1 Department of Craniofacial Development, Guy's, King's and St Thomas' School of
Dentistry, Floor 28 Guy's Tower, Guy's Hospital, London Bridge, London SE1
9RT, UK
2 Department of Molecular Biology, Kawasaki Medical School, Kurashiki,
Japan
3 Developmental Biology Institute, LGPD, Campus de Luminy, case 107, University
of Aix-Marseille II, 13288 Marseille Cedex 09, France
* Author for correspondence (e-mail: pfrancis{at}hgmp.mrc.ac.uk)
Accepted 6 September 2002
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Summary |
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Key words: Wnt, Ihh, Chondrocyte differentiation
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Introduction |
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Differentiation is controlled by signals from both the prehypertrophic
chondrocytes and perichondrium. The secreted factor Ihh expressed in
the prehypertrophic region negatively regulates chondrocyte differentiation
through the induction of PTHrP in the periarticular perichondrium
(Vortkamp et al., 1996). Thus,
overexpression of Ihh in the developing chick limb prevents chondrocyte
terminal differentiation (Vortkamp et al.,
1996
). However, experiments in vitro have shown that, in the
absence of the perichondrial signal, Ihh promotes terminal differentiation
suggesting that Ihh may signal directly to the chondrocytes to promote
differentiation (Stott and Chuong,
1997
; Akiyama et al.,
1999
). This has been supported by the observation that the
hedgehog receptor patched-1, which is induced by Ihh signalling, is
expressed in the chondrocytes adjacent to Ihh-expressing cells
(Vortkamp et al., 1998
). Ihh
can also act locally on the adjacent perichondrium to initiate bone collar
formation and can promote the proliferation of early differentiating
chondrocytes in a PTHrP-independent manner
(St-Jacques et al., 1999
;
Karp et al., 2000
;
Chung et al., 2001
).
Other factors that control chondrocyte differentiation include members of
the Wnt and fibroblast growth factor families. The Wnt family of proteins
consists of at least 22 cysteine-rich, secreted glycoproteins which
participate in a multitude of developmental processes (for reviews, see
Cadigan and Nusse, 1997;
Wodarz and Nusse, 1998
). Of
these, Wnt4, -5a, -5b and -14, together with the secreted
Wnt antagonist Frzb, are expressed in the developing skeleton and
have been shown to have distinct roles
(Hoang et al., 1996
;
Hoang et al., 1998
;
Kawakami et al., 1999
;
Wada et al., 1999
;
Yamaguchi et al., 1999
;
Ladher et al., 2000
;
Hartmann and Tabin, 2000
;
Hartmann and Tabin, 2001
).
Wnt4, which is expressed in the developing joints, has been shown to
accelerate hypertrophy whilst Wnt5a, which is expressed in the
perichondrium, delays hypertrophy
(Kawakami et al., 1999
;
Hartmann and Tabin, 2000
).
Finally, Wnt14, which is expressed in the developing joint interzone,
has been implicated in the initial steps of joint development
(Hartmann and Tabin, 2001
). In
addition, Wnt1 and -7a have been shown to block chondrogenesis in vitro and/or
in vivo (Rudnicki and Brown,
1997
; Stott et al.,
1999
; Tufan and Tuan,
2001
). Here we have further examined the role of Wnt signalling
during chondrogenesis using the in vitro micromass system and in vivo
misexpression studies in the developing chick limb.
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Materials and Methods |
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Production of retroviruses
Concentrated retroviral supernatants were prepared by the method of Logan
and Francis-West (Logan and Francis-West,
1999) and were used at a titre of greater than 108 pfu
for the in vitro micromass assays. The RCAS BP(A)-Wnt5b retrovirus encoded
mouse Wnt5b. The other retroviral constructs have been described previously:
ß-catenin (Kengaku et al.,
1998
), Wnt1 (Rudnicki and
Brown, 1997
), Wnt4 (Hartmann
and Tabin, 2000
), Wnt5a
(Kawakami et al., 1999
), Wnt11
(K. Anakwe, L. Robson, P. Buxton et al., unpublished) and Ihh
(Vortkamp et al., 1996
). For
control retroviruses we used RCAS BP(A) expressing enhanced-green fluorescent
protein (Clontech) or RCAS BP(A) vector.
Micromass cultures
Micromass cultures were prepared as described by Francis-West et al.
(Francis-West et al., 1999a).
However, the cells were resuspended at 2x107 cells/ml in
concentrated viral supernatant before plating in 10 µl aliquots in 24-well
dishes (Nunc). After 1 hour, cultures were flooded with 1 ml medium (DMEM
supplemented with 10% FBS, 1% chick serum, 50 U/ml penicillin, 50 µg/ml
streptomycin) and cultured at 37°C and 5% CO2/air in a
humidified atmosphere.
