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 1 August 2005
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SUMMARY |
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Key words: Ihh, Gli3, PTHrP (Pthlh), Wnt, ß-Catenin, Cartilage, Bone, Vascularization, Mouse
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Introduction |
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The role of vascularization in osteoblast formation during endochondral
skeletal development is not well understood. Cartilage anlagen that prefigure
the long bones are initially avascular; nascent vascularization coincides with
osteoblast differentiation in the perichondrium. Ihh-/-
mice exhibited profound defects in cartilage vascularization, and thus have
not been informative in previous analyses for assessing the role of
vascularization in osteoblast development in the endochondral skeleton.
However, the skull bones did form in the Ihh-/- embryo
(St-Jacques et al., 1999),
indicating the presence of an Ihh-independent osteogenic pathway in the
intramembranous bones.
Hh signaling is mediated through transcriptional regulation by the
zinc-finger transcription factors Gli1, Gli2 and Gli3 in mammals, or Cubitus
interruptus (Ci) in Drosophila. Mutant studies in the mouse
demonstrated crucial roles for Gli2 and Gli3 in the developing embryo, whereas
Gli1 appeared to be dispensable (Bai et
al., 2002; Ding et al.,
1998
; Matise et al.,
1998
; Mo et al.,
1997
; Motoyama et al.,
1998
; Park et al.,
2000
), although additional phenotypes were noted when
Gli1 was deleted in the Gli2-/- background
(Park et al., 2000
). In
particular, Gli2 appeared to function in vivo predominantly as a
transcriptional activator both in the central nervous system
(Ding et al., 1998
;
Komori et al., 1997
;
Matise et al., 1998
;
Sasaki et al., 1999
) and in
the developing hair follicle (Mill et al.,
2003
). Gli3, however, was shown to function primarily as a
transcription repressor of Hh targets. Removal of both Gli3 and
Shh in the mouse rescued a multitude of defects in specification of
the ventral neuron types, patterning of the limb and lung organogenesis caused
by the loss of Shh alone (Li et
al., 2004
; Litingtung and
Chiang, 2000
; Litingtung et
al., 2002
). However, additional studies have also revealed an
activator function for Gli3 in both the somite and the spinal cord
(Bai et al., 2004
;
Buttitta et al., 2003
), and a
repressor activity for Gli2 in the somite
(Buttitta et al., 2003
). These
studies underline the diverse roles of the Gli molecules in mediating Hh
signaling in different physiological contexts.
To determine the potential roles of Gli3 in mediating Ihh signaling during skeletal development, we have generated double mutant embryos of Ihh-/-; Gli3-/-, and have analyzed the skeletal phenotype. The results demonstrate that derepression of Gli3 accounts for multiple, but not all, aspects of Ihh signaling in the endochondral skeleton.
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Materials and methods |
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Analyses of mouse embryos
Whole-mount skeletal staining of embryos was based on McLeod
(McLeod, 1980). For analyses
on sections, 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. For histology or in situ hybridization on E17.5 and E18.5
embryos, limbs were decalcified in 14% EDTA in PBS (pH 7.4) for 48 hours after
fixation and prior to processing. In situ hybridization was performed as
described previously by using 35S-labeled riboprobes
(Long et al., 2001
). All in
situ probes were as previously described
(Hu et al., 2005
;
Long et al., 2004
;
Long et al., 2001
), with the
exception that the Pthlh probe was derived from a full-length cDNA
clone (ATCC, catalog no. 7061403). 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.
Western analyses
For western analyses of Gli3, whole-cell lysates were prepared using a RIPA
buffer [20 mM Tris (pH 8.0), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl,
0.1% SDS, 1 mM EGTA, 1 mM EDTA, protease inhibitor cocktail (Roche),
phosphatase inhibitor cocktails 1 and 2 (Sigma, St Louis, MO)] from the fore-
and hindlimb cartilage elements of E14.5 embryos. Cartilage was pulverized in
liquid nitrogen using a mini-pestle (Clontech) prior to protein extraction.
Total cell lysate (100 µg) was separated on a SDS-PAGE gel and a specific
antibody (Li et al., 2004) was
used to detect Gli3 by the ECL method (Amersham Biosciences). An antibody
against
-catenin (Santa Cruz Biotechnology) was used as a loading
control.
