1 Genetic Disease Research Branch, National Human Genome Research Institute,
National Institute of Health, Bethesda, MD 20892, USA
2 Department of Medicine (School of Medicine), University of Pennsylvania,
Philadelphia, PA 19104, USA
* Author for correspondence (e-mail: yyang{at}nhgri.nih.gov)
Accepted 4 December 2002
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SUMMARY |
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Key words: Wnt5a, Wnt5b, Chondrocyte, Proliferation, Mouse
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INTRODUCTION |
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It has been shown that the proliferative and articular/resting chondrocyte
region can be further divided into two zones (Zone I and Zone II) based on the
distinct cell morphologies, developmental fates and functions
(Abad et al., 2002;
Iwamoto et al., 2000
). Zone I
corresponds to the epiphysis of developing long bones. Zone I cells are round
and divide less frequently (Kobayashi et
al., 2002
; Long et al.,
2001
). They are articular chondrocytes that will become permanent
articular cartilage and resting chondrocytes in the growth plate
(Archer et al., 1994
;
Hunziker, 1994
;
Mitrovic et al., 1978
;
Pacifici, 1995
). Articular
cartilage maintains normal joint function throughout life, whereas resting
chondrocytes may contain cells similar to stem cells that are capable of
generating new clones of proliferative zone chondrocytes
(Abad et al., 2002
). Zone II
cells are highly proliferative chondrocytes in the growth plate that will exit
the cell cycle and undergo hypertrophy
(Howlett, 1979
;
Hunziker, 1994
;
Karp et al., 2000
). Zone II
cells are flattened and increase their cell size gradually the closer they are
to the hypertrophic zone. These cells divide more frequently and line up with
each other to form columns parallel to the long axis of the bone
(Dodds, 1930
;
Long et al., 2001
). Zone II
depends on Zone I for a constant supply of proliferative chondrocytes. In the
meantime, a population of undifferentiated chondrocytes remains in Zone I to
maintain joint function at all times. Thus, the transition between Zone I and
Zone II has to be carefully regulated. However, the underlying molecular
mechanism is still poorly understood.
Signaling molecules have been shown to play important roles in regulating
chondrocyte proliferation and differentiation (reviewed by
de Crombrugghe et al., 2001;
Karsenty, 2001
). Disruption of
the normal cell signaling during endochondral bone formation results in
skeletal anomalies in both humans and mouse mutants
(Gao et al., 2001
;
Li and Olsen, 1997
). Indian
hedgehog (Ihh) and parathyroid hormone related peptide (PTHrP; Pthlh
Mouse Genome Informatics) are two signaling molecules that play crucial roles
in regulating the pace of chondrocyte hypertrophy through forming a negative
feedback loop (Lanske et al.,
1996
; Vortkamp et al.,
1996
). Recent studies have shown that PTHrP and Ihh signaling may
also be involved in regulating the transition between Zone I and II, the two
chondrocyte zones before prehypertrophy
(Kobayashi et al., 2002
).
However, it was not clear which signaling molecules directly inhibit the
transition between Zone I and II.
The mechanism by which signaling molecules control cell proliferation and
differentiation involves key intracellular regulatory factors, which include
cell cycle regulators and transcription factors. Cell cycle inhibitors such as
p21, p57, p107 and p130, members of retinoblastoma (Rb) family, are involved
in regulating chondrocyte proliferation and differentiation
(Aikawa et al., 2001;
Cobrinik et al., 1996
;
Su et al., 1997
;
Yan et al., 1997
). These
studies have shown that chondrocyte hypertrophy is delayed when cell cycle
withdrawal is inhibited. Sox9 and Cbfa1 (Runx2 Mouse Genome
Informatics) are transcription factors that appear to have opposite activities
in regulating chondrocyte differentiation
(Bi et al., 2001
;
Takeda et al., 2001
;
Ueta et al., 2001
). Sox9
inhibits chondrocyte hypertrophy whereas Cbfa1 enhances it. Although it is not
clear how signaling molecules regulate Cbfa1 activity in the chondrocytes, it
has been shown that Sox9 transcriptional activity can be significantly
enhanced through phosphorylation by protein kinase A (PKA). Moreover, PTHrP
signals through activating PKA and may inhibit chondrocyte maturation by
increasing Sox9 activity (Huang et al.,
2001
). Sox9 is required for the expression of several
cartilage-specific extracellular matrix components and it directly regulates
Col2a1 (previously known as ColII) gene expression
(Bell et al., 1997
;
Lefebvre et al., 1996
;
Mukhopadhyay et al., 1995
;
Zhou et al., 1995
).