Retroviral transgene expression was verified by RT-PCR. RNA was extracted
from cultures and DNase-treated as described by Tufan and Tuan
(Tufan and Tuan, 2001) and
Tufan et al. (Tufan et al.,
2002
). One µg total RNA was reverse transcribed using 1.0 µl
M-MLV reverse transciptase (Promega) and 500 ng random hexamers (Promega)
according to the manufacturer's protocol. Retroviral transgene expression was
determined using primers (25 pmol of each) specific for the transgene
(Table 1), 1 µl cDNA, 1.5 mM
MgCl2, 0.2 mM each dNTP, 1x Thermo-reaction buffer (Promega)
and 1.0 U Taq polymerase (Promega) in a total volume of 50 µl. PCR
was performed in a Perkin-Elmer DNA Thermal Cycler using one cycle of 94°C
for 5 minutes, followed by 25 (GAPDH), 35 (transgenes after 48, 72 hours) or
40 (transgenes after 24 hours) cycles of one minute for denaturing (94°C),
one minute for annealing (TA;
Table 1), and one minute for
elongation (72°C). Negative control PCR reactions were performed in
parallel: these contained all components of the reaction except for cDNA.
|
To assess the effects of Wnt overexpression on the initiation of
chondrogenesis, micromass cultures were cultured for three days after which
matrix production was assessed by alcian blue staining
(Francis-West et al., 1999a).
The alcian blue dye was extracted with 4 M GuHCl and quantified by measuring
its absorbance at 590 nm. The cultures were then re-stained with alcian blue
dye and the number of nodules was counted.
Alternatively, for analysis of terminal differentiation, cultures were
grown for seven days and then fixed in 95% MeOH for 10 minutes. For
histological detection of alkaline phosphatase activity, the cultures were
washed in PBS and stained in Naphthol AS-MX phosphate in 0.4 M Tris-HCl (pH
8.3) and Fast Red Violet LB salt at 37°C in the dark for 10 to 15 minutes.
Additionally, ALP activity was determined using the method described by Leboy
et al. (Leboy et al., 1989).
Briefly, each micromass culture was lysed in 500 µl lysis buffer and the
ALP activity quantified in a 50 µl aliquot using the substrate
p-nitrophenyl phosphate. For type X collagen immunohistochemistry,
seven day cultures were fixed with MeOH as above, then incubated with type X
collagen antibody [diluted 1:500; a gift from Alvin Kwan, University of
Cardiff, UK (Kwan et al.,
1986
)] followed by incubation with Texas red-conjugated anti-mouse
IgG antibody (diluted to 20 µg/ml; Vector Labs). Fluorescent antibody
staining was visualised on an Axiovert 200 fluorescence microscope (Zeiss)
using exactly the same exposure time for all photomicrographs.
To determine the effects of Wnt overexpression on cell proliferation, 24-, 48- or 72-hour-old retrovirus-infected micromass cultures were incubated with 10 µM 5-bromo-2'-deoxy-uridine (BrdU) for 30 minutes at 37°C. The cultures were fixed with ethanol, overlaid with 1% agarose to facilitate removal from the tissue culture surface, and processed into paraffin wax and sectioned at 8 µm. Sections were rehydrated, acidified in 2N HCl for 30 minutes and digested with 0.1% type XXIV protease (Sigma) for 60 seconds. Sections were then incubated overnight with biotinylated anti-BrdU antibody (diluted 1:50; Caltag Labs) which was detected using the Vectastain® Elite ABC kit (Vector Labs) in conjunction with the DAB peroxidase substrate kit (Vector Labs) according to the manufacturer's instructions. Sections were counterstained with toluidine blue O and the number of BrdU-labelled and unlabelled cells was counted.
Retroviral infection of limbs
Chick embryonic (O-line) fibroblasts (CEFs) were transfected with
retroviral DNA (encoding Wnt5a, Wnt5b, Wnt11, Ihh or a control construct) as
described in Logan and Francis-West (Logan
and Francis-West, 1999). After seven days when the cells were
fully infected, the CEFs were trypsinised from the culture plate, pelleted and
allowed to consolidate for one hour (Logan
and Francis-West, 1999
). The cell pellet was grafted into the
right wing bud of stage 18/19 embryos and the embryos allowed to develop until
day 10, during which time the replication-competent retrovirus spreads
throughout the limb (Duprez et al.,
1996
). The embryos were fixed in 4% PFA/PBS, and were processed
into paraffin wax and sectioned for analysis by in situ hybridisation as
above.
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Results |
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Wnt5b is detected by stage 28 throughout the developing perichondrium and in the adjacent mesenchyme (Fig. 1A,B). The perichondrial expression continues until at least stage 37 (Fig. 1E,F,I,J). By stage 30, Wnt5b expression is also found in a subset of prehypertrophic chondrocytes (Fig. 1E,F,I,J and data not shown). In the stage 40 tibiotarsus, Wnt5b is more widely expressed being found throughout the element except in the distal part consisting of the proliferating chondrocytes (Fig. 3B). Initially, Wnt5a transcripts are present around the developing cartilage condensations between stages 24 and 26 (data not shown). As the element differentiates, Wnt5a transcripts become restricted to the developing perichondrium (Fig. 2E,F and data not shown). In addition to the perichondrium, Wnt5a is detected in the joint interzone in the digits between stages 30 to 32 but is downregulated as the joint cavitates (Fig. 2E,F and data not shown).
Wnt4 transcripts are found in the developing joint and in
differentiating chondrocytes (Fig.
2A-D; Fig. 3A)
(Kawakami et al., 1999;
Hartmann and Tabin, 2000
).
Initially, at stage 28 in the elbow, Wnt4 expression is clearly seen
in the joint interzone and in the mesenchyme surrounding the joints
(Fig. 2A,B). Between stages 30
to 40, Wnt4 transcripts become localised to the surface articular
chondrocytes and the joint capsule (Fig.
