1 Department of Medicine, Washington University Medical School, St. Louis, MO
63110, USA
2 Department of Molecular Biology and Pharmacology, Washington University
Medical School, St Louis, MO 63110, USA
Author for correspondence (e-mail:
flong{at}wustl.edu)
Accepted 3 November 2004
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SUMMARY |
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Key words: Hh, Wnt, Osteoblast, Mouse
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Introduction |
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Indian hedgehog (Ihh) signaling is indispensable for osteoblast development
in the endochondral skeleton. In developing cartilage, Ihh is
primarily expressed by prehypertrophic chondrocytes (chondrocytes immediately
prior to hypertrophy) as well as in early hypertrophic chondrocytes, and Ihh
signals to both immature chondrocytes and the overlying perichondrial cells
(St-Jacques et al., 1999;
Vortkamp et al., 1996
).
Ihh-/- mice completely lack osteoblasts in the
endochondral skeleton, at least in part because of the failure to express
Runx2 (St-Jacques et al.,
1999
). Furthermore, Ihh is required for the formation of ectopic
bone collar in growth plates containing both wild-type and PTH/PTHrP receptor
null chondrocytes (Chung et al.,
2001
). Genetic manipulation of Smoothened (Smo),
which encodes an obligatory component of the Hh signaling pathway, has
revealed that cells devoid of Smo, hence Hh signaling, fail to undergo
osteoblast differentiation (Long et al.,
2004
). Therefore, direct Ihh input in early osteogenic progenitors
is necessary for osteoblast differentiation. However, it is not known whether
Ihh interacts with other osteogenic signals in this regulation, or whether Ihh
also functions at later stages of osteoblast development.
Wnt signaling has been implicated in postnatal bone mass homeostasis. Wnt
molecules exert their functions by activating several distinct intracellular
pathways, including that mediated by ß-catenin, known as the canonical
pathway (Huelsken and Birchmeier,
2001; Wodarz and Nusse,
1998
). In this pathway, Wnt proteins signal through the Frizzled
family of receptors and the low-density lipoprotein receptor-related proteins
(Lrp5 and Lrp6, Arrow in Drosophila) as co-receptors, resulting in
stabilization of ß-catenin (Mao et
al., 2001b
; Pinson et al.,
2000
; Tamai et al.,
2000
; Wehrli et al.,
2000
). The stabilized ß-catenin transports to and accumulates
in the nucleus where it interacts with transcription regulators including the
lymphoid enhancer-binding factor 1 (Lef1) and T-cell factors (Tcf1, Tcf3 and
Tcf4), leading to transcriptional activation of downstream target genes
(Eastman and Grosschedl,
1999
). Recently, Lrp5 was found to be inactivated in patients with
the osteoporosis-pseudoglioma syndrome
(Gong et al., 2001
).
Conversely, an activating mutation in Lrp5 has been linked in two separate
cases to individuals with a high bone density syndrome
(Boyden et al., 2002
;
Little et al., 2002
).
Furthermore, mice deficient in Lrp5 were viable but postnatally
developed a low bone mass phenotype because of reduced osteoblast
proliferation and function (Kato et al.,
2002
). The endogenous Wnt ligand(s) that signal through Lrp5 have
not been identified. Moreover, the role of the Wnt/ß-catenin pathway in
the development of the osteoblast lineage remains unclear as mice deficient in
ß-catenin die because of gastrulation defects by E8.5 before skeletal
development occurs (Haegel et al.,
1995
; Huelsken et al.,
2000
). Interestingly, several studies have suggested that
activation of canonical Wnt signaling can induce osteoblast differentiation in
cell culture models (Bain et al.,
2003
; Gong et al.,
2001
; Rawadi et al.,
2003
).
Here, we examine the role of ß-catenin in osteoblast development by removing it from early osteogenic tissues using the Cre/LoxP-mediated gene inactivation. We demonstrate that Wnt signaling is essential for osteoblast development and acts downstream of Hh. We also identify Wnt7b as a potential ligand controlling osteogenesis in vivo.