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Results |
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Whole-mount skeletal preparations from E18.5 embryos revealed that most skeletal elements of the DKO embryos exhibited an intermediate length between wild-type and Ihh-/- littermates (Fig. 1). This was most evident with the forelimb skeleton whose overall length was markedly increased in the DKO (Fig. 1A3) over the Ihh-/- embryo (Fig. 1A5), although the hindlimb skeleton showed a more modest increase in length (Fig. 1, compare B3 with B5). Notably, both limb skeletons of DKO embryos remained considerably smaller than those of either the wild-type (Fig. 1A1,B1) or the Gli3-/- embryos (Fig. 1A2,B2). The increase over Ihh-/- mutants in overall size of the skeleton was not evident in embryos of Ihh-/-; Gli3+/- (I-/-; G+/-) in which one copy of Gli3 remained intact (Fig. 1, compare A4 with A5, B4 with B5). In addition to a slight reduction in the length of long bones (Fig. 1, compare A1 with A2, B1 with B2), the Gli3-/- embryos exhibited a number of characteristic patterning defects in the limbs, including the absence of the deltoid tuberosity (Fig. 1A1, asterisk) in the humerus and a varying degree of truncation of the distal tibia (Fig. 1B2, big arrowhead). In addition, the Gli3-/- embryos invariably exhibited bifurcation of phalange 1 (P1) of all digits (Fig. 1B2, small arrowhead, out of plane of focus in A2) as well as polydactyly in both the forelimb (Fig. 1A2, digits 1-7) and the hindlimb (Fig. 1B2, digits 1-6, 2* representing syndactyly at digit 2). Importantly, all defects characteristic of the Gli3-/- embryo were maintained in the DKO embryo, including polydactyly, bifurcation of P1 (Fig. 1A3,B3, small arrowheads) and truncation of the tibia (Fig. 1B3, big arrowhead). These results indicate that Gli3 functions antagonistically with and genetically downstream of Ihh to regulate linear growth of the long bones.
Examination of the whole-mount stained skeletons at E18.5 also revealed that the bone collar was absent in most long bones of the DKO embryo. As in the Ihh-/- mutant (Fig. 1A5,B5), the metacarpal (`mc'), metatarsal (`mt') and phalanges (`p') of the DKO embryo exhibited little or no mineralization (Fig. 1A3,B3). Also similar to those in the Ihh-/- mutant (Fig. 1A5,B5), the radius (`r') (Fig. 1A3) as well as the fibula (`fi') (Fig. 1B3) contained mineralized cartilage (stained dark red) but no bone collar. Interestingly, the ulna and the more proximal elements such as the humerus (`h') (Fig. 1A3) and the femur (`f') (Fig. 1B3) in the DKO embryo contained some abnormal bone (green arrowheads in insets) around the mineralized cartilage at the diaphysis of these elements. Overall, bone formation remained largely dysregulated in the DKO embryo.
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We next determined whether Ihh antagonizes Gli3 activity by regulating the
proteolytic processing of Gli3. Western analyses were performed with whole
cell lysates harvested from E14.5 limb cartilage, using a Gli3 antibody.
Although the truncated repressor form of Gli3 (Gli3R, 83 kDa) was
detectable in the normal cartilage (Fig.
1E, `WT'), its amount was much increased in the absence of
Ihh (Fig. 1E,
`Ihh-/-'). Interestingly, in both the wild type and the
Ihh-/- cartilage, the full-length activator form
(Gli3A, 190 kDa) was a minor form that could be detected only after
longer exposure (Fig. 1E,
inset). Moreover, the total amount of Gli3 in the Ihh-/-
cartilage appeared to more than that in the wild-type cartilage. Thus, the
loss of Ihh likely resulted in an increase in both Gli3 total production as
well as the relative levels of Gli3R. The specificity of the
Gli3R and Gli3A bands was confirmed by their absence in
the Gli3-/- sample
(Fig. 1E,
`Gli3-/-'). A non-specific 80 kDa band also recognized by
the antibody was conveniently used as a loading control (asterisk,
Fig. 1E). The similar loading
across the genotypes was confirmed by using an antibody against
-catenin, an abundant adherens junction protein
(Fig. 1E).