2C,D). At stage 40 in the tibiotarsus, Wnt4 is expressed
by hypertrophic chondrocytes in an area undergoing lateral resorption
(Fig. 3A).
Wnt11 transcripts are not detectable within the developing limb elements at stage 28 but are present in the developing humerus at stage 30; this expression continues until at least stage 37 (Fig. 1A,D,E,H,I,L and data not shown). Like Wnt5b, Wnt11 is expressed in a subset of prehypertrophic chondrocytes but, in contrast to Wnt5b, its expression is restricted to a subpopulation of prehypertrophic chondrocytes just beneath the perichondrium (compare Fig. 1F,J with 1H,L). At stage 40 in the tibiotarsus, when the element has fully differentiated, expression of Wnt11 within the prehypertrophic chondrocytes is highest adjacent to the perichondrial ring, a structure which marks the edge of the bone front (Fig. 3C).
The expression patterns of Wnt5b and Wnt11 correlate with
that of Ihh suggesting that there may be a relationship between Wnt
and Ihh signalling in developing cartilage. To investigate this, we compared
the timing and distribution of Wnt5b, Wnt11 and Ihh
expression. This revealed that Ihh expression is first apparent at
stage 26 as previously reported [data not shown
(Vortkamp et al., 1996;
Vortkamp et al., 1998
)] whilst
Wnt5b and Wnt11 transcripts are not detectable within the
developing humerus until stage 30 when they are expressed within the
Ihh-expressing domain (Fig.
1E-L).
Wnts have differential effects on chondrogenesis
To investigate how Wnts control chondrogenesis, Wnts were overexpressed
using the chick replication-competent retrovirus RCAS BP in the in vitro
micromass assay system. As the expression patterns of Wnt5b and
Wnt11 suggested a potential relationship with Ihh, we compared their
effects with that of Ihh.
Mesenchymal cells from stage 23/24 wing buds were mixed with concentrated retrovirus particles and plated in high-density micromass cultures. Expression of the Wnt transgenes at 24, 48 and 72 hours was investigated by RT-PCR analysis using PCR primer combinations specific to the transgene. This showed that there was either a low level (control, Wnt5b, -11) or undetectable levels (Wnt4, -5a) of transgene expression after 24 hours but that all transgenes were expressed within 48 hours of infection (Fig. 4A).
|
After three days, cultures were fixed and stained with alcian blue which
stains sulphated glycosaminoglycans in the matrix, and the number of nodules
was counted to determine the effect of Wnts on the initiation of
chondrogenesis. As reported previously, Wnt1 inhibited chondrocyte
differentiation as judged by the reduction in both alcian blue staining and
the number of cartilaginous nodules (Fig.
4B,C; P<0.0001)
(Rudnicki and Brown, 1997).
Likewise, ß-catenin, a downstream mediator of Wnt1 signalling, also
dramatically reduced chondrogenic differentiation (P<0.05; data
not shown). Wnt4 reduced alcian blue staining and the number of nodules to
approximately 70% of control (P<0.0005;
Fig. 4), while Wnt11 had no
detectable effect in these assays (Fig.
4B,C). Wnt5a, Wnt5b and Ihh enhanced alcian blue staining by
1.5-fold (P<0.005, P<0.0001 and P<0.0001,
respectively; Fig. 4B,C); however, while Wnt5a and -5b increased the number of nodules by 1.5-fold
(P<0.005 and P<0.0001, respectively;
Fig. 4B,C), Ihh had no
significant effect on nodule number (Fig.
4B,C).
Ihh has been shown to promote terminal differentiation in vitro
(Stott and Chuong, 1997;
Akiyama et al., 1999
). As
Ihh, Wnt5b and Wnt11 are co-expressed in the prehypertrophic
chondrocytes, with Ihh being expressed prior to Wnt5b and
Wnt11, this raised the possibility that these Wnts may mediate Ihh
activity to promote chondrocyte hypertrophy in vitro. To investigate this,
type X collagen expression and alkaline phosphatase (ALP) activity, which are
markers of chondrocyte hypertrophy, were analysed after seven days of culture.
We also investigated the effect of Wnt4 since this has been shown to
accelerate chondrogenesis when overexpressed in chick limbs in vivo
(Hartmann and Tabin,
2000
).
After seven days, Ihh promoted ALP activity
(Fig. 5A,B;
P<0.001) as shown previously for sonic hedgehog (Shh) in micromass
cultures (Stott and Chuong,
1997). Wnt4 also increased ALP activity but not to the extent of
Ihh cultures (Fig. 5A,B;
P<0.005). In contrast, Wnt5a, Wnt5b and Wnt11-infected cultures
did not detectably alter the levels of ALP activity
(Fig. 5A,B). Similarly,
whole-mount staining of the cultures revealed that type X collagen was
expressed by all cultures, including the control, but was upregulated in
cultures misexpressing Ihh and Wnt4. In contrast, type X collagen levels did
not appear to be changed by Wnt5a, -5b or -11 misexpression
(Fig. 5C; n=6 from two
experiments).