More recently, Wnt family members have been found to be involved in
regulating skeletal development. Wnt proteins are important signaling
molecules that have been shown to regulate cell proliferation and
differentiation during embryonic development and tumor formation (for reviews,
see Huelsken and Birchmeier,
2001; Peifer and Polakis,
2000
). Studies carried out in chick have shown that ectopic
Wnt5a expression delays chondrocyte differentiation, whereas
ectopicWnt4 expression promotes chondrocyte differentiation
(Hartmann and Tabin, 2000
). In
addition, overexpression of Wnt14 in the chick limb leads to ectopic
synovial joint formation (Hartmann and
Tabin, 2001
). However, direct evidence to show that endogenous Wnt
gene function is required for chondrocyte proliferation or differentiation is
lacking.
We have found that both Wnt5a and Wnt5b are expressed in
the chondrocyte of developing long bones in mice. To understand the mechanism
by which Wnt5a and Wnt5b regulate endochondral skeletal
development in vivo, we have analyzed the skeletal development defects in
mutant mice either that lack Wnt5a function
(Yamaguchi et al., 1999) or
overexpress Wnt5a or Wnt5b in chondrocytes. Our results
indicate that Wnt5a is required for regulating chondrocyte
proliferation and differentiation in both Zone I and Zone II, and Wnt5a
signaling directly inhibits the transition from Zone I to Zone II. In
addition, Wnt5b promotes cell proliferation and Zone II formation.
Thus, Wnt5a and Wnt5b appear to coordinate longitudinal long
bone growth by exerting opposite activities in regulating chondrocyte
proliferation and chondrocyte-specific Col2a1 expression.
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MATERIALS AND METHODS |
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Skeletal analysis
Embryos at E17.5 or E18.5 were dissected in phosphate-buffered saline
(PBS). The embryos were then skinned, eviscerated and fixed in 95% ethanol.
Skeletal preparation was performed as described previously
(McLeod, 1980)
Histological analysis, in situ hybridization and
immunohistochemistry
Embryos were dissected in PBS and fixed in 4% formaldehyde at 4°C
overnight, dehydrated with increasing ethanol concentration, and embedded in
paraffin wax. Serial sections of 6 µm were stained according to the
Weigert-Safranin staining procedure
(Prophet et al., 1994). BrdU
labeling and radioactive 35S RNA in situ hybridization were
performed on serial sections as described
(St-Jacques et al., 1999
).
Whole-mount in situ hybridization were performed as described
(Goodrich et al., 1996
). RNA
probes have been described previously: Ihh
(Bitgood and McMahon, 1995
);
Wnt5a and Wnt5b (Gavin
et al., 1990
); Ptch
(Goodrich et al., 1996
);
Osf2/Cbfa1 (Runx2 Mouse Genome Informatics)
(Ducy et al., 1997
);
osteocalcin (Desbois et al.,
1994
); and Pthlh, Pth/Pthrpr, Col2a1 and
Col110a1 (ColX) (Lee et
al., 1996
).
For immunohistochemistry, sections were digested with 0.1% hyaluronidase
for 15 minutes at 37°C, heated in citric buffer (pH 6.0, 10 mM) at
95°C for 25 minutes to enhance antigen accessibility. Slides were blocked
with 4% horse serum (Sigma) at room temperature for 2 hours, incubated
overnight at 4°C with primary antibodies: anti-cyclin D1 (Santa Cruz sc
8396) at 1:40; anti-p130 (BD Transduction Laboratories 610261) at 1:40; and
anti-Sox9 at 1:30 (Huang et al.,
2001). Signals were detected using the ABC kits (Vector
laboratories) and DAB (Sigma), or Alexa Fluor 594-conjugated secondary
antibodies purchased from Molecular Probes.
Primary chondrocyte isolation, culture, transfection and luciferase
assay
Primary mouse chondrocytes were prepared from the ventral rib cages of 0-
to 3-day-old mice, according to the previously published protocol
(Lefebvre et al., 1994). Cells
were transfected using LipofectAMINE-PLUS (Invitrogen) according to the
manufacturer's instructions. The amount of DNA in each transfection was kept
constant by addition of an appropriate amount of empty expression vector. The
Col2a1 reporter construct pKN159Ax6luc and the mutant Col2a1
reporter PKN159MTluc were gifts from Yoshihiko Yamada
(Krebsbach et al., 1996
;
Tanaka et al., 2000
). To
express Wnt5a or Wnt5b, the coding region of the genes were
inserted into pIRES-hrGFP-1a expression vector (Stratagene). To determine
Col2a1 reporter activity, 1.5x105 cells were
co-transfected with a reporter construct (0.33 µg), pRLSV40 (Promega) as an
internal control (0.016 µg) and the indicated plasmids in six-well plates.
The transfected cells were harvested 48 hours later and assayed for luciferase
activity using the Dual-Luciferase Reporter Assay System (Promega). All
measurements were made using a Lumat LB9507 luminometer (EG&G
Berthold).