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Normal proliferation and maturation of chondrocytes in the DKO embryo
Results from the whole-mount skeleton analyses prompted us to examine first
whether cartilage development was normalized in the DKO embryo. We first
analyzed chondrocyte maturation by histological analyses. At E18.5, the
Ihh-/- embryo exhibited only very rudimentary
vascularization at the center of the humerus (asterisk,
Fig. 2B) and contained a much
reduced growth region (red double-headed arrow,
Fig. 2B) as previously
described (St-Jacques et al.,
1999). By contrast, in the DKO embryo vascularization was more
advanced (asterisk, Fig. 2C),
and the growth region was markedly increased (red double-headed arrow,
Fig. 2C). Interestingly, both
the nonhypertrophic (green double-headed arrow) and the hypertrophy zone
(black double-headed arrow) were widened in the DKO embryo over the wild-type
counterpart (Fig. 2, compare A
and C), suggesting likely delays in both the onset and the removal of
hypertrophic chondrocytes in the DKO embryo.
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Analyses of the histological sections also revealed differences in the perichondrium among the various embryos. In the wild-type embryo, concurring with chondrocyte development, the perichondrium also underwent distinct morphological changes along the epiphyseal-diaphyseal axis. Specifically, in regions flanking the prehypertrophic chondrocytes (Fig. 2F1), the perichondrium (orange double-headed arrows) differentiated into an inner region with more and cuboidal cells (red arrows) contrasting with an outer region with fewer and elongated cells (green arrows). The morphological distinction between the layers became more pronounced in regions flanking the hypertrophy zone (Fig. 2G1) where the inner layer containing cuboidal cells (red arrows) directly lined the bone collar (yellow contour), whereas the outer layer (green contour) became more fibrous and contained fewer and much elongated cells (green arrows). In the Ihh-/- embryo, the perichondrium was generally hypoplastic (orange double-headed arrow, Fig. 2F2). At the diaphysis where rudimentary vascularization occurred (Fig. 2G2), although the perichondrium thickened with more cuboidal cells (red arrows) in the inner region and more elongated cells (green arrows) in the outer region (green contour), no distinct layers were established. In the DKO embryo, the width of the perichondrium (orange double-headed arrow, Fig. 2F3) was markedly increased over the Ihh-/- mutant and was similar to the wild-type counterpart. However, in contrast to that in the wild-type embryo, the perichondrium flanking the prehypertrophic chondrocytes in the DKO animal failed to differentiate into the morphologically distinct layers, but instead was uniformly populated with elongated cells (green arrows, Fig. 2F3). Differentiation of the perichondrium eventually occurred around the mid-region of the hypertrophic zone where bone formation began to occur (yellow contour, Fig. 2G3). Here, as in the wild-type animal, the cuboidal cells (red arrows) resided immediately adjacent to the bone collar, whereas the elongated cells (green arrows) populated the outer fibrous layer (green contour). Thus, the perichondrium in the DKO embryo exhibited normal growth but failed to undergo normal differentiation.
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We next examined the status of chondrocyte proliferation in the growth region cartilage. BrdU labeling experiments showed that the proliferation rate of chondrocytes in E14.5 DKO embryos was similar to the wild-type level, in contrast to a 50% reduction in Ihh-/- embryos (Fig. 3H).
Bone formation correlated with a partial rescue of vascularization in DKO embryos
The whole-mount skeletal staining experiment above revealed abnormal bone
formation in certain skeletal elements of the DKO embryo. To confirm this
finding, we stained tissue sections by the von Kossa method. At E18.5, the
wild-type embryo had developed a definitive bone collar (arrows) surrounding
the mineralized cartilage (red asterisks) in the long bones such as the ribs
(Fig. 4A1), the humerus
(Fig. 4B1) and the femur
(Fig. 4C1). The
Ihh-/- embryo, however, as previously reported, completely
lacked the bone collar (arrows) in all long bones although cartilage
mineralization occurred (asterisks, Fig.