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Overexpression of Wnts does not change proliferation in vitro
Overexpression of Wnt5a and Wnt5b in vitro increased the number of nodules
and hence initiated the first steps of chondrogenesis
(Fig. 4B,C). This may occur by
an increase in cell proliferation and/or cell adhesion. Conversely, Wnt4
decreased the number of cartilage nodules. To investigate the possible
mechanism, we pulse-labelled Wnt4-, Wnt5a- and Wnt5b-infected micromass
cultures at 24, 48 and 72 hours with bromodeoxyuridine (BrdU) to label the
proliferating cells. We also analysed the effect of Wnt11 in parallel. This
revealed that, following Wnt misexpression, there were no detectable changes
in the overall proliferation in any of the Wnt-infected micromass cultures
when compared to the control cultures (Fig.
6 and data not shown).
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Ihh and Wnt signalling controls chondrocyte differentiation in
parallel pathways in vivo
The in vitro analyses suggested that Ihh promotes terminal differentiation
through Wnt5b- and Wnt11-independent pathways. However, it is possible that
these Wnts may mediate the other functions of Ihh. To investigate this we
misexpressed Ihh in the developing limb bud using the replication-competent
retrovirus RCAS BP to determine whether Ihh induces either Wnt5b or
-11 expression. To infect limbs, fibroblasts infected with the Ihh
retrovirus were grafted into the developing limb bud between stages 18 and 19,
24 hours prior to the condensation of the developing cartilage elements. This
resulted in ectopic expression of Ihh around the cartilage elements
and in small domains within the cartilage itself (n=7;
Fig. 7C,D)
(Duprez et al., 1996;
Vortkamp et al., 1996
).
Embryos were allowed to grow to day 10 at which time they were analysed
histologically for changes in skeletal development and by in situ
hybridisation analysis on tissue sections using molecular markers which are
expressed at specific stages of differentiation.
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Ihh misexpression resulted in truncation and dysmorphology of the skeletal
elements which consisted of disorganised chondrocytes
(Fig. 7A,B) (Vortkamp et al., 1996).
Chondrocyte differentiation was delayed prior to prehypertrophy as indicated
by the lack of endogenous Ihh and type X collagen expression
(Fig. 7C,D and data not shown)
(Vortkamp et al., 1996
); in
addition, PTHrP was upregulated in the periarticular region (data not
shown) (Vortkamp et al.,
1996
). These phenotypes were not associated with ectopic induction
of Wnt5b or -11; in fact their expression was reduced or
absent (Fig. 7E,F). This
suggests that these Wnts are not downstream targets of Ihh signalling at least
in its ability to prevent terminal differentiation.
To further determine the function and mechanism of action of Wnt5a, -5b and
-11, these Wnts were misexpressed in vivo in the developing chick limb. Again,
as with Ihh, retroviral infection resulted in patches of ectopic expression in
and around the skeletal elements (Fig.
8C,D and data not shown). Wnt5a misexpression resulted in
truncated limbs and occasionally fused joints (n=5;
Fig. 8A and data not shown)
(Kawakami et al., 1999;
Hartmann and Tabin, 2000
).
Histological and molecular analysis showed that these truncations may result
from delayed differentiation of the hypertrophic zone which was either absent
or smaller (Fig. 8A,I). Formation of the hypertrophic zone is the main mechanism for elongation of the
skeletal element and thus its loss would significantly decrease the length of
the skeletal elements. In addition, the prehypertrophic zone was smaller as
determined by Ihh expression (FIg.
8G). However, the rounded zone of proliferating cells appeared
normal (Fig. 8A). Hence, Wnt5a
appeared to block or delay differentiation during or before the
prehypertrophic step. Wnt5a was also observed to induce ectopic Wnt5b
expression within the cartilage and upregulate the expression of
Wnt5b in the perichondrium (Fig.
8E). Although not as pronounced an effect as for Wnt5a,
misexpression of Wnt5b likewise resulted in shortened limbs (data not shown).
Again, this appeared to be due to a delay in terminal chondrocyte
differentiation as suggested by the smaller domain and/or lower levels of
type X collagen expression (n=11; data not shown).
|
Similarly, Wnt11-infected limbs were slightly truncated and the joints were fused in two out of four cases (data not shown). However, in contrast to Wnt5a, overexpression of Wnt11 (n=4; Fig. 8D) did not appear to delay chondrocyte differentiation as determined by the expression of Ihh, Wnt5b and type X collagen (Fig. 8F,H,J). The proximity of Wnt11 expression to the perichondrial ring suggested a possible link between Wnt11 function and formation of the bone collar. However, there was no apparent change in expression of the bone marker osteocalcin in Wnt11-infected limbs compared to control limbs (data not shown).
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Discussion |
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To examine how Wnt signalling affects the initiation of chondrogenesis we
used a micromass assay system which revealed that Wnt5a and -5b, but not Wnt4
and -11, can promote the first steps of chondrogenesis. Thus, there was an
increase in nodule number in Wnt5a- and Wnt5b-infected micromass cultures. In
contrast, Wnt4 decreased the number of nodules. These effects are consistent
with the expression patterns of Wnt4 and -5a during skeletal
development. Wnt5a is initially expressed in the mesenchyme around the
developing condensations where it may act to promote chondrogenesis by
recruitment of the mesenchymal cells into the chondrogenic lineage. Later,
Wnt5a is expressed in the perichondrium which contributes to
appositional growth. The ability of Wnt5a to recruit cells into the
chondrogenic lineage is further supported by the widening of the diaphysis
observed in Wnt5a-infected limbs. In contrast, Wnt4 expression is
associated with the developing joint
(Kawakami et al., 1999;
Hartmann and Tabin, 2000
). The
joint develops within a precartilaginous condensation and requires the
de-differentiation of these early prechondrocytes (reviewed by
Francis-West et al., 1999b
).