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RESULTS |
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To understand the molecular mechanism by which Wnt5a regulates
endochondral skeletal morphogenesis, Wnt5a expression was examined in
the sections of developing long bones by in situ hybridization. Wnt5a
was expressed at the boundary of proliferative and prehypertrophic
chondrocytes and perichondrium/periosteum
(Fig. 1C). Its expression
domain overlapped with that of Ihh in the prehypertrophic region and
flanked the hypertrophic chondrocytes that express Col10a1. In both
the perichondrium and prehypertrophic chondrocytes, the expression of
Wnt5a overlapped with that of Cbfa1, a transcription factor
that determines osteoblast cell fate and regulates chondrocyte differentiation
(Ducy et al., 1997;
Kim et al., 1999
;
Komori et al., 1997
;
Otto et al., 1997
;
Takeda et al., 2001
;
Ueta et al., 2001
). The
expression pattern of Wnt5a, together with the histological analysis
of the Wnt5a-/- embryos indicates that Wnt5a is a
signaling molecule required for both chondrocyte and osteoblast
differentiation during mouse endochondral skeletal morphogenesis.
Delayed Ihh expression and chondrocyte hypertrophy in
Wnt5a mutant limbs
The defect in chondrogenesis in the Wnt5a mutant limb was detected
by in situ hybridization from 11.5 dpc, when chondrogenesis has just started
(Fig. 2A). We found that the
expression of Sox9, the earliest marker for mesenchymal condensation,
was not detected in the distalmost limb mesenchyme in the Wnt5a
mutant. At 12.5 dpc, the distal zone that failed to express Sox9 expanded,
indicating that Wnt5a activity is required for mesenchymal
condensation in the distal limb bud.
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Part of Wnt5a transcript was still produced in the Wnt5a mutant. It is worth noting that Wnt5a expression was still detected in the distal limb but missing in the Wnt5a mutant at 12.5 dpc, suggesting that Wnt5a may be required for its own expression in chondrocytes (Fig. 2B).
As Wnt5a expression overlapped with that of Ihh in the
prehypertrophic region, and Ihh plays a key role in coordinating
chondrocyte proliferation and differentiation, we examined whether
Ihh expression was affected in the Wnt5a-/- limbs
(Fig. 2A,B). At 11.5 dpc,
Ihh expression was detected in the future humerus, radius and ulna in
the wild-type embryo. In the Wnt5a mutant, Ihh expression
was only weakly detected in the humerus and absent in the radius and ulna
(Fig. 2A), although mesenchymal
condensation occurred normally in the proximal limb. At 12.5 dpc, shortly
after cartilage formation in the forelimb, Ihh was expressed strongly
in the developing cartilage in the wild-type embryos, but no expression was
detected in the Wnt5a mutant, and only weak expression was observed
at 13.5 dpc (Fig. 2B and data
not shown). At 15.5 dpc, strong expression of Ihh was observed in the
humerus of the Wnt5a-/- embryo, but only patchy expression
was detected in the ulna (Fig.
2C). Ihh expression correlated with the upregulation of
patched (Ptch), a transcriptional target of hedgehog signaling
(Goodrich et al., 1996)
Fig. 2C).
As limb skeletal morphogenesis progresses from the proximal to distal part
of the limb, Ihh expression was first detected in the proximal limb.
It is likely that the weaker expression of Ihh earlier in development
was an indirect result of delayed chondrocyte differentiation and the
proximodistal sequence of skeletal development was preserved in the
Wnt5a mutant. This was confirmed by our observation that the
expression of Pthrpr and Cbfa1 in the prehypertrophic
chondrocytes was reduced and delayed and the expression of Col10a1, a
hypertrophic chondrocyte marker, was absent in the
Wnt5a-/- limb from 12.5 dpc to 15.5 dpc
(Fig. 2B,C). Together, these
results indicate that Wnt5a is required for the transition from
proliferative chondrocytes to prehypertrophic chondrocytes. Moreover, as the
phenotype of chondrocyte differentiation in the Wnt5a-/-
limb is different from that in the Ihh-/- limb
(St-Jacques et al., 1999), it
is unlikely that Wnt5a acts through Ihh in regulating
chondrocyte differentiation.
As PTHrP signaling suppresses chondrocyte differentiation at multiple steps
(Kobayashi et al., 2002;
Lanske et al., 1996
), we
examined Pthlh expression in Wnt5a mutants. Pthlh
expression in the periarticular region of the Wnt5a-/-
mutant was comparable with that in the wild-type embryo at 13.5 dpc
(Fig. 2B). As Ihh signaling is
required for the expression of Pthlh, this result suggests that
normal Pthlh expression is maintained by very low levels of Ihh
signaling in the Wnt5a mutant.
