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First published online August 25, 2006
doi: 10.1242/10.1242/dev.02546
1 Genetic Disease Research Branch, National Human Genome Research Institute,
Bethesda, MD 20892, USA.
2 Cardiovascular Research Institute, University of California, San Francisco, CA
94143, USA.
* Authors for correspondence (e-mail: pao-tien.chuang{at}ucsf.edu; yingzi{at}mail.nih.gov)
Accepted 19 July 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Wnt, ß-catenin, Ihh, Patched, Cartilage, Endochondral bone, Joint, Chondrocyte hypertrophy, Osteoblast differentiation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Karsenty and
Wagner, 2002; Kronenberg,
2003; Zelzer and Olsen,
2003). Wnt and hedgehog signaling pathways control cell
proliferation, differentiation, survival and migration in both developing
embryos and adult animals. In the developing skeletal systems, several aspects
of endochondral bone formation are critically regulated by both
Wnt/ß-catenin and Ihh signaling pathways. Inactivation of ß-catenin
or Ihh signaling leads to ectopic chondrocyte differentiation at the expense
of bone formation (Day et al.,
2005; Hill et al.,
2005; Hu et al.,
2005; Long et al.,
2004). Wnt/ß-catenin and Ihh signaling pathways also regulate
chondrocyte hypertrophy and maturation
(Akiyama et al., 2004;
Day et al., 2005;
St-Jacques et al., 1999;
Vortkamp et al., 1996). In the
developing cartilage, Ihh is expressed in prehypertrophic
chondrocytes. It controls osteoblast differentiation in the perichondrium by
controlling the expression of Runx2, which encodes a transcription
factor required for osteoblast differentiation
(St-Jacques et al., 1999). Ihh
is also a major driving force for chondrocyte proliferation
(Karp et al., 2000), and it
sets the pace for chondrocyte hypertrophy by activating the expression of
parathyroid hormone related peptide (Pthrp; Pthlh - Mouse
Genome Informatics), which inhibits chondrocyte hypertrophy
(Lanske et al., 1996;
St-Jacques et al., 1999;
Vortkamp et al., 1996). In
contrast to what is known about the functional mechanism of Ihh signaling, the
molecular mechanism underlying the Wnt/ß-catenin functions in
endochondral skeletal development remains to be elucidated. Deciphering the
genetic relationship between Wnt/ß-catenin and Ihh signaling pathways
will provide significant new insight into the molecular mechanisms by which
both signaling pathways control endochondral bone formation.
It has been proposed that Ihh may signal upstream of Wnt signaling because
the expression of Wnt7b and Tcf1 in the perichondrium is
lost in the Ihh mutant (Hu et
al., 2005). However, instead of being regulated by Ihh
directly, the expression of Wnt7b and Tcf1 may be simply
associated with osteoblast differentiation in the perichondrium, which is
abolished in the Ihh mutant. Consistent with this, Tcf1 is
not required for osteoblast differentiation in mice
(Glass et al., 2005) and there
is no evidence to support that Wnt7b is required for osteoblast
differentiation in vivo. Another unsettled issue is the role of
Wnt/ß-catenin signaling in regulating the expression of the transcription
factor osterix (Osx; Sp7 - Mouse Genome Informatics) that
acts downstream of Runx2 in controlling osteoblast differentiation
(Nakashima et al., 2002).
Although Osx expression was clearly detected in the
perichondrium/periosteum when ß-catenin was inactivated by Cre-mediated
recombination (Day et al.,
2005), its expression was missing in another similar study
(Hu et al., 2005). Therefore,
the control of Osx expression by the Wnt/ß-catenin signaling,
and the genetic interactions between Wnt/ß-catenin and Ihh signaling
await further investigation by rigorous genetic tests.
Another important aspect of endochondral skeletal development is synovial
joint formation, which is controlled by Wnt/ß-catenin signaling
(Guo et al., 2004;
Hartmann and Tabin, 2001).
Although joint formation is not disrupted in Ihh mutant mice
(St-Jacques et al., 1999), it
is likely that Ihh signals to the joint region, and it is possible that Ihh
signaling needs to be kept at a low level to allow normal joint formation.