Therefore, one function of Wnt4 may be to act together with Wnt14, which is
also expressed in the developing joint and inhibits chondrogenesis in vitro
and in vivo, to control joint formation
(Hartmann and Tabin,
2001
).
Initiation of chondrogenesis, and hence the formation of nodules in vitro,
is associated with an increase in cell proliferation and/or adhesion which can
be modulated by Wnt signalling. For example, Wnt1 and -7a increase cell
adhesion by maintaining the expression of cell adhesion molecules such as
NCAM, N-cadherin and ß1-integrin during chondrogenesis
(Stott et al., 1999;
Tufan and Tuan, 2001
). This is
associated with the arrest of chondrocyte differentiation at the early cell
aggregation step (Rudnicki and Brown,
1997
; Stott et al.,
1999
). As other studies have shown that Wnt5a does not appear to
change cell adhesion, as assessed by the expression of N-cadherin, a factor
crucial for the condensation step, we determined whether Wnt overexpression
affected cell proliferation (Oberlender
and Tuan, 1994
; Tufan and
Tuan, 2001
). However, although Wnt5a controls the proliferation of
undifferentiated limb mesenchymal cells in vivo, BrdU-labelling studies showed
that the overexpression of Wnt5a did not significantly affect the levels of
cell proliferation in micromass culture
(Yamaguchi et al., 1999
).
Similarly, Wnt4 and -5b did not change cell proliferation. Although it is
still possible that Wnts specifically change proliferation in the forming
aggregates/nodules, which form a subpopulation of the culture, these data do
suggest that other mechanisms may be involved. For example, these Wnts could
affect the expression of other cell adhesion molecules which have not yet been
analysed. It is also possible that Wnts affect cell sorting which occurs
during chondrogenesis in micromass culture
(Cottrill et al., 1987
).
The changes in nodule number following overexpression of Wnt4, -5a and -5b
were paralleled by changes in the levels of matrix, as assessed by staining
for sulphated glycosaminoglycans with alcian blue, suggesting that Wnt4, -5a
and -5b signalling does not significantly change matrix
production/degradation. However, this analysis does not rule out effects of
Wnt signalling on other matrix molecules. For example, Wnt4 is expressed in
regions of hyaluronan synthesis and thus it is possible that Wnt4 regulates
hyaluronan production. High levels of hyaluronan prevent chondrogenesis by
inhibiting the early steps of cell aggregation, and hyaluronan is also a key
component of joint cavitation (Toole,
1981; Dowthwaite et al.,
1998
).
Wnt4, which accelerates terminal differentiation in vivo, also promoted
terminal differentiation in vitro
(Hartmann and Tabin, 2000).
Therefore, Wnt4 is likely to modulate the rate of chondrocyte differentiation.
It has been previously proposed, as for other signalling factors such as
GDF-5, that Wnt4 signals from the joint to control the development of the
adjacent skeletal elements (Francis-West
et al., 1999a
; Hartmann and
Tabin, 2000
). However, in addition to its expression in the
developing joints we also found Wnt4 transcripts in a subpopulation
of hypertrophic cells at stage 40 in some but not all developing bones. Thus,
it is possible that Wnt4 may directly promote differentiation rather than
acting through a relay of signals or controlling cell cycle progression from
the developing joint.
The expression of Wnt5b and Wnt11 overlapped with
Ihh and its receptor patched-1
(Vortkamp et al., 1998). As
Ihh is expressed prior to both Wnt5b and Wnt11,
this raised the possibility that these Wnts may mediate the effects of Ihh.
However, the in vitro and in vivo studies suggested that these Wnts are
components of distinct pathways. First, unlike Ihh, Wnt5b and -11 did not
promote terminal differentiation in vitro. Second, overexpression of Wnt5b and
-11 did not block differentiation prior to the prehypertrophic step. Finally,
Ihh overexpression did not induce the ectopic expression of either
Wnt5b or -11.
The expression of Wnt11 in a subset of prehypertophic chondrocytes
just underlying the presumptive bone collar suggested that Wnt11 has a role in
bone collar formation. Furthermore, in the tibiotarsus at stage 40,
Wnt11 expression clearly demarcates the perichondrial ring, which is
the edge of the developing cortical bone. Wnt5b is also expressed in
this region. However, these Wnts are unlikely to be involved in the
Ihh-regulation of bone collar formation as the hedgehog receptor,
patched-1, is expressed in the perichondrium and periosteum
suggesting that Ihh signals directly to the cells involved in bone collar
formation and not through a relay in the prehypertrophic zone
(St-Jacques et al., 1999).
Consistent with this, overexpression of Wnt11 (or Wnt5a and -5b) did not
induce development of the bone collar, although this does not rule out other
roles for these Wnts such as controlling vascularisation. The joint fusions
observed in Wnt11-misexpressed limbs may result from defects in the
musculature, since Wnt11 misexpression in vivo affects myogenic
differentiation (K. Anakwe, L. Robson, P. Buxton et al., unpublished).