4A2-C2). Like the Ihh-/- embryo, the DKO
embryo also lacked a bone collar at the normal position in all long bones
(arrows, Fig. 4A3-C3) despite
ample mineralization in the cartilage (red asterisks,
Fig. 4A3-C3). Interestingly,
however, in the humerus (Fig. 4B3 and
B4) and the femur (Fig. 4C3 and
C4), as well as certain ribs
(Fig. 4A3 and A4), a varying
amount of bone deposition (arrows, Fig.
4A4-C4) was detected at the diaphysis. A similar observation was
made in the ulna (data not shown). The bone deposition was often asymmetrical
and not adherent to the underlying cartilage (double-headed arrows,
Fig. 4B4,C4). An additional
important feature of the ectopic bone was that it invariably correlated with
vascularization of the cartilage, as evidenced by the presence of red blood
cells (purple asterisk, Fig.
4A3). Moreover, the degree of ossification also correlated with
that of vascularization. For example, the humerus
(Fig. 4B3) often exhibited more
ossification and was also more advanced in vascularization, whereas the femur
(Fig. 4C3) lagged in both
aspects. Thus, in the DKO embryo, the normal bone collar failed to form but
ectopic bone developed at the diaphysis of long bones where vascularization
occurred.
Cartilage vascularization in the long bones of the DKO embryo was partially rescued over the Ihh-/- embryo, although it remained defective compared with the wild-type embryo. This was exemplified in a rib of an E18.5 DKO embryo by the appearance of a rudimentary marrow cavity (red asterisk, Fig. 4D3), whereas no vascularization was noted in any ribs of an Ihh-/- embryo at same age (Fig. 4D2). The vascularization in the DKO embryo was noticeably delayed compared to the wild-type embryo, resulting in a wider hypertrophic zone (Fig. 4, double-headed arrows, compare D1 with D3).
A partial rescue of orthotopic osteoblast development in DKO embryos
The lack of normal bone collars in the DKO embryo prompted us to
investigate in detail the development of the osteoblast lineage. To this end,
we examined the expression of a panel of markers activated at various stages
of osteoblast development. At E15, in a wild-type humerus, Runx2 as
well as low levels of Col10a1 and alkaline phosphatase (Ap;
Akp - Mouse Genome Informatics) were characteristically expressed in
the perichondrial cells (red arrows, Fig.
5A1-C1) towards the epiphysis (`e',
Fig. 5A1), representing early
stages of the osteoblast lineage. Towards the diaphysis (`d',
Fig. 5A1) where cells of the
lineage became progressively more mature, the levels of Col10a1 and
Ap were elevated in the perichondrium (green arrows,
Fig. 5A1,C1), whereas
Runx2 expression (green arrow,
Fig. 5B1) remained relatively
constant. Meanwhile, Osx (Sp7 - Mouse Genome Informatics)
and subsequently bone sialoprotein (Bsp) were activated in the more
mature perichondrium (green arrows, Fig.
5D1,E1). In addition to the expression in the osteoblast lineage,
high levels of Runx2, Ap and Bsp were also detected in
hypertrophic chondrocytes (green asterisks,
Fig. 5B1,C1,E1, respectively),
whereas Osx was also in the prehypertrophic cells (green asterisk,
Fig. 5D1). Moreover, a lower
level of Runx2 was also detected in the immature chondrocytes (red
asterisk, Fig. 5B1). In the
Ihh-/- embryo, consistent with previous reports
(Hu et al., 2005;
St-Jacques et al., 1999
), none
of the osteoblast markers were expressed in the perichondrium at E15 (red
arrows, Fig. 5A2-E2), although
a number of molecules were diffusedly expressed in the cartilage, reflecting
the dysregulation of chondrocyte maturation (green asterisks,
Fig. 5B2-E2). Interestingly in
the DKO embryo, although Col10a1 and Runx2 were expressed in
the perichondrium (red arrows, Fig.
5A3,B3, respectively), Ap, Osx or Bsp could not
be detected there (red arrows, Fig.
5C3-E3, respectively). Moreover, the level of Col10a1
expression remained low throughout the perichondrium (red arrows,
Fig. 5A3). In the cartilage,
expression of Ap and Osx (green asterisks,
Fig. 5C3 and D3, respectively)
became restricted to discrete domains similar those in the wild-type embryo.