All mammalian hedgehogs (Hhs), including Ihh, transduce their signals
through two multipass transmembrane proteins, smoothened (Smo) and patched 1
(Ptch1) (reviewed by Huangfu and Anderson,
2006; Lum and Beachy,
2004). The current model proposes that, in the absence of the Hh
ligand, the signaling activity of Smo is inhibited by Ptch1. Hh binding to
Ptch1 relieves its inhibition of Smo, allowing Smo to transduce the Hh signal
to the intracellular components. It is predicted that Hh signaling can be
maximally activated in a cell-autonomous manner by inactivating
Ptch1. The Wnt/ß-catenin signaling is the canonical Wnt
signaling transduced through stabilizing ß-catenin (reviewed by
Logan and Nusse, 2004;
Moon, 2005). In the absence of
Wnt ligands, ß-catenin is phosphorylated by glycogen synthase kinase 3
(Gsk3) and then targeted for degradation in the proteasome. When Wnt ligands
bind to their receptors frizzled and low-density lipoprotein receptor-related
protein 5/6 (Lrp5/6), ß-catenin phosphorylation is inhibited. This causes
the cytoplasmic accumulation and nuclear translocation of ß-catenin,
which activates downstream gene transcription by binding to Lef/Tcf factors.
Therefore, the strength of the canonical Wnt signal can be manipulated by
altering the protein levels of ß-catenin. Canonical Wnt signaling has
been blocked cell autonomously by tissue-specific inactivation of
ß-catenin in many developmental processes.
Here, we have generated a floxed allele of Ptch1 and activated Hh signaling cell autonomously in osteochondral progenitor cells during endochondral bone formation. We found that upregulation of Hh signaling uncoupled chondrocyte hypertrophy and osteoblast differentiation. Endochondral ossification occurred in the absence of chondrocyte hypertrophy. In addition, upregulation of Hh signaling led to mild joint fusion. Furthermore, when canonical Wnt signaling was blocked at the same time that Ihh signaling was upregulated, we observed cell-context-dependent genetic interactions between the canonical Wnt and Ihh signaling pathways. In osteoblast differentiation, ß-catenin acts, not only downstream of the Ihh pathway, but also downstream of Osx. In chondrocyte survival, Wnt/ß-catenin is upstream of Ihh signaling when inhibiting chondrocyte apoptosis. Finally, during chondrocyte hypertrophy and synovial joint formation, Wnt/ß-catenin and Ihh signaling pathways exert opposite activities and there is a synergistic interaction between them during synovial joint formation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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loxP allele (333 bp
band size). Oligonucleotides Ptch1b and Ptch1f
(5'-GCTACAAGGAGGCTCTAGGTGC-3') were used to genotype the
Ptch1 wild-type allele (200 bp band size). ß-catenin alleles
were genotyped as described (Guo et al.,
2004).
Skeletal analysis
Embryos were dissected in PBS. The embryos were then skinned, eviscerated,
and fixed in 95% ethanol. Skeletal preparations were performed as described
(McLeod, 1980).
Histology, in situ hybridization, immunohistochemistry and TUNEL assay.
Embryos were fixed in 4% paraformaldehyde at 4°C overnight. Fixed
samples were embedded in paraffin and sectioned at 5 µm thickness.
Histological analysis, BrdU labeling, immunohistochemistry and radioactive
35S-RNA in situ hybridization were performed as described
(Yang et al., 2003). Primary
antibodies included anti-p21 mouse monoclonal IgG (Santa Cruz) at 1:40,
anti-p27 mouse monoclonal IgG (Santa Cruz) at 1:40, anti-p57 rabbit polyclonal
IgG at 1:160 (Santa Cruz) and anti-cyclin D1 mouse monoclonal IgG (Santa Cruz)
at 1:80. Signals were detected using biotin-conjugated secondary antibodies
and the ABC Kit (Vectorstain). Cell death was detected as described
(Barrow et al., 2003). Probes
for in situ hybridization have been described previously: ColX, ColII,
Ihh and Runx2 (Yang et al.,
2003); Osx (Nakashima
et al., 2002); Mmp13
(Day et al., 2005);
Gli1 (Lewis et al.,
2001); and Hip1
(Chuang and McMahon, 1999). An
EST clone (Clone ID 5346064, from Invitrogen) was used to generate
Pthrp probes.