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Materials and methods |
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Analyses of mouse embryos
Embryonic tissues were collected in PBS, fixed in 10% formalin overnight at
room temperature, then processed and embedded in paraffin prior to sectioning
at 6 µm. For detection of mineralization, sections were stained with 1%
silver nitrate (von Kossa method) and counterstained with Methyl Green. In
situ hybridization was performed as described previously, using
35S-labeled riboprobes (Long et
al., 2001) or using digoxigenin-labeled riboprobes
(Brent et al., 2003
). For BrdU
analysis, pregnant females were injected with BrdU at 0.1 mg/g body weight at
2 hours prior to harvest. Embryos were collected in ice-cold PBS, processed
and sectioned as above. BrdU detection was performed using a kit from Zymed
Laboratories (South San Francisco, CA) as per instructions. Labeling index was
scored for a least four sections from various planes of section through the
cartilage. Multiple wild-type and mutant pairs of littermates were analyzed
and results from a representative pair are reported here. For
immunohistochemistry, sections were prepared as above and stained with a
monoclonal antibody against ß-catenin (BD Biosciences/Pharmingen, San
Diego, CA) following antigen retrieval using SDS (BD Biosciences/Pharmingen
protocol). Signal was detected using the Vectorstain Elite ABC kit (Vector
Laboratories, Burlingame, CA) and DAB as chromagen (Zymed Laboratories).
Cell lines and osteogenesis assays
The C3H10T1/2 cells, the Wnt3a-expressing cell line and the control cell
line were obtained from ATCC (Manassas, VA) and maintained as per
instructions. Wnt3a-conditioned medium and the control conditioned medium were
harvested as per instructions. The recombinant N-terminal fragment of Shh
(N-Shh) was purchased from R&D Systems (Minneapolis, MN) and used at 1
µg/ml. C3H10T1/2 cells were cultured to confluence prior to N-Shh or Wnt3a
stimulation. AP expression was detected either by substrate staining or a
chemical assay as previously described
(Katagiri et al., 1994). In
experiments where both GFP fluorescence and AP staining were visualized, BCIP
and NBT were used as substrates for AP (Roche Diagnostics Cooperation,
Indianapolis, IN). To assess the effects of cycloheximide on Hh-induced gene
expression, C3H10T1/2 cells were treated with N-Shh for 24 hours with the
addition of either DMSO or 10 µg/ml cycloheximide (Sigma, dissolved in
DMSO) before harvested for real-time PCR (see below).
Expression constructs and retroviral infections
Retroviral expression constructs were generated by a two-step procedure.
The various cDNA fragments were first cloned into the vector pCIG
which contains the GFP sequence following the internal ribosomal
entry site (IRES) and the nuclear localization signal (NLS)
(Megason and McMahon, 2002).
The fragment containing the cloned cDNA along with the IRES-NLS-GFP
sequence was subsequently cloned into the retroviral vector pSFG
(Ory et al., 1996
). The
Smo* cDNA was as previously described
(Long et al., 2001
).
Dkk1 was donated by Dr Christopher Niehrs
(Glinka et al., 1998
). The
dominant-negative form of Tcf4 (dnTcf4) was originally from
Dr McCormick (Tetsu and McCormick,
1999
). A control virus was also generated by cloning the
NLS-GFP sequence into pSFG. Viruses were packaged as
previously described (Ory et al.,
1996
). Viral titers were determined by counting the GFP-positive
cells under a fluorescence microscope. For infections, cells
50%
confluent were incubated with the virus-containing medium for 24 hours, and
then cultured in regular complete medium for an additional 4 days before
harvest for either AP assay or RNA extraction. For bone nodule formation
assays, C3H10T1/2 cells were infected with either the Smo* or the
control virus for 24 hours and then cultured in regular complete medium in the
presence of ascorbic acid and ß-glycerophosphate for 2 weeks with medium
changed as needed. The cells were finally fixed in 10% formalin and stained
with Alizarin Red. For transient transfections of Wnt genes, full-length
sequences of Wnt5a, Wnt7b or Wnt9a (ATCC) were cloned into
pCIG and then transfected with Lipofectamine (GibcoBRL, Gaithersburg,
MD).