Bsp and high levels of Runx2, both characteristic of later
hypertrophic chondrocytes, were not yet expressed in the cartilage at this
stage (Fig. 5D3 and B3, respectively), consistent with a delay in chondrocyte maturation in the DKO
embryo, as noted earlier. However, similar to the wild-type embryo, a low
level of Runx2 expression was detected in the immature chondrocytes
in the DKO embryo (red asterisk, Fig.
5B3). Thus, development of the osteoblast lineage was initiated
but was later stalled in the DKO embryo.
We next further investigated the molecular basis for the arrest of
osteoblast development in the DKO embryo. Our previous work identified that
Wnt signaling through the canonical pathway was crucial for osteoblast
development and that this signaling was abolished in the
Ihh-/- embryo (Hu et
al., 2005). We therefore examined whether canonical Wnt signaling
was rescued in the DKO embryo. At E14.5, in the wild-type embryo Tcf1
and Dkk1, both direct transcriptional targets of Wnt canonical
signaling (Chamorro et al.,
2005
; Gonzalez-Sancho et al.,
2005
; Niida et al.,
2004
; Roose et al.,
1999
), were upregulated in the perichondrium (red arrows,
Fig. 5F1,G1). In the DKO
embryo, however, as in the Ihh-/- mutant
(Hu et al., 2005
),
Tcf1 and Dkk1 were not detected in the perichondrial cells
(Fig. 5F2,G2, respectively).
Importantly, the canonical Wnt pathway remained quiescent even at E18.5, when
Tcf1 was normally expressed in the perichondrium flanking the
prehypertrophic chondrocytes (red arrows,
Fig. 5H1) but was not
detectable in a similar region in the DKO embryo (red arrows,
Fig. 5H2). Notably,
Tcf1 expression was activated in the periosteum overlying the ectopic
bone (asterisk, Fig. 1H2),
indicating that canonical Wnt signaling was coupled with bone formation at the
ectopic site. Thus, in the DKO embryo, osteoblast development at the
orthotopic position was arrested prior to the activation of canonical Wnt
signaling in the progenitors.
We next investigated whether the defect in orthotopic osteoblastogenesis was merely a temporary delay that was corrected at later stages. At E18.5, in the wild type humerus Ap and Bsp (Fig. 6A1,B1, respectively) were expressed highly in cells of the osteoblast lineage associated with the primary spongiosa (`PS'), the bone collar (`BC') as well as the perichondrium (green arrows) flanking the prehypertrophic chondrocytes (`PH'), in addition to their expression in the hypertrophic chondrocytes (`H'). Similarly, Osx (Fig. 6C1) was expressed in the primary spongiosa (`PS'), the bone collar (`BC') and the perichondrium (arrows). Osteocalcin (Oc; Bglap1 - Mouse Genome Informatics) (Fig. 6D1), a specific marker for the mature osteoblast, was detected in the primary spongiosa (`PS') as well as the bone collar (`BC') that extended to flank the mid-region (green arrows) of the hypertrophic zone (`H'). By contrast, in the DKO humerus (Fig. 6A2-D2) none of the markers were detected in the corresponding regions of the perichondrium (green arrows), indicating a persistent lack of orthotopic bone formation. However, all the markers were expressed in the ectopic bone formed around the diaphysis (green asterisks, Fig. 6A2-D2). Thus, orthotopic osteoblast development was persistently arrested in the DKO embryo, even though ectopic bone formed at the diaphysis.
Distinct fates for perichondrial mesenchymal cells in the absence of either Ihh or canonical Wnt signaling
Our earlier observation that less bone formed at the femur than at the
humerus in the DKO embryo (Fig.
4, compare B3 with C3) prompted us to examine the fate of the
perichondrial mesenchymal cells at the diaphysis of the femur. Interestingly,
at E18.5 in areas of the femur where no overt ossification had occurred,
mesenchymal cells around the diaphysis (red asterisks,
Fig. 7A,B) accumulated under
the perichondrium (`PC', Fig.