Micromass and limb cultures
Micromass cultures and Cre-Adenovirus infection were performed as
previously described (Guo et al.,
2004). Recombinant mouse Bmp2 and noggin were purchased from
R&D Systems. Bmp2 (0.5 µg/ml) and noggin (1.5 µg/ml) were added to
the micromass culture 24 hours after the cells were plated at high density. In
the limb culture, the BGJB (Invitrogen) medium, containing noggin (1.5
µg/ml), was changed every 2 days.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
We found that in the Ptch1c/-; Col2a1-Cre embryos, long
bones in the limb were shorter, bone mineralization was enhanced and the
posterior skull failed to form (Fig.
2L-N). These results are consistent with previous findings when
Ihh was overexpressed during endochondral bone formation in mice
(Long et al., 2004;
Long et al., 2001). It appears
that failure of posterior skull formation in the Ptch1c/-;
Col2a1-Cre embryos may be due to enhanced antichondrogenic
Wnt/ß-catenin signaling in the skull mesenchyme, which resulted from
abnormal brain development. In support of this, loss of ß-catenin
completely rescued skull formation in the Ptch1c/-;
Catnbc/-; Col2a1-Cre double mutant embryos
(Fig. 3A). In the limb sections
of the Ptch1c/-; Col2a1-Cre embryos, we found that
chondrocyte hypertrophy was inhibited (Fig.
2A,B) and Pthrp expression was upregulated
(Fig. 2C,D), suggesting that
Ihh signaling was activated in the developing cartilage. Indeed, the
expression of Hh signaling transcriptional targets such as Gli1 and
Hip1 was activated in both chondrocytes and perichondrium, with the
strongest sites of activation observed in the perichondrium and joint region
(Fig. 3B). These results
demonstrate that the Ptch1 allele was inactivated by Cre-mediated
recombination, which led to Hh signaling activation in the
Ptch1c/-; Col2a1-Cre embryos. As a result of Hh signaling
activation, we found that mineralized periosteum extended ectopically to the
joint region, where mineralization was never found in wild-type controls
(Fig. 2E,F). Mild joint fusion
was also observed in the Ptch1c/-; Col2a1-Cre embryo
(Fig. 2G,H), which is
consistent with previous observation that Shh overexpression in the
cartilage resulted in joint fusion
(Tavella et al., 2004). These
results suggest that, to maintain joint integrity, Ihh signaling in the joint
needs to be kept sufficiently low not to allow ectopic osteoblast
differentiation and cartilage fusions.
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). It was not clear whether the residual expression of
Runx2 and Osx was a direct consequence of residual
ß-catenin-positive cells caused by incomplete recombination, or an
indirect result of reduced Ihh signaling and thinner perichondrium caused by
reduced Ihh expression in the Catnbc/-;
Col2a1-Cre embryo (Day et al.,
2005), as Ihh is required for cell proliferation and the
expression of Runx2 and Osx
(Karp et al., 2000;
Long et al., 2004). To clarify
this issue, we took advantage of the thicker perichondium and the
ligand-independent cell autonomous upregulation of Hh signaling in
Ptch1c/-; Col2a1-Cre mouse embryos and investigated
whether the removal of Wnt signaling in these embryos would affect
Runx2 and Osx expression. In the Ptch1c/-;
Col2a1-Cre mouse embryo at 14.5 dpc, we found that Runx2
expression was significantly upregulated throughout the thickened
perichondrium, including the joint region, in a similar pattern to
Gli1 and Hip1 expression
(Fig. 4A). Importantly, in the
Ptch1c/-; Catnbc/-; Col2a1-Cre double mutant
embryo, we still observed strong ectopic expression of Runx2 and
Osx in the perichondrium, although the perichondrium was slightly
thinner (Fig. 4A). The
persistent and strong expression of Runx2 and Osx cannot be
due to ß-catenin expression in the perichondrium because ossification,
which requires ß-catenin, was almost completely inhibited in the
Ptch1c/-; Catnbc/-; Col2a1-Cre double mutant
(Fig. 3A,
Fig. 4B). These results
indicate that like Gli1 and Hip1 expression, Runx2
expression may be directly controlled by Ihh signaling. Furthermore,
ß-catenin is not required for the expression of Runx2 and
Osx, two downstream targets of Ihh signaling, in early osteoblasts in
the developing long bones. Our results demonstrate that ß-catenin is
required downstream of Ihh signaling and Osx expression during early
osteoblast differentiation.