Whilst these overexpression studies suggested that Wnt5b is not part of the Ihh signalling pathway, they did suggest that it has a role in controlling the rate of differentiation. In Wnt5a- and Wnt5b-infected limbs, chondrocyte differentiation was delayed as shown by either the absence or smaller domain of type X collagen expression. Consistent with its expression in prehypertrophic chondrocytes, the function of Wnt5b may be to delay terminal differentiation, and thus act to balance Ihh signalling, which can promote terminal differentiation. These studies also showed that Wnt5b may autoregulate its own expression or be controlled by Wnt5a in the adjacent perichondrium. Strikingly, ectopic Wnt5b expression was even found in the developing muscles following Wnt5a misexpression.
Bothe Wnt5a and -5b led to different effects on terminal chondrocyte
differentiation in vivo and in vitro: differentiation was delayed or blocked
in vivo whilst it was not affected in vitro (see also
Kawakami et al., 1999;
Hartmann and Tabin, 2000
). As
demonstrated by others and also shown here, hedgehog signalling has different
effects in vivo and in vitro, blocking and promoting differentiation
respectively (Vortkamp et al.,
1996
; Stott and Chuong,
1997
). The ability of hedgehog to promote differentiation in
vitro, but not in vivo, has been attributed to the absence of perichondrial
signals in early micromass cultures (Stott
and Chuong, 1997
). Thus, like Ihh, the ability of Wnt5a/5b to
block or delay terminal differentiation in vivo may be mediated through the
perichondrium. Alternatively, there may be other factors expressed in vivo,
but not in vitro, which modify the effect of Wnt5a/5b signalling.
The Wnts have been simply divided into two families based on their ability
to transform the C57MG mammary epithelial cell line and their effect when
injected into Xenopus embryos: the Wnt1 class which includes Wnt1,
-3a, -7a and -8, and the Wnt5a class which contains Wnt4, -5a and -11.
However, recent data has suggested that this classification is biased and has
raised the complexity of subdivision of the Wnt family which may be dependent
on the receptor profile. For example, Wnt5a can induce a secondary axis in
Xenopus when co-injected with the Wnt receptor Frizzled5, whilst on
its own Wnt5a cannot (He et al.,
1997). Furthermore, the Wnt1 class does not always signal through
the canonical Wnt pathway (Kengaku et al.,
1998
). Indeed, in our study, members of the Wnt5a class exhibited
distinct effects of chondrogenesis which may be related to different
signalling pathways. Analyses in zebrafish and Xenopus embryos have
shown that Wnt5a signals through the phosphatidyl-inositol/Ca2+ or
protein kinase C (PKC) pathways whereas Wnt11 is suggested to signal via JNK
(Slusarski et al., 1997
;
Djiane et al., 2000
;
Kuhl et al., 2000
;
Tada and Smith, 2000
). PKC is
required for chondrogenesis of mesenchymal stem cells and, therefore, Wnt5a
may initiate chondrogenesis by PKC activation
(Chang et al., 1998
;
Yang et al., 1998
). In
contrast, ß-catenin has been implicated in the Wnt4 signalling pathway
(Hartmann and Tabin, 2000
).
Finally, it is clear that the Wnt family of growth factors are fundamental
players in the control of skeletal development and degenerate abnormalities.
The challenge will be to unravel the diverse effects of Wnt signalling and how
they interact with other signalling pathways. Recent advances have already
emphasised the increasing complexity of these networks showing that Wnt3a
interacts with the BMP pathway to promote chondrogenesis in the C3H10T1/2 cell
line (Fischer et al.,
2002
).
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Acknowledgments |
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Akiyama, H., Shigeno, C., Iyama, K., Ito, H., Hiraki, Y., Konishi, J. and Nakamura, T. (1999). Indian hedgehog in the late-phase differentiation in mouse chondrogenic EC cells, ATDC5: upregulation of type X collagen and osteoprotegerin ligand mRNAs. Biochem. Biophys. Res. Commun. 257,814 -820.[CrossRef][Medline]
Cadigan, K. M. and Nusse, R. (1997). Wnt
signaling: a common theme in animal development. Genes
Dev. 11,3286
-3305.
Chang, S. H., Oh, C. D., Yang, M. S., Kang, S. S., Lee, Y. S.,
Sonn, J. K. and Chun, J. S. (1998). Protein kinase C
regulates chondrogenesis of mesenchymes via mitogen- activated protein kinase
signaling. J. Biol. Chem.
273,19213
-19219.
Chung, U. I., Schipani, E., McMahon, A. P. and Kronenberg, H.
M. (2001). Indian hedgehog couples chondrogenesis to
osteogenesis in endochondral bone development. J. Clin.
Invest. 107,295
-304.
Cottrill, C. P., Archer, C. W. and Wolpert, L. (1987). Cell sorting and chondrogenic aggregate formation in micromass culture. Dev. Biol. 122,503 -515.[Medline]
Djiane, A., Riou, J., Umbhauer, M., Boucaut, J. and Shi, D.
(2000). Role of frizzled 7 in the regulation of convergent
extension movements during gastrulation in Xenopus laevis.