Since the skeletal elements in the Wnt5a mutant limb were
significantly shortened, BrdU labeling experiments were performed in 14.5 dpc
mouse embryos to asses chondrocyte proliferation in the distal humerus
(Fig. 3A,B). Undifferentiated
chondrocytes are divided into two zones according to the cell shape,
developmental fate and the known Ihh signaling domain
(Abad et al., 2002;
Iwamoto et al., 2000
;
Long et al., 2001
)
(Fig. 3A). In the wild-type
embryo, Zone II is more highly proliferative than Zone I
(Fig. 3B), as has been shown
before (Long et al., 2001
). In
the Wnt5a mutant, the proliferation in Zone I was slightly increased.
By contrast, the proliferation in Zone II was reduced by about 30% to a level
similar to Zone I. These results indicate that Wnt5a is required for
chondrocyte proliferation in Zone II where it is expressed. It is possible
that Wnt5a acts indirectly by affecting Ihh expression as Ihh is a
major positive factor for chondrocyte proliferation and Ihh
expression was much weaker in Wnt5a-/- long bones
(Fig. 2A,B). In addition to a
proliferation deficiency, the cartilage morphology was severely distorted and
the diameter of Zone I was reduced; the diameter of Zone II was increased in
Wnt5a mutants (Fig.
3A).
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Delayed osteoblast differentiation in Wnt5a mutants
To investigate bone development in Wnt5a-/- limbs, we
examined expression of Cbfa1, a transcription factor essential for
osteoblast differentiation. Cbfa1 expression was significantly
reduced in both chondrocytes and perichondrium in the Wnt5a mutants
(Fig. 2B,C). In addition, the
expression of osteocalcin, a marker for mature osteoblasts
(Desbois et al., 1994), was
not detected in Wnt5a mutants at 15.5 dpc, whereas its expression was
readily detected in wild-type long bones
(Fig. 2C). These results
demonstrate that osteoblast differentiation is delayed in Wnt5a
mutants, possibly as a result of delayed chondrocyte differentiation and
Ihh expression.
As Wnt5a expression in chondrocytes and the perichondrium overlaps
with that of Cbfa1, it is also possible that Wnt5a regulates
osteoblast development directly. However, endochondral ossification is
dependent on Ihh. Thus, reduced Ihh signaling activity may
secondarily result in delayed osteoblast differentiation. In the later case,
restoring Ihh activity in the absence of Wnt5a function may be
expected to rescue the osteoblast deficiency. To this end, Ihh was
ectopically expressed in chondrocytes under the control of Col2a1
promoter/enhancer using the UAS-Gal4 system
(Long et al., 2001). When
these mice were mated to Wnt5a mutants
(Fig. 4), we found that
although the limb in the Wnt5a-/-; Col2a1-Gal4;
UAS-Ihh mouse was slightly longer than the Wnt5a-/-
limb, it was still much shorter than the limb of Col2a1-Gal4; UAS-Ihh
mice. Osteoblast differentiation in the Wnt5a-/- limb was
rescued by Ihh overexpression Thus, Wnt5a may regulate
osteoblast differentiation through indirectly affecting Ihh
expression in chondrocytes. In the cartilage, the gross phenotype of the
Wnt5a-/-; Col2a1-Gal4; UAS-Ihh mouse was the
combination of Wnt5a-/- and Col2a1-Gal4; UAS-Ihh
mice. As ectopic Ihh expression is able to rescue
Ihh-/- mice (A. McMahon, personal communication) to a
level similar to Col2a1-Gal4; UAS-Ihh mice, our results indicate that
Wnt5a and Ihh act in separate pathways in regulating
chondrocyte proliferation and differentiation in the developing long
bones.
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Wnt5a delays chondrocyte differentiation in a dose-dependent
manner
To understand better how Wnt5a regulates the differentiation of
chondrocytes, Col2a1-Wnt5a transgenic mice were generated
(Fig. 5A). In these transgenic
mice, Wnt5a was expressed in non-hypertrophic chondrocytes under the
control of rat Col2a1 promoter and enhancer
(Nakata et al., 1993). When
compared with the wild-type littermates, the Col2a1-Wnt5a transgenic
mice showed severe skeletal defects. Skeletal elements in the limb were short
and ossification was delayed. The cartilage was thicker and chondrocyte
hypertrophy was significantly delayed in the developing long bones of the
transgenic mouse (Fig. 5A).