Interestingly, osteoblast maturation, indicated by von Kossa staining and
the expression of mature osteoblast markers osteopontin (Opn;
Spp1 - Mouse Genome Informatics) and matrix metalloproteinase 13
(Mmp13), was not detected in the Ptch1c/-;
Col2a1-Cre mouse embryo at 14.5 dpc when ossification had just started in
the wild-type embryo, although the expression of Runx2 and
Osx was strongly activated (Fig.
4A). Osteoblast maturation caught up later at 16.5 dpc
(Fig. 4B). Furthermore, in the
humerus of the Ptch1c/-; Col2a1-Cre embryo, chondrocyte
hypertrophy indicated by ColX expression was missing, and yet
osteoblast differentiation occurred at 16.5 dpc
(Fig. 4B). Therefore, activated
Ihh signaling is sufficient to induce osteoblast differentiation and
maturation without chondrocyte hypertrophy. Because Ihh is also
required for Runx2 expression and osteoblast differentiation
(St-Jacques et al., 1999), our
results further indicate that during normal endochondral bone formation, the
coupling of chondrocyte hypertrophy with osteoblast differentiation and
maturation is mediated by Ihh signaling. The slight delay in osteoblast
maturation in the Ptch1c/-; Col2a1-Cre embryo is likely to
be due to the severely delayed chondrocyte hypertrophy
(Fig. 4A,B), because factors
such as Vegf and Mmps are expressed in hypertrophic
chondrocytes and are required to promote osteoblast maturation
(Inada et al., 2004;
Stickens et al., 2004;
Zelzer et al., 2002). We also
observed that the Mmp9 expression was ectopically activated in both
the Ptch1c/-; Col2a1-Cre and Ptch1c/-;
Catnbc/-; Col2a1-Cre double mutant mouse embryos in a pattern
similar to that of Gli1 and Hip1 expression
(Fig. 4A), suggesting that
Mmp9, but not Mmp13, expression may also be directly
controlled by Hh signaling. In contrast to the short delay in osteoblast
maturation in the Ptch1c/-; Col2a1-Cre mutant, osteoblast
differentiation was severely blocked in the Ptch1c/-;
Catnbc/-; Col2a1-Cre double mutant in which there was almost
no von Kossa staining or expression of Opn and Mmp13 at 16.5
dpc, although the expression of Osx was still stronger than that in
the Catnbc/-; Col2a1-Cre mutant
(Fig. 4B). These results
confirm that ß-catenin is required downstream of Osx for the
progression of osteoblast maturation.
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;
Yan et al., 1997;
Zhang et al., 1997;
Cobrinik et al., 1996), we
examined the status of chondrocyte proliferation by BrdU labeling in
Ptch1 and ß-catenin mutant embryos. In the developing cartilage,
chondrocytes are organized into two zones with distinct proliferation rates
before hypertrophy (Long et al.,
2001; Yang et al.,
2003). The resting zone chondrocytes are located at the articular
end and proliferate slowly. The proliferative zone is composed of fast
proliferating chondrocytes, and is located between the resting zone and the
prehypertrophic region where Ihh is expressed
(Fig. 6A). We found that, as
expected, in the Ptch1c/-; Col2a1-Cre embryo, chondrocyte
proliferation in the presumed resting zone was significantly increased to a
level similar to that in the proliferative zone by activated Hh signaling
(Fig. 6A,B). In contrast to a
previous study (Akiyama et al.,
2004), we did not detect a reduction in chondrocyte proliferation
in the Catnbc/-; Col2a1-Cre mouse embryo
(Fig. 6B). Interestingly,
chondrocyte proliferation in Ptch1c/-; Catnbc/-;
Col2a1-Cre double mutant embryos resembles that in the
Catnbc/-; Col2a1-Cre mouse embryo
(Fig. 6A,B), particularly in
the resting zone, indicating that Wnt/ß-catenin signaling may be required
for the activity of Hh signaling in promoting cell proliferation in resting
zone chondrocytes. As both Hh and Wnt/ß-catenin signaling can regulate
cell proliferation through regulating cyclin D1 expression
(Long et al., 2001;
Tetsu and McCormick, 1999), we
next examined the expression of cyclin D1, and p21, p27 and p57, members of
the Cip/Kip family of cyclin-cyclin dependent kinase (CDK) inhibitors, in
mutant embryos at 14.5 dpc.
