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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: e.mackie@vet.unimelb.edu.au
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.
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