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At 14.5 dpc, Col10a1 expression domain was either reduced (Col2a1-Wnt5a number 17) or absent (Col2a1-Wnt5a number 25). The loss of Col10a1 expression correlated with higher levels of Wnt5a expression in individual transgenic embryos and a resulted increase in the severity of the phenotype (Fig. 5B). In addition, the prehypertrophic chondrocyte zone where Ihh and Pthrpr are normally expressed was either not detected (Col2a1-Wnt5a number 25) or remained at the midline of the skeletal element (Col2a1-Wnt5a number 17) (Fig. 5B). Thus, Wnt5a overexpression caused a delay in chondrocyte differentiation before hypertrophic development, and ectopic expression of Wnt5a alone was not sufficient to activate ectopic Ihh expression. Consequently, the region of undifferentiated chondrocytes was lengthened in Col2a1-Wnt5a transgenic embryos, even though the entire skeletal element was shorter (Fig. 5B). However, cell proliferation was decreased in both Zone I and II in the Col2a1-Wnt5a transgenic mice, as indicated by BrdU labeling (Fig. 5C). Thus, surprisingly, both loss and gain of Wnt5a activity resulted in a similar perturbation of chondrocyte proliferation and differentiation.
Wnt5b was also expressed in the cartilage, and ectopic
expression of Wnt5b delayed chondrocyte differentiation
Wnt5b is the closest Wnt relative to Wnt5a (amino acid
identity 79%). Interestingly, Wnt5b was also expressed in
chondrocytes in a region between prehypertrophic and hypertrophic chondrocytes
(Fig. 6A). To test whether
Wnt5b plays a similar role as Wnt5a in regulating
chondrocyte proliferation and differentiation, Col2a1-Wnt5b
transgenic mice were generated. Examination of these transgenic mice at 17.5
dpc indicates that chondrocyte hypertrophy was delayed and bone ossification
was also reduced (Fig. 6B). In
the most severe examples, the skull was open, a phenotype that correlated with
Col2a1 expression in the brain
(Cheah et al., 1991) and was
not observed in any of the Col2a1-Wnt5a transgenic mouse. When the
long bone phenotype of Col2a1-Wnt5b was further analyzed, we found
that expression of both Ihh and ColX was undetectable at
15.5 dpc (Fig. 6C) indicating
chondrocyte differentiation was delayed prior to hypertrophic development as
observed in Col2a1-Wnt5a mice. However, in contrast to
Col2a1-Wnt5a mice, chondrocyte proliferation was increased in both
Zone I and II in the Col2a1-Wnt5b transgenic mice
(Fig. 6D).
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Wnt5a and Wnt5b act differently in regulating
chondrocyte proliferation and Col2a1 expression
Chondrocyte differentiation in Wnt5a-/-,
Col2a1-Wnt5a and Col2a1-Wnt5b mice was similarly delayed
before chondrocyte hypertrophy. However, chondrocyte proliferation appeared to
have been altered differentially by Wnt5a and Wnt5b
overexpression. As chondrocytes have two zones with different properties of
cell proliferation before hypertrophy, we examined whether chondrocyte
differentiation had been arrested in different zones in
Wnt5a-/-, Col2a1-Wnt5a and Col2a1-Wnt5b
mice. To achieve this, we first analyzed the expression of several cell cycle
regulators that have been shown to control chondrocyte proliferation and
differentiation. p130, one of the Rb proteins, is involved in cartilage
development (Cobrinik et al.,
1996). Rb proteins impede G1/S transition by inhibiting the
S-phase-promoting transcription factor E2F
(Dyson, 1998
;
Weinberg, 1995
). Cyclin D1
acts as an intracellular sensor for mitogens
(Sherr and Roberts, 1999
) and
has been shown to be a transcriptional target of Wnt signaling
(Megason and McMahon, 2002
).
Cyclin D1-CDK4 complex promotes G1/S transition partly through phosphorylating
and inactivating Rb (Weinberg,
1995
). We found that in the wild-type cartilage, cyclin D1 and
p130 protein expression was complementary to each other in Zone I and Zone II
(Fig. 7A). Zone I expressed a
higher level of P130 but a lower level of cyclin D1. Conversely, Zone II
expressed a higher level of cyclin D1 but a lower level of p130. p130 was also
expressed at a higher level in differentiating chondrocytes when cells exit
Zone II. These expression patterns correlate well with the different cell
cycling rates in these two zones. In the Wnt5a mutant, cyclin D1
expression was decreased. This was at least partly due to reduced and delayed
Ihh expression, as Ihh overexpression was able to increase
cyclin D1 expression in the Wnt5a mutant
(Fig. 7A). Zone I, which
expressed higher levels of p130, was reduced in size in the Wnt5a
mutant. In addition, p130 expression was decreased in chondrocytes undergoing
hypertrophy in the Wnt5a mutant
(Fig. 7A). These data suggest
that Wnt5a may exert two effects by upregulating p130 expression: holding
cells in Zone I and promoting the exit from Zone II to the hypertrophic zone.