Consistent with the status of cell proliferation revealed by BrdU labeling,
cyclin D1 expression is normally expressed highly in proliferative
chondrocytes and we found that its expression was greatly upregulated in the
presumed resting chondrocytes in the Ptch1c/-; Col2a1-Cre
mutant embryo (Fig. 7A). In
addition, we did not observe a reduction in cyclin D1 expression in the
Catnbc/-; Col2a1-Cre mutant embryo, and the cyclin D1
expression pattern was similar in Catnbc/-; Col2a1-Cre and
Ptch1c/-; Catnbc/-; Col2a1-Cre mutant embryos.
These results suggest that although ß-catenin is required for cell
proliferation and cyclin D1 expression downstream of Ihh signaling in the
resting zone, there may be a ß-catenin-independent pathway in the
proliferative zone to promote cyclin D1 expression and cell proliferation. P27
was expressed in chondrocytes of all zones and its expression was not altered
by Hh or Wnt/ß-catenin signaling (Fig.
7A). P57 is strongly expressed in prehypertrophic chondrocytes and
is required to promote chondrocyte hypertrophy
(Yan et al., 1997;
Zhang et al., 1997). The
expression pattern of p57 was not altered in the Catnbc/-;
Col2a1-Cre mutant embryo. But in the Ptch1c/-;
Col2a1-Cre and especially the Ptch1c/-;
Catnbc/-; Col2a1-Cre mutant embryo, strong expression of p57
was detected throughout the cartilage even though chondrocyte hypertrophy was
significantly delayed (Fig.
7A). Interestingly, p21 was expressed almost exclusively in
prehypertrophic chondrocytes in the wild-type embryo. Only very few cells in
the proliferative zone expressed p21 at both 14.5 and 18.5 dpc
(Fig. 7A,B). In the
Ptch1c/-; Col2a1-Cre double mutant embryo, few
chondrocytes expressed p21, similar to what was observed in the proliferative
region of the wild-type cartilage at 14.5 and 18.5 dpc
(Fig. 7A,B). In the
Catnbc/-; Col2a1-Cre mouse embryo, slightly more cells in
the resting and proliferative zones expressed p21 at 14.5 dpc
(Fig. 7A). Strikingly, at 18.5
dpc, significantly more cells in the proliferative and resting zones of the
Catnbc/-; Col2a1-Cre embryo expressed p21
(Fig. 7B). But in the double
mutant embryo, p21 expression was reduced to a level similar to that in the
Ptch1c/-; Col2a1-Cre embryo at both 14.5 and 18.5 dpc
(Fig. 7A,B), indicating that
p21 expression is inhibited by both the Wnt/ß-catenin and Hh signaling
pathways in proliferative and resting chondrocytes, and that Hh signaling acts
downstream of Wnt/ß-catenin signaling. It should be noted here that, in
contrast to cyclin D1 expression, changes in p57 and p21 expression did not
correlate with those of chondrocyte proliferation in the mutants. p57 or p21
expressing cells were increased in either Ptch1c/-;
Col2a1-Cre or Catnbc/-; Col2a1-Cre embryos,
respectively, but there was no corresponding reduction in cell proliferation
(Figs 6,
7). As p21 acts, not only as an
inhibitor for cell proliferation, but also as a regulator of cell death with
both anti- and pro-apoptotic function depending on the cellular context
(Gartel and Radhakrishnan,
2005; Tsao et al.,
1999; Wang et al.,
2005; Yamamoto and Nishioka,
2005), we examined cell death by TUNEL assay. In the wild-type
embryo, cell death was mostly observed in the joint and the most mature
hypertrophic chondrocytes at 18.5 dpc. Cell death was not evident in the
proliferative and resting chondrocytes
(Fig. 7B). By contrast, in the
Catnbc/-; Col2a1-Cre embryo at 18.5 dpc, extensive cell
death was detected in both proliferative and resting chondrocytes, and this
cell death phenotype was completely suppressed in the Ptch1c/-;
Catnbc/-; Col2a1-Cre embryo
(Fig. 7B). These data
demonstrate that Wnt/ß-catenin acts upstream of the Hh signaling pathway
in promoting chondrocyte survival, and further suggest that p57 and p21 may
act downstream of canonical Wnt and Hh signaling in this process.