Development 127,3091
-3100.
Dowthwaite, G. P., Edwards, J. C. and Pitsillides, A. A.
(1998). An essential role for the interaction between hyaluronan
and hyaluronan binding proteins during joint development. J.
Histochem. Cytochem. 46,641
-651.
Duprez, D., Bell, E. J., Richardson, M. K., Archer, C. W., Wolpert, L., Brickell, P. M. and Francis-West, P. H. (1996). Overexpression of BMP-2 and BMP-4 alters the size and shape of developing skeletal elements in the chick limb. Mech. Dev. 57,145 -157.[CrossRef][Medline]
Fischer, L., Boland, G. and Tuan, R. S. (2002). Wnt-3A enhances BMP-2 mediated chondrogenesis of murine C3H10T1/2 mesenchymal cells. J. Biol. Chem. (in press).
Francis-West, P. H., Tatla, T. and Brickell, P. M. (1994). Expression patterns of the bone morphogenetic protein genes Bmp-4 and Bmp-2 in the developing chick face suggest a role in outgrowth of the primordia. Dev. Dyn. 201,168 -178.[Medline]
Francis-West, P. H., Abdelfattah, A., Chen, P., Allen, C.,
Parish, J., Ladher, R., Allen, S., MacPherson, S., Luyten, F. P. and Archer,
C. W. (1999a). Mechanisms of GDF-5 action during skeletal
development. Development
126,1305
-1315.
Francis-West, P. H., Parish, J., Lee, K. and Archer, C. W. (1999b). BMP/GDF-signalling interactions during synovial joint development. Cell Tissue Res. 296,111 -119.[CrossRef][Medline]
Gerber, H. P. and Ferrara, N. (2000). Angiogenesis and bone growth. Trends Cardiovasc. Med. 10,223 -228.[CrossRef][Medline]
Hartmann, C. and Tabin, C. J. (2000). Dual
roles of Wnt signaling during chondrogenesis in the chicken limb.
Development 127,3141
-3159.
Hartmann, C. and Tabin, C. J. (2001). Wnt-14 plays a pivotal role in inducing synovial joint formation in the developing appendicular skeleton. Cell 104,341 -351.[Medline]
He, X., Saint-Jeannet, J. P., Wang, Y., Nathans, J., Dawid, I.
and Varmus, H. (1997). A member of the Frizzled protein
family mediating axis induction by Wnt-5A. Science
275,1652
-1654.
Hoang, B., Moos, M., Jr, Vukicevic, S. and Luyten, F. P.
(1996). Primary structure and tissue distribution of FRZB, a
novel protein related to Drosophila frizzled, suggest a role in skeletal
morphogenesis. J. Biol. Chem.
271,26131
-26137.
Hoang, B. H., Thomas, J. T., Abdul-Karim, F. W., Correia, K. M., Conlon, R. A., Luyten, F. P. and Ballock, R. T. (1998). Expression pattern of two Frizzled-related genes, Frzb-1 and Sfrp-1, during mouse embryogenesis suggests a role for modulating action of Wnt family members. Dev. Dyn. 212,364 -372.[CrossRef][Medline]
Karp, S. J., Schipani, E., St-Jacques, B., Hunzelman, J.,
Kronenberg, H. and McMahon, A. P. (2000). Indian hedgehog
coordinates endochondral bone growth and morphogenesis via parathyroid hormone
related-protein-dependent and -independent pathways.
Development 127,543
-548.
Kawakami, Y., Wada, N., Nishimatsu, S. I., Ishikawa, T., Noji, S. and Nohno, T. (1999). Involvement of Wnt-5a in chondrogenic pattern formation in the chick limb bud. Dev. Growth Differ. 41,29 -40.[CrossRef][Medline]
Kengaku, M., Capdevila, J., Rodriguez-Esteban, C., de la Pena,
J., Johnson, R. L., Belmonte, J. C. and Tabin, C. J. (1998).
Distinct WNT pathways regulating AER formation and dorsoventral polarity in
the chick limb bud. Science
280,1274
-1277.
Kuhl, M., Sheldahl, L. C., Malbon, C. C. and Moon, R. T.
(2000). Ca(2+)/calmodulin-dependent protein kinase II is
stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in
Xenopus. J. Biol. Chem.
275,12701
-12711.
Kwan, A. P., Freemont, A. J. and Grant, M. E. (1986). Immunoperoxidase localization of type X collagen in chick tibiae. Biosci. Rep. 6,155 -162.[Medline]
Kwan, A. P., Dickson, I. R., Freemont, A. J. and Grant, M. E. (1989). Comparative studies of type X collagen expression in normal and rachitic chicken epiphyseal cartilage. J. Cell Biol. 109,1849 -1856.[Abstract]
Ladher, R. K., Church, V. L., Allen, S., Robson, L., Abdelfattah, A., Brown, N. A., Hattersley, G., Rosen, V., Luyten, F. P., Dale, L. and Francis-West, P. H. (2000). Cloning and expression of the Wnt antagonists Sfrp-2 and Frzb during chick development. Dev. Biol. 218,183 -198.[CrossRef][Medline]
Leboy, P. S., Vaias, L., Uschmann, B., Golub, E., Adams, S. L.
and Pacifici, M. (1989). Ascorbic acid induces alkaline
phosphatase, type X collagen, and calcium deposition in cultured chick
chondrocytes. J. Biol. Chem.