Real-time PCR
Total cellular RNA was isolated using Trizol reagent (GibcoBRL). The cDNA
was prepared using reagents from Roche Diagnostics Cooperation. Real-time PCR
was performed on GeneAmp5700 (Applied Biosystems, Foster City, CA) as per
instructions. All primers were designed using the ABI software Primer Express
and sequences are available upon request. Three independent samples were
analyzed for each condition and results were normalized to GAPDH in each
sample. For analyses of cartilage samples, cartilage elements were isolated
from the hindlimbs of E14.5 embryos and collected in liquid nitrogen. The
cartilage was pulverized in liquid nitrogen with a small pestle fit in 1.5 ml
tubes, and then extracted for RNA using the Trizol reagent.
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Results |
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Consistent with the lack of bone at the histological level, molecular
analyses by in situ hybridization demonstrated that mature osteoblasts failed
to develop in Dermo1-Cre; ß-cateninc/c
embryos. Collagen type I (Col1a1) was expressed at a lower level in
early osteoblast progenitors but was subsequently upregulated with further
differentiation. In the humerus of E18.5 wild-type embryos, robust expression
of Col1(I) was detected in the perichondrium
surrounding the prehypertrophic and hypertrophic cartilage
(Fig. 1F, arrow), in the bone
collar flanking the marrow cavity (Fig.
1F, `M'), as well as in the primary spongiosa
(Fig. 1F, asterisk). In CKO
littermates however, only a low level of Col1a1 expression was
detected in the perichondrium (Fig.
1F', arrow), with no Col1a1 expression in the
marrow cavity (Fig. 1F',
`M'). Like Col1a1, bone sialoprotein (Bsp;
Spp1 - Mouse Genome Informatics) was normally expressed in the
perichondrium surrounding the prehypertrophic and hypertrophic cartilage
(Fig. 1G, arrow), as well as in
the primary spongiosa (Fig. 1G,
asterisk). In CKO embryos however, Bsp was not detectable in either
the perichondrium or the marrow cavity
(Fig. 1G', arrow,
`M'). Finally, osteocalcin (OC; Bglap1 - Mouse
Genome Informatics) was normally expressed by mature osteoblasts present
either in the bone collar (Fig.
1H, arrow) or in the primary spongiosa
(Fig. 1H, arrowhead), but no
OC was detectable in CKO embryos
(Fig. 1H', arrow).
Similarly in the skull, whereas Col1a1, Bsp and OC are
normally all expressed in the frontal bone at E18.5
(Fig. 1I-K, arrows), none of
these markers was detected in the equivalent regions of CKO littermates
(Fig. 1I'-K', arrows). Thus, removal of ß-catenin by Dermo1-Cre disrupted
osteoblast differentiation.
Histological analyses of the developing long bones also revealed a significant delay in chondrocyte maturation in CKO embryos. At E14.5, chondrocytes at the diaphysis of a normal humerus became hypertrophic (Fig. 1L); this, however, was not the case in the CKO embryo where all chondrocytes remained small (Fig. 1L'). The delay in hypertrophy in the CKO mutant continued through E15.5, when only a small cluster of chondrocytes at the core of the humerus initiated hypertrophy (Fig. 1M', magenta arrow), whereas the vast majority of cells remained immature (Fig. 1M', orange arrows). By contrast, in E15.5 wild-type littermates, blood vessels invaded the hypertrophic cartilage of the humerus (Fig. 1M, inset). Extensive hypertrophy did eventually occur in the CKO mutant by E17.5 and this was accompanied by vascular invasion as evidenced by red blood cells in the core of the humerus (Fig. 1N'). However, no bone was detected in the mutant humerus at either E17.5 (Fig. 1N') or E18.5 (Fig. 1O') even though the primary spongiosa (Fig. 1N, arrows) and a bone collar (Fig. 1O, arrows) were evident in the wild-type littermate. Instead, the humerus cartilage in CKO mutants was surrounded by thin layers of perichondrial cells at E18.5 (Fig. 1O', arrows). Thus, ß-catenin is required for both the normal schedule of chondrocyte hypertrophy, as well as bone formation.
Molecular basis for osteoblast defect in Dermo1-Cre; ß-cateninc/c embryo
To characterize further the arrest of osteoblast development in
Dermo1-Cre; ß-cateninc/c (CKO) embryos,
additional molecular analyses were performed on the humerus of E14.5 embryos.