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). For
instance, the elbow joint was normal in both Ptch1c/-;
Col2a1-Cre and Catnbc/-; Col2a1-Cre single mutant
embryos, but was completely fused in the double mutant
(Fig. 8B). To test whether the
joint ever forms in the double mutant, we examined the earliest elbow joint
development at 12.5 dpc when the elbow joint interzone has just formed (see
Fig. S1 in the supplementary material). We found that the elbow joint
interzone failed to form only in the double mutant embryos, based on both
morphological and molecular criteria (see Fig. S1 in the supplementary
material). These results indicate that the inhibition of canonical Wnt
signaling was strongly enhanced by the activation of Ihh signaling, thus
causing synovial joint fusion.
Consistent with previous findings in Col2a1-Gal4; UAS-Ihh mouse
embryos (Minina et al., 2002),
we observed that the expression of Bmp7, and to a lesser extent,
Bmp2 and Bmp4, but not the joint marker Gdf5, was
activated in chondrocytes, perchondrium and the forming joint region in both
Ptch1c/-; Col2a1-Cre and Ptch1c/-;
Catnbc/-; Col2a1-Cre mutant embryos
(Fig. 8C). Because Bmp
signaling acts to inhibit synovial joint formation
(Brunet et al., 1998;
Tsumaki et al., 2002;
Zou et al., 1997), it is
likely that the Bmp genes mediate the activity of Ihh signaling in regulating
synovial joint formation and that canonical Wnt signaling interacts with Ihh
signaling indirectly through Bmps. We tested this possibility directly by
culturing the limb buds from 12.5 dpc wild-type and Ptch1c/-;
Col2a1-Cre mutant embryos. Bmp signaling was inhibited in the culture by
the addition of noggin, a Bmp inhibitor. The humerus-scapula joint was fused
in the Ptch1c/-; Col2a1-Cre mutant embryos in vivo and in
vitro, and cells in the presumptive joint area maintained chondrocyte-specific
Safranin O staining. Importantly, in the noggin-treated
Ptch1c/-; Col2a1-Cre embryos (n=3), many cells in
the humerus-scapula joint were elongated and had much lighter Safranin O
staining, thereby resembling those joint cells found in the wild-type embryos
(Fig. 8D). Furthermore, we
found that the expression of the joint marker Gdf5 was lost in the
humerus-scapula joint of the Ptch1c/-; Col2a1-Cre mutant,
but was significantly rescued by noggin treatment (see Fig. S2 in the
supplementary material), demonstrating that joint formation was rescued in the
Ptch1c/-; Col2a1-Cre mutant by blocking Bmp signaling.
Noggin did not affect joint formation in the wild-type limbs (n=3;
Fig. 8D). These results
demonstrate that joint fusion in Ptch1c/-; Col2a1-Cre
mutant embryos is caused by upregulated Bmp signaling.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Canonical Wnt signaling is required downstream of Ihh signaling and
Osx in regulating osteoblast differentiation during endochondral
ossification Previous studies have uncovered the requirement of the Ihh
and canonical Wnt signaling pathways in osteoblast differentiation during
endochondral bone formation (Day et al.,
2005; Hill et al.,
2005; Hu et al.,
2005; Long et al.,
2004). In this study, by activating the Hh signaling pathway while
inactivating ß-catenin simultaneously in the same cells in
Ptch1c/-; Catnbc/-; Col2a1-Cre double mutant
embryos, we clarified two issues that were not settled before. First, the
genetic relationship between Ihh signaling and ß-catenin in promoting
osteoblast differentiation was not clearly demonstrated in vivo. It has been
suggested that ß-catenin may act downstream of Ihh signaling based on
correlations of gene expression. In particular, expression of Tcf1
and Wnt7b was suggested as a downstream target of Ihh signaling in
activating the canonical Wnt signaling (Hu
et al., 2005). However, the lack of Tcf1 or
Wnt7b expression in the perichondrium of Ihh-/-
embryos could be a consequence of a loss of the right cell types in which
Tcf1 and Wnt7b are normally expressed. Second, it was not
clear whether ß-catenin could act downstream of Osx in
controlling osteoblast differentiation, as Osx expression was
reduced, but not abolished, in Catnbc/-; Dermo1-Cre and
Catnbc/-; Col2a1-Cre mutant embryos
(Day et al., 2005) (this
study). The reduced expression of Osx could be a result of reduced
Ihh signaling and thinner perichondrium in the mutant embryos. In this study,
the thickness of perichondrium, the expression of both Runx2 and
Osx, and the final bone formation were greatly increased by
activating Hh signaling in the Ptch1c/-; Col2a1-Cre
mutant. Yet, further removal of ß-catenin in the Ptch1c/-;
Catnbc/-; Col2a1-Cre double mutant reduced bone formation to
the same degree as that in the Catnbc/-; Col2a1-Cre
mutant, despite the fact that expression of Runx2 and Osx in
the perichondrium of the double mutant was similar to that in the wild type
and stronger than that in the Catnbc/-; Col2a1-Cre mutant.