7B), forming an asymmetrical `mesenchymal collar'. Remarkably, the
mesenchymal cells appeared to have migrated through the late hypertrophic
chondrocytes splitting the hypertrophic zone (`H',
Fig. 7A,C). Moreover, the
mesenchymal cells embedded between the hypertrophic zones appeared to have
died, resulting in an empty canal (green asterisk,
Fig. 7C). The cell death
correlated with the paucity of cartilage vascularization, which was reduced to
small areas of the hypertrophic cartilage
(Fig. 7D). More importantly,
the viable mesenchymal cells beneath the perichondrium expressed markers of
the osteoblast lineage, including Ap, Osx
(Fig. 7B1-B2, respectively) and
Runx2 (data not shown), none of which was detected in the
perichondrium (`PC'). The expression of these markers was not associated with
hypertrophic chondrocytes as the cells did not express Col10a1
(Fig. 7B3). Thus, in the DKO
embryo, perichondrial mesenchymal cells accumulated around the diaphysis of
the femur developed along the osteoblast lineage.
|
Finally, we examined the fate of the perichondrial cells in the E18.5 Ihh-/- embryo. As noted earlier (Fig. 2), the perichondrium in the Ihh-/- embryo was generally hypoplastic but slightly thickened at the diaphysis of the humerus (boxed region, Fig. 7G), where rudimentary vascularization occurred (asterisk, Fig. 7G). Moreover, the perichondrial cells within this region underwent morphological differentiation (Fig. 7G1), as indicated by the cuboidal cells (red arrows) within the inner region (`1') versus the elongated cells (yellow arrows) at the outer region (`2'). Importantly, the inner cells expressed Runx2, Osx and Ap (Fig. 7H1-J1, respectively) but not Col10a1 (data not shown). Thus, in the Ihh-/- embryo, rudimentary vascularization correlated with early stages of osteoblastogenesis from the perichondrium.
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Discussion |
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The identity of GliA is presently unknown. Although
Gli2A has been shown to possess activator activities necessary for
Shh signaling in both the central nervous system
(Ding et al., 1998;
Komori et al., 1997
;
Matise et al., 1998
;
Sasaki et al., 1999
) and the
developing hair follicle (Mill et al.,
2003
), Gli2-/- embryos exhibited only quantitative
defects in bone formation (Miao et al.,
2004
). Thus, multiple Gli activators may function in combination
to control progression of osteogenesis.
Notably, despite the rescue of cartilage proliferation and maturation, the
limb skeletal elements of the DKO embryos remained considerably shorter than
their wild-type counterparts. This finding underscores the important
contribution of a normal bone collar (which failed to develop in the DKO
embryo) to the overall linear growth of skeletal elements. Similarly, our
previous studies showed that the Col2-Cre; Smon/c mice, which had a
similar proliferation defect as the Ihh-/- mutant but maintained a
largely normal bone collar, exhibited a much milder growth defect than the
Ihh-/- embryo (Long et al.,
2001). Thus, development of the bone collar, together with growth
of the cartilage, contributes to the linear length of a long bone.
The recovery of a low level of Ptch1 in the DKO embryo is consistent with a
dual mechanism by which Ihh controls Ptch1 transcription: whereas a low level
expression can be achieved by derepression of Gli3R, the full
activation of Ptch1 expression may require the activator forms of Gli
molecules in response to robust Ihh signaling. Ptch1 was recently shown to be
a direct transcription target of Gli proteins
(Agren et al., 2004).
Despite the defect in cartilage vascularization in the DKO embryo,
perichondrial mesenchymal cells independently invaded the hypertrophic
cartilage. Thus, invasion of the mesenchymal cells can be uncoupled from
cartilage vascularization. The molecular basis underlying the mesenchymal
invasion is presently unknown but appears to be distinct from that for the
blood vessel invasion, as Mmp9
(Vu et al., 1998) was not
expressed by the invading mesenchyme (data not shown).
Ihh signals via distinct transcriptional effectors at different stages of
osteoblast development (Fig.
8B). Initially, Ihh activates expression of Runx2 and low levels
of Col1a1 by counteracting the repressor function of Gli3 (Gli3R)
(`1'). Further progression of the lineage, however, requires additional Ihh
signaling, most probably transcriptional activation via GliA (`2').
Previously, we showed that Ihh signaling activated canonical Wnt signaling
which was in turn required for the activation of Osx expression
(Hu et al., 2005). Thus,
GliA is likely required to activate the canonical Wnt signaling,
which remained silent in the DKO embryo (`3'). Alternatively, defective Wnt
signaling could be secondary to earlier defects in the osteoblast lineage
(e.g. absence of Aplow-positive cells) in the DKO embryo.