Consistent with the analyses at E18.5, Col1a1 was normally expressed
at a low level in the perichondrium towards the epiphysis
(Fig. 2A, arrowhead) but was
strongly upregulated in regions surrounding the hypertrophic cartilage
(Fig. 2A, arrow). In CKO
embryos however, a low level of Col1a1 was maintained throughout the
perichondrium without any increase at the diaphysis
(Fig. 1A', arrowhead).
Alkaline phosphatase (AP) was expressed in a similar pattern as
Col1a1 in the wild-type embryo (compare Fig.
2A with
2B, arrowheads and arrows),
except that AP was also expressed by early hypertrophic chondrocytes
(Fig. 2B, `H'). In the
CKO embryo, only a low level of AP was detected in the perichondrium
(Fig. 2B', arrowhead). No
AP expression was evident in the chondrocytes of the E14.5 humerus,
consistent with a delay in chondrocyte maturation with the removal of
ß-catenin (Fig.
2B'). The failure to upregulate Col1a1 and
AP in the perichondrium of the CKO mutant was not secondary to a
delay in chondrocyte hypertrophy, as both markers remained expressed at low
levels even at E18.5 after hypertrophy had occurred
(Fig. 1F'; data not
shown). These data indicate that in the absence of ß-catenin, osteoblast
development was arrested after low levels of Col1a1 and AP
were expressed in the early progenitors.
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Compared with the Dermo1-Cre; ß-cateninc/c mutant, Ihh-/- embryos exhibited an earlier defect in osteoblast development. Specifically, at E14.5 Col1a1 was not detectable in the perichondrium of long bones of the Ihh-/- mutant (Fig. 2A'', arrowhead), although Col1a1 was expressed in the ligament (Fig. 2A'', `L'). Similarly, no AP-expressing cells were present in the perichondrium of the Ihh-/- mutant (Fig. 2B'', arrowhead). Moreover, Runx2 was not expressed in the perichondrium of the Ihh-/- mutant (Fig. 2C'', arrow) although scattered expression was detected in chondrocytes (Fig. 2C'', asterisk). Similarly, the Ihh-/- mutant did not exhibit any expression of Osx in the perichondrium (Fig. 2D'', arrow), although a low level of expression in chondrocytes was detectable (Fig. 2D'', asterisk). These results demonstrate that Ihh signaling is required for the initiation of the osteogenic program.
ß-Catenin promotes chondrocyte maturation and proliferation
Consistent with observations at the histological level as described
earlier, molecular analyses confirmed that ß-catenin was necessary for
the proper maturation of chondrocytes. At E15.5 in the wild-type humerus,
strong expression of collagen type X Col10a1, a specific marker for
hypertrophic chondrocytes (Linsenmayer et
al., 1991), characteristically defines two hypertrophic zones
(Fig. 3A, `H')
separated by a nascent marrow cavity (Fig.
3A, `M'). In the CKO embryo, however, expression of
Col10a1 had just begun in a small cluster of cells in the center of
the humerus (Fig. 3A', arrow), indicating a marked delay in chondrocyte maturation. Consistent with
the delay, Ihh expression, which delineates two discreet
prehypertrophic domains in the wild-type humerus at E15.5
(Fig. 3B), remained in a
contiguous region of the diaphysis in a CKO littermate
(Fig. 3B'). Patched 1
(Ptch1), which encodes the receptor for Hh proteins, is also a
transcriptional target of the Hh pathway
(McMahon et al., 2003
). In the
wild-type embryo, Ptch1 was characteristically upregulated in the
immature proliferating chondrocytes (Fig.
3C, `P') adjacent to the Ihh-expressing domain,
at the primary spongiosa within the marrow cavity
(Fig. 3C, `M'), as
well as in the perichondrium flanking the Ihh-expressing chondrocytes
(Fig. 3C, arrow). Remarkably,
in the CKO embryo, although Ptc1h expression in the chondrocytes was
similar to the wild-type level, expression in the perichondrium was much
reduced (Fig. 3C',
arrow). Thus, the osteoblast defect in the CKO embryo correlates with a
suppressed response of the perichondrial cells to Ihh signaling.