Indeed, when Wnt5a was overexpressed in chondrocytes, cyclin D1
expression was suppressed, but p130 expression remained high
(Fig. 7B,C), indicating that
most chondrocytes in Col2a1-Wnt5a mice were held in Zone I. As Zone I
to Zone II transition precedes Zone II to hypertrophic zone transition,
delayed Zone I to Zone II transition will postpone Zone II to hypertrophic
zone transition; thus, chondrocyte hypertrophy was delayed in
Col2a1-Wnt5a mice. By contrast, when Wnt5b was
overexpressed, cyclin D1 expression was upregulated in all undifferentiated
chondrocytes, whereas Zone I marked by high p130 expression was reduced in
size (Fig. 7B,C). These results
showed that more chondrocytes in Col2a1-Wnt5b mice were in Zone II,
which is highly proliferative. Thus, Wnt5a overexpression appears to
prevent chondrocytes from entering Zone II, whereas Wnt5b
overexpression promotes entrance to Zone II and prevents cell cycle withdrawal
of Zone II chondrocytes.
|
As Wnt5a and Wnt5b exhibited opposite activities in
regulating chondrocyte proliferation, we then examined whether they also
differentially regulated chondrocyte-specific gene expression. The
transcription factor Sox9 has been shown to play an important role in
chondrocyte hypertrophy (Bi et al.,
2001; Huang et al.,
2001
) and to control the expression of several
chondrocyte-specific genes (Bi et al.,
1999
; Lefebvre and de
Crombrugghe, 1998
). To test whether Wnt5a or
Wnt5b regulates Sox9 pathway, we first examined Sox9 protein
expression in chondrocytes using immunohistochemistry. Sox9 protein levels
were decreased in both Wnt5a-/- and Col2a1-Wnt5a
mice, and no alteration was observed in Col2a1-Wnt5b mice when
compared with the wild-type littermate
(Fig. 8A). As Col2a1
is a direct target of Sox9, we probed the transcription activity of Sox9 by
examining Col2a1 expression in Wnt5a-/-,
Col2a1-Wnt5a and Col2a1-Wnt5b mice. Col2a1
expression was increased in the cartilage of the Wnt5a-/-
and Col2a1-Wnt5b mice, but decreased in the Col2a1-Wnt5a
mice (Fig. 8B). To obtain a
more quantitative assessment, luciferase assay of the Col2a1 reporter
was performed in mouse primary chondrocytes
(Fig. 8C). Col2a1-luciferase
activity was suppressed by Wnt5a but increased by Wnt5b in a
dose-dependent manner. As many Wnts, including Wnt1, signal through
activiating LEF/TCF-mediated transcription
(van Noort and Clevers, 2002
),
we examined the effect of TCF1 and Wnt1 on the Col2a1 reporter. We
found that surprisingly, Wnt1 inhibited Col2a1 reporter activity.
TCF1 had no effect on the Col2a1 reporter or a mutant
Col2a1-reporter, which has mutated Sox9-binding site
(Tanaka et al., 2000
)
(Fig. 8D). As TCF1 activated
LEF/TCF reporter (driven by LEF/TCF binding sites) in the primary chondrocytes
(data not shown), these results suggest the effect of Wnt5a or Wnt5b was
independent of LEF/TCF factors. However, these data indicate that
Wnt5a and Wnt5b have opposite activities in regulating
Col2a1 expression, which suggests that increased Sox9 activity may
also contribute to the delay in chondrocyte differentiation in
Wnt5a-/- and Col2a1-Wnt5b mice.
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DISCUSSION |
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The cartilage of a developing long bone in the limb has a characteristic
morphology, with distinct spatial organization of proliferative and
differentiated chondrocyte zones (Fig.
9). This morphology is established and maintained during normal
development through molecular interactions that control transitions between
different chondrocyte zones. Mutations that block or delay such transitions
will result in abnormal accumulation of chondrocytes in one of the two zones
before chondrocyte hypertrophy, which often leads to short and thick
cartilages. This phenomenon has been observed in many mouse mutants, including
those with abnormal FGF (Iwata et al.,
2000; Naski et al.,
1998
), PTHrP (Schipani et al.,
1997
) and Wnt signaling described here. We show that one function
of Wnt5a signaling is to keep the chondrocytes in a state that is fated to
become articular/resting chondrocytes. Thus, Wnt5a may play a pivotal role in
the development and maintenance of articular/resting chondrocytes in the joint
and cartilage growth plates.
Wnt5a signaling is required for chondrocyte differentiation and may
downregulate Sox9 transcription activity
The sequential proliferation and differentiation of chondrocytes are
tightly regulated by multiple signaling molecules and transcription factors.
Chondrocyte differentiation can be regulated at two stages according to
previous studies: the transition from proliferative to prehypertrophic
chondrocytes; and the transition from prehypertrophic to hypertrophic
chondrocytes (Crowe et al.,
1999; Vortkamp et al.,
1996
). Ihh expression serves as a marker for
prehypertrophic chondrocytes and Col10a1 is a marker for hypertrophic
chondrocytes. Wnt5a is expressed in proliferative and prehypertrophic
chondrocytes and is required for the first transitional event, as chondrocyte
differentiation was significantly delayed and both Ihh and
Col10a1 expression were greatly reduced in the Wnt5a mutant.
