressed at all of the sites ofincipient joint formation in the developing limb, suggest-ing it may be involved in the onset of joint differentia-tion (Gould et al., 1995; Koyama et al., 1995). Althoughthe role of syndecan-3 in joint formation is not clear, assuggested above, one possibility is that it might beinvolved in modulating the activity of signalling mol-ecules that regulate the process. It is noteworthy thatthe heparin-binding BMP-like signalling molecules Gdf5plays a crucial role in regulating the onset of jointformation (Storm and Kingsley, 1996).

THE POSSIBLE ROLE OF SYNDECAN-3IN REGULATING THE ONSET OF BONE

DIFFERENTIATION IN THEDEVELOPING LIMB

The perichondrium located along the diaphyses of thecartilage models becomes the periosteum, which is thesource of osteogenic cells that deposit a layer of bonearound the cartilage rudiment (Scott-Savage and Hall,1979; Holder, 1978; Pechak et al., 1986; Bruder andCaplan, 1989). The early periosteum consists of anouter stacked cell layer consisting of osteoprogenitorcells that give rise to an inner layer of osteoblasts thatsecrete the periosteal bony collar (Pechak et al., 1986;Bruder and Caplan, 1989). Thus, the periosteal bonycollar, which is the first bone formed in the limb,develops by intramembranous ossification in whichosteoblasts directly differentiate from osteogenic precur-sor cells.

Syndecan-3 transcripts and protein are very highlyexpressed in the inner layers of the periosteum whereosteoblasts are differentiating and depositing the peri-osteal bony collar (Fig. 3) (Gould et al., 1995; Koyama etal., 1995, 1996), suggesting it may be involved in theonset of bone differentiation (Gould et al., 1995). Synde-can-3 is also expressed in developing calvaria (Gould etal., 1995) and at the onset of the osteogenic differentia-tion of primary cultures of embryonic chick calvarialcells (Lictler and Kosher, unpublished). The onset ofsyndecan-3 expression in calvaria cell cultures corre-lates with the onset of expression of transcripts for

several bone-specific proteins including osteocalcin,bone sialoprotein, osteopontin, and alkaline phospha-tase. These studies indicate that syndecan-3 may in-deed play an important role in regulating osteoblastdifferentiation.

One manner in which syndecan-3 might be involvedin osteogenesis is by modulation or mediation of theactivity of heparin-binding growth factors that areinvolved in regulating the process. A heparin-bindingsignalling molecule that, like syndecan-3, is highlyexpressed by differentiating osteoblasts during theformation of the periosteal bony collar is HB-GAM(Dreyfus et al., 1998). HB-GAM also called pleiotrophinis a member of a small family of developmentallyregulated heparin-binding signalling molecules, theother member of which is midkine also known asretinoic acid-induced heparin-binding factor (RIHB)with which it shares 50% sequence identity (Li et al.,1990; Muramatsu and Muramatsu, 1991; Raulais et al.,1991). HB-GAM exhibits discrete temporally- and spa-tially-regulated domains of expression during embryo-genesis that suggest it plays a regulatory role in themorphogenesis and differentiation of several develop-ing organs, particularly during epithelial-mesenchy-mal tissue interactions (Mitsiadis et al., 1995b; Naka-moto et al., 1992; Muramatsu, 1993; Vanderwinden etal., 1992). Its pattern of expression in the developingbrain and its ability to promote neurite outgrowthsuggest it is involved in the growth and development ofthe nervous system (Li et al., 1990; Rauvala et al., 1994;Nolo et al., 1995).

Syndecan-3 has been identified as a high affinity cellsurface receptor for HB-GAM (Raulo et al., 1994), andmediates the neurite promoting outgrowth of HB-GAMby influencing an intracellular signal transductionpathway (Kinnunen et al., 1998). Thus, the co-expres-sion of syndecan-3 and HB-GAM by differentiatingosteoblasts during periosteal bone formation suggeststhat interactions between them may play an importantrole in regulating the onset of osteogenesis. HB-GAM isalso expressed by differentiating osteoblasts during theintramembranous ossification of the mandibular, maxil-lary, and cranial bones (Mitsiadis et al., 1995a), furthersuggesting it may play an important role in regulatingthe onset of bone differentiation.

Fig. 3. Expression of syndecan-3 transcripts (A8) and syndecan-3protein during periosteal bone formation. (A) bright field; (A8) darkfield.

127SYNDECAN-3 IN LIMB SKELETAL DEVELOPMENT

THE ROLE OF SYNDECAN-3 INCHONDROCYTE MATURATION DURING

ENDOCHONDRAL OSSIFICATIONAs the periosteal bony collar is being deposited

around the diaphyses of the cartilage models, chondro-cytes in the middle of the diaphyses undergo differentia-tion to prehypertrophic chondrocytes which then be-come hypertrophic and initiate expression of type Xcollagen (Mundlos and Olsen, 1997; Erlebacher et al.,1995). As the area of hypertrophy enlarges, the matrixsurrounding the hypertrophic chondrocytes calcifies,and blood vessels arising from the periosteum invadethe area of hypertrophy. Some of the invading cellsdifferentiate into marrow cells including hematopoieticstem cells and others into osteoblasts and osteoclastswhich replace the calcified cartilage with bone, a pro-cess called endochondral ossification. This primarycenter of ossification in the middle of the cartilagemodel expands towards the epiphyseal ends of thecartilage models. Several zones of chondrocytes indifferent phases of maturation are present in theepiphyses and subsequently in the growth plate. Areserve zone of small relatively inactive chondrocytes isadjacent to a proliferating zone of actively dividingchondrocytes. The proliferation of chondrocytes and therecruitment of reserve chondrocytes to take part inproliferation may be regulated at least in part bymembers of the IGF and FGF families of growth factors(Baker et al., 1993; Liu et al., 1993; Shiang et al., 1994;Deng et al., 1996; Naski et al., 1996).

Several observations suggest that syndecan-3 mayplay a role in chondrocyte proliferation during endochon-dral bone formation (Shimazu et al., 1996). Syndecan-3mRNA and protein expression is spatially restricted toimmature proliferating chondrocytes in the top zone ofthe growth plate in chick embryo tibia, and little or noexpression is detectable in either articular chondro-cytes or mature hypertrophic chondrocytes (Shimazu etal., 1996). Chondrocytes proliferating in vitro exhibithigh syndecan-3 expression, and its expression is mark-edly down-regulated upon induction of maturation byvitamin C treatment (Shimazu et al., 1996). Further-more, heparinase I or III treatment which shouldremove the heparan sulfate GAG chains of syndecan-3inhibits the ability of FGF-2 to stimulate both synde-can-3 expression and proliferation in chondrocytes invitro (Shimazu et al., 1996). These studies suggest thatsyndecan-3 participates in the maturation of chondro-cytes during endochondral ossification by regulatingthe proliferative phase of the process.

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