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Canonical Wnt signaling is disrupted in Ihh-/- embryos
We next examined the potential relationship between Ihh and canonical Wnt
signaling in osteoblast development. Immunohistochemistry was performed on
sections of long bones to determine the distribution of ß-catenin. At
E14.5, ß-catenin was normally detected in both prehypertrophic and early
hypertrophic chondrocytes (Fig.
4A, `PH' and `EH' respectively) as well as in
the perichondrium (Fig. 4A,
box). Immature and proliferating chondrocytes, on the other hand, were largely
negative for the ß-catenin signal
(Fig. 4A, `P'). At higher
magnification, ß-catenin clearly accumulated in the nucleus of the
perichondrial cells flanking the prehypertrophic chondrocytes
(Fig. 4A', arrows). Some
staining was also observed at the boundaries of the cells
(Fig. 4A', arrowheads),
probably reflecting ß-catenin in association with the adherens junction.
Remarkably, in the Ihh-/- mutant, no nuclear
ß-catenin was detectable in perichondrial cells although low levels of
ß-catenin were detected at the cell boundaries
(Fig. 4B', arrowheads).
In addition, in Ihh-/- embryos, no signal was detected in
chondrocytes (Fig. 4B, asterisk), consistent with the absence of hypertrophic chondrocytes at this
stage.
|
We next examined canonical Wnt signaling in the long bones of wild type versus Ihh-/- embryos by assaying the expression of target genes Tcf1 and Dkk1. As described earlier, in E14.5 wild-type embryos, both Tcf1 and Dkk1 were expressed in the perichondrium flanking the prehypertrophic and the hypertrophic regions (Fig. 5A,B, arrows). Tcf1 was also expressed at a lower level in prehypertrophic and proliferating chondrocytes (Fig. 5A, `PH' and `P' respectively). In the Ihh-/- littermate, however, no expression was detected in the perichondrium for either Tcf1 or Dkk1 (Fig. 5A',B', arrows), although expression of Tcf1 in the chondrocytes was maintained (Fig. 5A', `C'). Other members of the Lef/Tcf family were not detectable in the perichondrium of the E14.5 wild-type tibia (Fig. 5C-E, arrows), but were expressed in chondrocytes in distinct patterns. In particular, Lef1 expression was largely restricted to proliferating chondrocytes (Fig. 5C, `P'); Tcf3 was detected throughout the cartilage proper (Fig. 5D); Tcf4 was expressed both in the proliferating chondrocytes and at a higher level in hypertrophic chondrocytes (Fig. 5E, `P' and `H' respectively). In the Ihh-/- littermate, whereas Tcf3 and Tcf4 remained expressed in the chondrocytes (Fig. 5D',E', `C'), Lef1 was no longer detectable (Fig. 5C', `C'). Thus, Tcf1 is the primary member of the Lef/Tcf family expressed in the perichondrium. Moreover, the canonical Wnt signaling pathway is impaired in the Ihh-/- mutant.
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We next investigated osteogenesis induced by Wnt signaling in C3H10T1/2 cells. Wnt3a-conditioned medium induced AP as early as day 2 of treatment, and AP expression reached a maximum after 4 days of stimulation (Fig. 7A). Like AP, other osteoblast markers, including Col1a1, osteopontin (OP; Spp1 - Mouse Genome Informatics) and OC were all significantly induced within 48 hours of Wnt3a stimulation (Fig. 7D-F, respectively). However, neither Runx2 nor Osx was induced during this time (Fig. 6B,C, respectively). Thus, in C3H10T1/2 cells Wnt3a can induce osteogenesis without changes in either Runx2 or Osx. These results therefore reveal a second role for Wnt signaling that is downstream of Runx2 and Osx, and is in addition to its requirement for Osx expression as described earlier.
|
Wnt7b is a potential endogenous signal controlling osteogenesis
As an initial step to identify endogenous Wnt ligands that mediate
Hh-induced osteogenesis, we examined the expression of Wnt genes in relation
to Hh signaling. We first surveyed all 19 murine Wnt molecules by real-time
PCR in C3H10T1/2 cells following Hh stimulation. These experiments revealed
that Wnt5a, Wnt7b and Wnt9a were consistently induced over
control levels following 24 or 48 hours of Hh treatment
(Fig. 8A,B, respectively).