Consistent with this, it has been shown in chick that overexpression of a
dominant-negative Frizzled leads to delayed chondrocyte differentiation,
suggesting that in chick, some endogenous Wnt signals through Frizzled to
promote chondrocyte differentiation
(Hartmann and Tabin,
2000
).
Ihh expression was significantly reduced in the
Wnt5a-/-, Col2a1-Wnt5a and Col2a1-Wnt5b
mice described here because of delayed chondrocyte differentiation. However,
the phenotypes in the Wnt5a-/-, Col2a1-Wnt5a and
Col2a1-Wnt5b mice are opposite to those in Ihh-/-
and Prthrpr-/- mice in which chondrocyte differentiation
is accelerated. Moreover, overexpression of Ihh in chondrocytes did
not rescue the phenotype of skeletal shortening in Wnt5a mutants to
an extent comparable with the wild-type mice. Thus, it is unlikely that Wnt5a
or Wnt5b signal is directly mediated by Ihh function. It is likely
that Wnt5a, Wnt5b and Ihh-PTHrP signals act in parallel pathways to regulate a
common downstream factor that plays a key role in chondrocyte proliferation
and differentiation. One such common downstream factor might be Sox9, an HMG
box transcription factor that is found to regulate chondrocyte differentiation
as well as being required for chondrocyte cell fate determination
(Bi et al., 2001). Prior
studies suggest that PTHrP signaling may inhibit chondrocyte differentiation
through upregulating Sox9 activity (Huang
et al., 2001
). We have shown that the expression of
Col2a1, a direct transcriptional target of Sox9 is increased in the
Wnt5a mutant but decreased in Col2a1-Wnt5a transgenic mice.
Thus, we conclude that Wnt5a may promote chondrocyte hypertrophy in
part through decreasing the transcriptional activity of Sox9. Interestingly,
we find Wnt5b, which is expressed at the boundary of prehypertrophic
and hypertrophic chondrocytes, exhibits opposite activities in regulating
Col2a1 expression, suggesting that Wnt5b may enhance Sox9
transcription activity in a dose dependent manner
(Fig. 8). It is possible that
Wnt5b plays a similar role to Pthlh in inhibiting
chondrocyte hypertrophy through enhancing Sox9 transcription activity, which
may influence chondrocyte differentiation by controlling the expression of
chondrocyte specific genes (Bi et al.,
1999
; Lefebvre and de
Crombrugghe, 1998
). It will be interesting to investigate how Sox9
transcriptional activity is altered by Wnt5a and Wnt5b signaling and how Wnt
and PTHrP signaling pathways interact with each other in chondrocytes.
Proliferative chondrocytes can be divided into two zones with
distinct proliferation properties and cell morphology
Chondrocyte proliferation and differentiation are tightly associated with
cell cycle progression and withdrawal. Zone I and Zone II chondrocytes have
distinct morphology, developmental fate and cell cycling rate
(Abad et al., 2002;
Iwamoto et al., 2000
;
Long et al., 2001
), which has
prompted us to search for the underlying molecular mechanism. We find that a
negative cell cycle regulator p130 and a positive regulator cyclin D1, both of
which play important roles in regulating chondrocyte proliferation, exhibit
complimentary expression patterns in Zone I and II. These expression patterns
correlate well with the distinct proliferation properties in these two zones
(Fig. 7A). Chondrocytes divide
less frequently in Zone I and they express a higher level of p130 but a lower
level of cyclin D1. Conversely, Zone II cell divide more often and they
expresses a higher level of cyclin D1 but a lower level of p130. The higher
level of cyclin D1 expression in Zone II may be a direct result of
Ihh expression in the adjacent prehypertrophic zone, as it has been
shown that Ihh signaling is required for chondrocyte proliferation at least in
part through regulating the expression of cyclin D1
(Long et al., 2001
). Moreover,
it has been demonstrated in Drosophila that CyclinD mediates the
distinct ability of hedgehog in promoting cellular growth as well as cell
proliferation (Duman-Scheel et al.,
2002
). Therefore, it is likely that the lower cell cycle rate in
Zone I is maintained by higher level of Rb gene activity and that
cyclin D1 mediates the function of Ihh in establishing and maintaining Zone II
function. Thus, Wnt5a and Wnt5b may regulate transitions between different
chondrocyte zones through controlling the expression of the cell cycle
regulators.