The perichondrial mesenchymal cells are bi-potential for either osteoblasts
or chondrocytes (Fig. 8B, `4').
In embryos of either Dermo1Cre; ß-cateninc/c
or DKO, the cells accumulated beneath the perichondrium at the diaphysis,
forming a characteristic `wedge-shape' mesenchyme. In the absence of
ß-catenin, the cells failed to express Osx and subsequently
developed into chondrocytes, in a manner similar to that in the
Osx-/- embryo
(Nakashima et al., 2002). In
the DKO embryo, Osx expression was activated in these cells,
presumably by a vasculature-derived signal, and osteogenesis ensued. Thus, Osx
appears to function as a molecular switch between the osteoblast and
chondrocyte fates.
Chondrogenesis is unlikely a mere default fate for the perichondrial
progenitors, as no chondrogenesis was observed in the perichondrium of the
Ihh-/- embryo even though osteogenesis was impaired. Here,
the perichondrium was generally hypoplastic, and no `wedge-shape' mesenchyme
was present at the diaphysis. In agreement with the absence of Runx2
expression in the perichondrial cells prior to vascularization, the
perichondrium phenotype here is similar to that in the
Runx2-/- embryo
(Komori et al., 1997;
Otto et al., 1997
),
implicating Runx2 in the proper growth of the perichondrium. Similarly, in the
Runx2-/- embryo, the perichondrial cells failed to undergo
chondrogenesis, despite the defect in osteoblast development. These findings
could indicate that Runx2 activity is required for the perichondrial cells to
achieve their capacity for chondrocyte differentiation. Alternatively, the
failure in chondrogenesis could be secondary to the perichondrium hypoplasia
associated with Runx2 deficiency. Regardless of the mechanism, the absence of
chondrogenesis in the Ihh-/- perichondrium seemed to be at
odds with our previous finding that Smo-/- perichondrial
cells (nonresponsive to Ihh) developed into chondrocytes
(Long et al., 2004
). However,
the difference could be because the Ihh-nonresponsive cells in the previous
study resided among wild-type cells in which Ihh signaling occurred. It is
possible that secondary factors from the normal neighboring cells steered the
Ihh-nonresponsive cells towards chondrogenesis.
Our results also indicate an osteogenic role for the vasculature
(Fig. 8B). In this model, a
signal from the vasculature could promote bone formation from
Col1a1low- and Runx2-postive cells (the `stalled'
cells in the DKO embryo) by activating Ap expression (`6') and the
canonical Wnt/ß-catenin pathway (`5'). Moreover, a
vascularization-dependent signal could initiate osteogenesis independent of
Ihh signaling (as in the Ihh-/- embryo) (`7'). It should
be noted that we could not discern at present which mechanism contributed to
the `ectopic' bone in the DKO embryo. In addition, it is presently unknown
whether mature osteoblasts can eventually develop in the
Ihh-/- embryo as neonatal lethality of the mutant
precludes analyses at later stages. However, a recent study reported that an
Ihh-/- cartilage element, when implanted into the kidney
capsule, became vascularized and developed bone, apparently without activating
hedgehog signaling (Colnot et al.,
2005). The nature of the vascularization-dependent signal is
presently unknown but previous studies have implicated signals for the
endothelium in development of the liver
(Matsumoto et al., 2001
) and
the pancreas (Lammert et al.,
2001
). A definitive link between bone formation and endothelial
signaling, however, awaits further studies.
The concept that vascularization functions as an osteogenic signal independent of Ihh has interesting implications. It follows that normally two distinct processes (Ihh dependent versus vascularization dependent) contribute to bone formation in the endochondral skeleton. Disruption of both in the Ihh-/- embryo resulted in a complete loss of bone. This notion could also explain the relatively normal intramembranous bones in the skull of the Ihh-/- embryo. As the skull bones are evolved earlier, does vascularization represent a more primitive and universal mechanism for bone formation, whereas Ihh dependence could be a recent invention to accommodate bone formation from a cartilage template?
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ACKNOWLEDGMENTS |
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