264,17281
-17286.
Logan, C. and Francis-West, P. (1999). Gene transfer in avian embryos using replication-competent retroviruses. Methods Mol. Biol. 97,539 -551.[Medline]
Morgan, B. A. and Fekete, D. M. (1996). Manipulating gene expression with replication-competent retroviruses. Methods Cell Biol. 51,185 -218.[Medline]
Oberlender, S. A. and Tuan, R. S. (1994).
Expression and functional involvement of N-cadherin in embryonic limb
chondrogenesis. Development
120,177
-187.
Rudnicki, J. A. and Brown, A. M. (1997). Inhibition of chondrogenesis by Wnt gene expression in vivo and in vitro. Dev. Biol. 185,104 -118.[CrossRef][Medline]
Sen, M., Lauterbach, K., El-Gabalawy, H., Firestein, G. S.,
Corr, M. and Carson, D. A. (2000). Expression and function of
wingless and frizzled homologs in rheumatoid arthritis. Proc. Natl.
Acad. Sci. USA 97,2791
-2796.
Sen, M., Chamorro, M., Reifert, J., Corr, M. and Carson, D. A. (2001). Blockade of Wnt-5A/frizzled 5 signaling inhibits rheumatoid synoviocyte activation. Arthritis Rheum. 44,772 -781.[CrossRef][Medline]
Slusarski, D. C., Yang-Snyder, J., Busa, W. B. and Moon, R. T. (1997). Modulation of embryonic intracellular Ca2+ signaling by Wnt-5A. Dev. Biol. 182,114 -120.[CrossRef][Medline]
St-Jacques, B., Hammerschmidt, M. and McMahon, A. P.
(1999). Indian hedgehog signaling regulates proliferation and
differentiation of chondrocytes and is essential for bone formation.
Genes Dev. 13,2072
-2086.
Stott, N. S. and Chuong, C. M. (1997). Dual
action of sonic hedgehog on chondrocyte hypertrophy: retrovirus mediated
ectopic sonic hedgehog expression in limb bud micromass culture induces novel
cartilage nodules that are positive for alkaline phosphatase and type X
collagen. J. Cell Sci.
110,2691
-2701.
Stott, N. S., Jiang, T. X. and Chuong, C. M. (1999). Successive formative stages of precartilaginous mesenchymal condensations in vitro: modulation of cell adhesion by Wnt-7A and BMP-2. J. Cell. Physiol. 180,314 -324.[CrossRef][Medline]
Tada, M. and Smith, J. C. (2000). Xwnt11 is a
target of Xenopus Brachyury: regulation of gastrulation movements via
Dishevelled, but not through the canonical Wnt pathway.
Development 127,2227
-2238.
Tanda, N., Kawakami, Y., Saito, T., Noji, S. and Nohno, T. (1995). Cloning and characterization of Wnt-4 and Wnt-11 cDNAs from chick embryo. DNA Seq. 5, 277-281.[Medline]
Toole, B. P. (1981). Glycosaminoglycans in morphogenesis. New York: Plenum Press.
Tufan, A. C. and Tuan, R. S. (2001). Wnt
regulation of limb mesenchymal chondrogenesis is accompanied by altered
N-cadherin-related functions. FASEB J.
15,1436
-1438.
Tufan, A. C., Daumer, K. M. and Tuan, R. S. (2002). Frizzled-7 and limb mesenchymal chondrogenesis: effect of misexpression and involvement of N-cadherin. Dev. Dyn. 223,241 -253.[CrossRef][Medline]
Volk, S. W. and Leboy, P. S. (1999). Regulating the regulators of chondrocyte hypertrophy. J. Bone Miner. Res. 14,483 -486.[Medline]
Vortkamp, A., Lee, K., Lanske, B., Segre, G. V., Kronenberg, H. M. and Tabin, C. J. (1996). Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273,613 -622.[Abstract]
Vortkamp, A., Pathi, S., Peretti, G. M., Caruso, E. M., Zaleske, D. J. and Tabin, C. J. (1998). Recapitulation of signals regulating embryonic bone formation during postnatal growth and in fracture repair. Mech. Dev. 71,65 -76.[CrossRef][Medline]
Wada, N., Kawakami, Y., Ladher, R., Francis-West, P. H. and Nohno, T. (1999). Involvement of Frzb-1 in mesenchymal condensation and cartilage differentiation in the chick limb bud. Int. J. Dev. Biol. 43,495 -500.[Medline]
Wodarz, A. and Nusse, R. (1998). Mechanisms of Wnt signaling in development. Annu. Rev. Cell. Dev. Biol. 14,59 -88.[CrossRef][Medline]
Yamaguchi, T. P., Bradley, A., McMahon, A. P. and Jones, S.
(1999). A Wnt5a pathway underlies outgrowth of multiple
structures in the vertebrate embryo. Development
126,1211
-1223.
Yang, M. S., Chang, S. H., Sonn, J. K., Lee, Y. S., Kang, S. S., Park, T. K., and Chun, J. S. (1998). Regulation of chondrogenic differentiation of mesenchymes by protein kinase C alpha. Mol. Cells 8,266 -271.[Medline]