Wnt5a and Wnt5b regulate chondrocyte proliferation
through distinct pathways
It is likely that cell cycle regulators are also common targets for
signaling molecules in regulating chondrocyte proliferation. Chondrocyte
differentiation is delayed in both Col2a1-Wnt5a and
Col2a1-Wnt5b transgenic mice. However, the differentiation delay in
these two transgenic mice is caused by different regulatory activities of
Wnt5a and Wnt5b in chondrocytes (summarized in
Fig. 9). p130 and cyclin D1
expression is regulated by Wnt5a and Wnt5b in distinct
manners. Wnt5a increases p130 expression but suppresses cyclin D1
expression. Thus, Zone I is expanded at the expense of Zone II and chondrocyte
proliferation is decreased in Col2a1-Wnt5a mice. In addition,
decreased p130 expression may also lead to delayed transition from
proliferative to hypertrophic chondrocytes in the Wnt5a-/-
embryo. Wnt5b appears to have some redundant activities with both Ihh
and PTHrP signaling in promoting cyclin D1 expression and Sox9 transcription
activity. As a result, entrance to Zone II is enhanced, whereas Zone II to
hypertrophic chondrocyte transition was delayed. We conclude that
Wnt5a overexpression delays chondrocyte hypertrophy indirectly as a
result of prolonged residence of chondrocytes in Zone I, whereas
Wnt5b overexpression directly delays chondrocyte hypertrophy by
inhibiting cell cycle withdrawal and activating Sox9 activity.
In the Wnt5a-/- limb, chondrocytes enter Zone II prematurely but terminal differentiation is delayed as a combined result of decreased p130 expression and enhanced Sox9 activity in differentiating chondrocytes. This may cause reduced diameter of Zone I and enlarged diameter of Zone II (Fig. 3A). It is likely that the parallel columnar structure of chondrocytes in the proliferative chondrocyte zone ensures the normal longitudinal long bone growth and cartilage diameter. This process may be regulated by both Wnt5a and Wnt5b as suggested by the phenotypes of disrupted columnar chondrocyte organization, and short and thickened cartilages in Wnt5a-/-, Col2a1-Wnt5a and Col2a1-Wnt5b mice (Fig. 3A, Fig. 5C, Fig. 6D).
Taken together, Wnt5a and Wnt5b play a pivotal role in regulating the proper longitudinal lone bone growth by setting a proper pace during chondrocyte transition between different zones and controlling cell shape and polarity. We propose that when Zone I cells enter zone II, they express Wnt5a which signals back to maintain a stronger expression of p130 and a lower expression of cyclin D1 to prevent more cells from leaving Zone I. In addition, Wnt5a activates p130 expression and may suppress Sox9 activity to allow chondrocytes to leave Zone II and undergo hypertrophy. The newly formed hypertrophic chondrocytes express Wnt5b, which signals back to Zone II to prevent more cells from leaving Zone II (Fig. 9). It is likely that proper regulation of the transition between Zone I and II by Wnt5a is important in cartilage and bone development, and is essential in maintaining the articular cartilage throughout life.
The functional difference of Wnt5a and Wnt5b may rely on
which of the Wnt receptors, namely Frizzleds, are expressed in chondrocytes.
Vertebrate Wnt family comprises at least 18 different members and there are at
least 10 different Frizzleds in the human genome
(http://www.stanford.edu/~rnusse/wntgenes/mousewnt.html).
So far, three distinct Wnt signaling pathways have been identified
(Niehrs, 2001;
Winklbauer et al., 2001
). It
has been shown that the canonical Wnt pathway, which is mediated by
ß-catenin-LEF/TCF transcription complex, positively regulates cyclin D1
expression (Shtutman et al.,
1999
; Tetsu and McCormick,
1999
). Our data indicate that Wnt5b may signal through the canonic
pathway, whereas Wnt5a antagonizes it. This is supported by recent studies
that show a Wnt/Ca2+ pathway mediated by the Xenopus Wnt5a
decreases ß-catenin protein level
(Saneyoshi et al., 2002
). It
is interesting to note that Wnt1, a canonic Wnt in other systems, acted
similarly to Wnt5a in regulating Col2a1 reporter in the primary
chondrocytes. It is likely that a single Wnt, for example Wnt5a or Wnt1, can
elicit different responses depending on which Frizzled is expressed
(He et al., 1997
;
Slusarski et al., 1997a
;
Slusarski et al., 1997b
). We
find that when human Frizzled 5 is expressed together with either
Wnt5a or Wnt5b in the early Xenopus embryos, a
secondary axis is induced (data not shown), indicating Wnt5a and
Wnt5b both act as Wnt1 when Frizzled 5 is available. As Frizzled 5
shows little specificity with different Wnts
(Deardorff et al., 2001
), it
is unlikely that Frizzled 5 is a major Frizzled involved in transducing
Wnt5a/Wnt5b signals in chondrocytes. Since several different Frizzled genes
are expressed in the developing cartilage
(Xu et al., 2001
), Wnt5a and
Wnt5b signaling in chondrocytes may be very complex, and involve both
canonical and non-canonical pathways at the same time.
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ACKNOWLEDGMENTS |
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