Therefore, Osx-expressing cells fail to give rise to bone-forming
mature osteoblasts in the absence of ß-catenin. ß-catenin is
required not only downstream of the Ihh signaling pathway, but further
downstream of Osx in osteoblast maturation. Our results, together
with previous studies, indicate that Runx2 is a transcriptional
target of Ihh signaling. As Osx is downstream of Runx2,
ß-catenin is required at least three steps downstream of Ihh signaling.
Because ectopic chondrocyte differentiation occurred in the periosteum of the
developing long bones of the Catnbc/-; Col2a1-Cre and
Osx-/- embryo (Day et
al., 2005; Nakashima et al.,
2002), it is likely that loss of ß-catenin not only blocked
osteoblast differentiation, but also caused Osx-expressing cells to
lose Osx expression later and become chondrocytes. Consistent with
this, we found that in the absence of ß-catenin, Osx expression
was much weaker in older Catnbc/-; Col2a1-Cre embryos than
in wild type (Fig. 4A,B),
suggesting that ß-catenin may even be required for the maintenance of
Osx expression.
Ossification in the Ptch1 conditional mutant is directional along the longitudinal axis
During normal long bone development, mature ossification is initiated from
the middle of the cartilage. This is thought to be a result of the chondrocyte
hypertrophy that is required for ossification and which occurs in the middle
segment first (reviewed by Kronenberg,
2003). In the Ptch1c/-; Col2a1-Cre mouse
embryo, hedgehog signaling was activated cell autonomously in all chondrocytes
and throughout the perichondrium independently of Ihh ligand and chondrocyte
hypertrophy. However, mature ossification was still initiated from the middle
of the long bones, although ectopic ossification did occur throughout the
perichondrium, including the joint, eventually. The directional ossification
along the longitudinal axis cannot be a consequence of localized chondrocyte
hypertrophy as, in the developing humerus of the Ptch1c/-;
Col2a1-Cre mouse embryo, chondrocyte hypertrophy was inhibited by
upregulated Pthrp expression (Fig.
4). The directional ossification was not a result of a
differential response to Hh or Bmp signaling along the longitudinal axis of
the developing long bones either, as we found that the expression of Hh
signaling targets, Runx2 and Bmps was upregulated strongly throughout
the perichondrium, including the joint region. The directional osteoblast
maturation might be caused by some unidentified inhibitor(s) of osteoblast
differentiation localized in the joint region. The earliest sign of
directional ossification in the Ptch1c/-; Col2a1-Cre
mutant is the expression of Osx that was expressed at the strongest
level in the middle segment (Fig.
4A). Because osteoblast maturation eventually occurred in the
joint, we favor another explanation: that the directional osteoblast
maturation may simply reflect the time required by the Runx2
expressing cells to proceed to the next stages of osteoblast maturation,
including activating the expression of Osx. In the
Col2a1-Cre mice, Cre is expressed in early osteochondral
progenitor cells and chondrocytes, and not in late perichondral cells in the
middle segment of the long bones. However, the effects of Cre-mediated
recombination, which is inactivation of Ptch1, are inherited in all
descendants of the early Cre expressing cells. As the development of
chondrocytes and osteoblasts proceed from the joint to the middle segment of
long bones, many osteoblasts in the middle segment are descendants of the
earlier Col2a1-Cre expressing cells, and they have been activated by
Hh signaling for a longer period of time.