Moreover, induction of Wnt7b and Wnt9a did not require de
novo protein synthesis, as cycloheximide did not inhibit it although it did
repress Wnt5a induction. The mechanism for the induction of
Wnt7b and Wnt9a expression by cycloheximide is not
understood but a similar stimulatory effect has been previously reported
(Mullor et al., 2001).
|
We next determined whether Wnt5a, Wnt7b or Wnt9a could induce osteoblast differentiation. AP activity was assayed following transient transfection of constructs expressing one of the Wnt ligands in C3H10T1/2 cells. Wnt7b, but neither Wnt5a nor Wnt9a, increased AP expression approximately fourfold over the control (Fig. 8J). Thus, Wnt7b can induce pluripotent mesenchymal cells to undergo osteoblast differentiation.
Sequential roles of Hh and Wnt signaling in osteoblast development
Overall, these data suggest a model in which Hh and Wnt signals control
osteoblast development in a sequential manner
(Fig. 9). On the one hand, Hh
acts on very early progenitors to initiate the osteogenic program by
activating expression of Runx2, low levels of Col1a1 and
AP (Fig. 9, `1'). This
phase of signaling appears to require only a weak Hh response, as indicated by
low levels of Ptch1 expression. On the other hand, Hh induces
expression of Wnt ligands that signal through ß-catenin, which is in turn
required for Osx expression and further osteoblast differentiation
(Fig. 9, `2'). Whereas
Wnt/ß-catenin signaling is not sufficient to induce Osx, Hh can
directly stimulate Osx expression
(Fig. 9, `3'), and this phase
of signaling correlates with a strong Hh response, as reflected by robust
Ptch1 expression. Finally, Wnt signaling can promote osteoblast
differentiation without inducing Osx expression
(Fig. 9, `4'). In summary, Hh
and Wnt signals orchestrate the progression of osteoblast development.
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Discussion |
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Despite its role in the adherens junctions, removal of ß-catenin did
not cause any noticeable changes in the morphology and organization of cells.
Moreover, immunostaining with an antibody against -catenin, which
normally interacts with ß-catenin, did not show any changes in the
distribution in either chondrocytes or the perichondrial cells of the
Dermo1Cre; ßcatc/c mutant (data not shown).
It is likely that plakoglobin, which is closely related to ß-catenin
substitutes for ß-catenin to maintain the integrity of adherens
junctions, as previously reported in the ß-catenin-/- embryo
(Huelsken et al., 2000
).
Similarly, deletion of ß-catenin from the apical ectodermal ridge (AER)
of the limb bud did not change the distribution of E-cadherin in the ectoderm
(Barrow et al., 2003
). Thus,
the osteoblast defect observed in the present study is most likely due to
disruption of canonical Wnt signaling. In keeping with this notion,
ß-catenin normally accumulates at high levels in the nucleus of potential
osteogenic cells in the developing long bone, and expression of the Wnt target
genes Tcf1 and Dkk1 was abolished in the Dermo1Cre;
ßcatc/c mutant.
Although bone was largely absent throughout the Dermo1Cre;
ßcatc/c embryo, some bone formation was observed in
the scapula of the mutant. Interestingly, expression of Tcf1 and
Dkk1 was detected in the scapula of the mutant embryo at E15.5 (data
not shown). This most probably reflects incomplete removal of ß-catenin
in the scapula by Dermo1Cre, resulting in residual Wnt signaling in
this tissue. Consistent with the crucial role of ß-catenin in
osteogenesis, a previous study showed that removal of ß-catenin from the
cranial neural crest cells by Wnt1-Cre resulted in agenesis of most
craniofacial bones (Brault et al.,
2001).
Whereas Wnt/ß-catenin is required for osteogenesis throughout the
skeleton, Ihh is required only for the endochondral bones. Recently,
Smo, which encodes an obligatory component of the Hh pathway, was
removed with Wnt1-Cre to eliminate Hh response in cranial neural
crest cells (CNCC) that normally give rise to many of the craniofacial bones
via intramembranous ossification (Jeong et
al., 2004). This resulted in the absence of most CNCC-derived head
bones. However, owing to the early growth and patterning defects associated
with an increase in apoptosis and a decrease in proliferation, it is difficult
to conclude whether osteogenesis per se was impaired by the removal of Hh
responsiveness. Thus, it remains to be determined whether Hh or other signals
act upstream of Wnt/ß-catenin signaling in the head skeleton.