Opposite and independent functions of canonical Wnt and Ihh signaling in chondrocyte hypertrophy
We observed that the phenotype of delayed chondrocyte hypertrophy in the
Ptch1c/-; Col2a1-Cre mutant embryo was enhanced by loss of
ß-catenin. As canonical Wnt signaling promotes chondrocyte hypertrophy,
and Ihh signaling regulates chondrocyte hypertrophy mainly indirectly through
activating and maintaining the expression of Pthrp, the canonical Wnt
signaling pathway may also antagonize Pthrp signaling in controlling
chondrocyte hypertrophy. It is likely that the canonical Wnt and Pthrp
signaling pathways exert opposite activities in regulating common targets
required for the execution of chondrocyte hypertrophy. The common targets may
be Runx2 or Sox9, or both, as they are both required to
control chondrocyte hypertrophy (Bi et al.,
2001; Enomoto et al.,
2000; Kim et al.,
1999). Consistent with the role of Runx2 in promoting
chondrocyte hypertrophy, Runx2 expression was reduced in chondrocytes
in Ptch1c/-; Col2a1-Cre embryos, although its expression
was upregulated in the perichondrium (Fig.
4A). But a reduction of Runx2 expression in chondrocytes
was not obvious in the Catnbc/-; Col2a1-Cre mouse
(Fig. 4A). It is also possible
that the reduction of Runx2 expression in Ptch1c/-;
Col2a1-Cre cartilage is only secondary to the initial delay of
chondrocyte maturation caused by a Runx2-independent mechanism. This
is supported by a recent finding that Pthrp, a major target of Hh
signaling in controlling chondrocyte hypertrophy, regulates chondrocyte
hypertrophy through both Runx2-dependent and -independent pathways
(Guo et al., 2006). In
contrast to Runx2, Sox9 inhibits chondrocyte hypertrophy
(Bi et al., 2001). Loss of
ß-catenin increases Sox9 activity in chondrocytes
(Akiyama et al., 2004), whereas
Pthrp potentiates Sox9 activity by activating PKA, which phosphorylates Sox9
(Huang et al., 2001). As
Sox9 is expressed in all Col2a1 expressing chondrocytes at
all developmental stages, the regulation of Sox9 activity is at least one of
the underlying mechanisms for the opposite activities of canonical Wnt and
Pthrp signaling in regulating chondrocyte hypertrophy.
Canonical Wnt and Hh signaling inhibit chondrocyte apoptosis
Our results indicate that both the canonical Wnt and Hh signaling pathways
regulate chondrocyte survival. It has been demonstrated that the
oxygen-sensitive hypoxia-inducible factor 1 alpha (Hif1
) and Vegfa are
required for chondrocyte survival and the regulation of chondrocyte
proliferation in cartilage, which is an avascular tissue
(Schipani et al., 2001;
Zelzer et al., 2004). The
phenotypes of Catnbc/-; Col2a1-Cre embryos bear some
similarities to those of Hif1a and Vegfa cartilage mutants
in chondrocyte survival, although they are not as severe. It will be
interesting to further investigate whether the canonical Wnt and Hh signaling
pathways interact with the Hif1 and Vegfa pathway in controlling
chondrocyte survival.
We have previously shown that inactivation of ß-catenin promotes
chondrocyte differentiation (Day et al.,
2005; Guo et al.,
2004). Our finding here that ß-catenin inactivation also
resulted in a dramatically increased cell death in differentiated chondrocytes
further indicates that just inhibiting Wnt/ß-catenin signaling alone is
not sufficient to maintain healthy cartilage. The rescue of chondrocyte
apoptosis and the inhibition of chondrocyte hypertrophy by Hh signaling in
Ptch1c/-; Catnbc/-; Col2a1-Cre mouse embryos is
significant from the therapeutic standpoint: to maintain healthy joint
cartilage or to treat degenerating joint cartilage, inhibiting canonical Wnt
signaling needs to be combined with the manipulation of other signaling
pathways, such as activating Hh signaling or perhaps its downstream Pthrp
signaling.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/18/3695/DC1
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ACKNOWLEDGMENTS |
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