Interestingly, Hh response in the perichondrium is compromised in the Dermo1Cre; ßcatc/c embryo. Ptch1 was expressed at a much-reduced level in the perichondrial cells in the absence of ß-catenin. It is possible that Wnt/ß-catenin signaling directly regulates the amplitude of Hh response in osteogenic cells. Alternatively, strong Hh response may reflect a certain stage of osteoblast development and the loss of a strong response could simply reflect a developmental arrest in the absence of ß-catenin. The present study does not discern these possibilities. Regardless of the mechanism, Osx expression requires ß-catenin and correlates with robust Hh signaling.
The transcriptional effectors of canonical Wnt signaling in the osteoblast
lineage remain to be determined. In situ hybridization experiments identified
Tcf1 as the predominant member of the Lef/Tcf family expressed in the
perichondrium of embryonic long bones. Tcf1-/- mice,
however, developed a normal skeleton
(Verbeek et al., 1995). It is
not known whether other family members were upregulated and thus compensated
for the loss of Tcf1 in the Tcf1-/- mouse. It is also
possible that other members, although expressed at a much lower level, either
alone or in combination are responsible for normal osteogenesis in the
Tcf1-/- mutant. Tcf1 is unique in the family in
the sense that it is itself a target of the canonical Wnt pathway. Its high
level of expression in perichondrial cells could simply reflect robust
Wnt/ß-catenin signaling. Moreover, Tcf1 encodes a number of
isoforms including both transcriptional activators and repressors
(Van de Wetering et al.,
1996
). Thus, it is conceivable that deletion of both forms may
maintain the overall osteogenic program without causing any obvious phenotype.
Further insight on this subject will require analyses of various compound
mutants among the Lef/Tcf family members.
Osteogenesis induced by Smo* in C3H10T1/2 cells was not
completely inhibited by either Dkk1 or dnTcf4. The partial inhibition was not
due to lower titers of Dkk1 or dnTcf4 viruses, as
deliberately higher titers for these viruses did not change the degree of
inhibition (data not shown). This result could indicate that other pathways in
addition to canonical Wnt signaling contribute to Hh-induced osteogenesis. Of
note, other groups reported that Hh-induced osteogenesis in C3H10T1/2 cells
required BMP signaling (Spinella-Jaegle et
al., 2001; Yuasa et al.,
2002
).
The interaction between Hh and Wnt signaling is probably complex. Our
studies demonstrate that nuclear localization of ß-catenin as well as
expression of target genes for the Wnt canonical pathway were abolished in the
perichondrium in Ihh-/- embryos. This could, among other
possibilities, be due to the downregulation of Wnt expression in the absence
of Hh signaling. Indeed, expression of Wnt9a and Wnt7b was
either reduced or abolished in the perichondrium in Ihh-/-
embryos. In addition, both genes were induced by Hh signaling in C3H10T1/2
cells. Interestingly, a previous study showed that the Gli molecules,
transcriptional effectors of the Hh pathway, induced expression of several
Wnt genes in frog embryos (Mullor
et al., 2001). Alternatively, the Hh and Wnt signaling pathways
could intersect intracellularly via common regulators such as Suppressor of
fused [Su(fu)] (Meng et al.,
2001
) and GSK3 (Jia et al.,
2002
; Price and Kalderon,
2002
).
The expression studies as well as in vitro osteogenesis assays indicate
that Wnt7b may function as an osteogenic signal in vivo. Interestingly, a
recent study reported that Wnt7b was induced during osteogenesis in
primary cultures of bone marrow stromal cells
(Zhang et al., 2004).
Wnt7b-/- mice have been generated by two different
targeting strategies. Whereas one group reported placental defects
(Parr et al., 2001
) that
resulted in death by E11.5, the other concluded that defects in lung
development caused perinatal death (Shu et
al., 2002
). The latter case probably represents a hypomorph of
Wnt7b. It remains to be determined whether Wnt7b is required for osteogenesis
in vivo.
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ACKNOWLEDGMENTS |
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Footnotes |
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