1 Department Craniofacial Development, Guy's, King's and St Thomas' School of
Dentistry, Guy's Hospital, London Bridge, London SE1 9RT, UK
2 Department of Biology, Dalhousie University, Halifax, NS B3H 4J1, Canada
3 Connective Tissue Biology Laboratories, School of Biosciences, Cardiff
University, Museum Avenue, Cardiff CF10 3US, UK
* Author for correspondence (e-mail: archer{at}cardiff.ac.uk)
Accepted 15 May 2003
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SUMMARY |
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Key words: Cbfa-1, Ihh, Sox9, Hypertrophy, Chondrocyte, Membrane bone, Proliferation, Chick
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Introduction |
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Skeletogenic tissues are highly responsive to applied forces, and hence are
uniquely shaped by epigenetic influences (which we define as cellular
responses to macroscopic influences); an example in development is the
evocation of secondary cartilage. Secondary cartilage arises after membrane
bone formation at a variety of sites in birds and mammals (reviewed by
Fang and Hall, 1997;
Beresford, 1981
). In the chick,
secondary cartilage has been shown to occur at articulations between membrane
bones and cartilage elements of the head
(Murray, 1963
). Muscle action
across these joints leads to the induction of cartilage within the bounds of
the periosteum (Murray, 1963
),
and this tissue is believed to be the source of the chondrocytes. This
evocation process can be mimicked in explant culture by mechanical
articulation (Hall, 1967
), or
prevented through in ovo paralysis (Murray
and Smiles, 1965
). In the mouse temporo-mandibular joint (TMJ),
immunohistochemical data indicate a rapid hypertrophy of secondary
chondrocytes (Silbermann et al.,
1990
; Shibata et al.,
1997
). Despite the unusual epigenesis, the lifetime of a secondary
chondrocyte is short, and follows the normal course for a chondrocyte:
resorption of matrix by chondroclasts and replacement with endosteal bone
(Hall, 1972
).
The identification and study of two transcription factors, Sox9 and
Cbfa1/Runx2, has greatly enhanced our understanding of the skeletal tissues.
Sox9 is recognised as the key regulator of chondrocyte differentiation: it is
obligate for chondrocyte specification (Bi
et al., 1999), regulates cartilage specific genes
(Bell et al., 1997
;
Lefebvre et al., 1997
;
Sekiya et al., 2000
;
Xie et al., 1999
) and can
induce ectopic chondrogenesis when misexpressed
(Bell et al., 1997
;
Healy et al., 1999
). Its
transcriptional regulation is consistent with these roles
(Wright et al., 1995
;
Zhao et al., 1997
), and
excludes any putative role in osteoblast development.
The runt-box-containing transcription factor Cbfa1/Runx2
is similarly essential for osteoblast maturation
(Ducy et al., 1997;
Komori et al., 1997
);
haploinsufficiency leads to the human skeletal disorder cleidocranial
dysplasia (Mundlos et al.,
1997
). Hypertrophy of chondrocytes in the Cbfa1-knockout
mouse is also defective in some long 'bone' elements
(Inada et al., 1999
;
Kim et al., 1999
), which is
consistent with upregulation of Cbfa1 expression in chondrocytes
preceding hypertrophy (Kim et al.,
1999
). The requirement for Cbfa1 in chondrocyte
maturation has been further investigated using the Col2 promoter to
drive Cbfa1 expression in immature chondrocytes and in those that
would not normally hypertrophy (Takeda et
al., 2001
). Cbfa1 is sufficient in these cells to promote
hypertrophy and to rescue the chondrocyte phenotype of
Cbfa1-deficient mice. Moreover, these studies identify Cbfa1 as the
first transcription factor to regulate both chondrocyte and osteoblast
differentiation (Takeda et al.,
2001
).
As mentioned above, Cbfa1 plays an important role in the
attainment of hypertrophy by some chondrocytes; an example of the regulatory
integration between bone and cartilage. The co-dependence of these tissues has
also been illustrated by elucidation of the role of Indian hedgehog (Ihh) in
skeletogenesis. Overexpression of Ihh in the chick limb leads to
suppressed hypertrophy and an increase in bone collar formation
(Vortkamp et al., 1996). The
former effect results from a feedback loop involving Pthrp
upregulation. Ihh expression in the pre-hypertrophic chondrocytes
coincides with osteogenesis in the adjacent perichondrium; a link shown to be
causal by the failure of osteogenesis in the long bones of
Ihh-/- mice
(St-Jacques et al., 1999
).
Thus, in conjunction with its role in chondrocyte proliferation, achieved
directly (Karp et al., 2000
)
and indirectly (Vortkamp et al.,
1996
), Ihh couples cartilage differentiation with differentiation
of adjacent osteoblasts (Chung et al.,
2001
). It can be seen, therefore, that a crucial step in
osteoblast induction is chondrocyte exit from the cell cycle leading to
pre-hypertrophy and Ihh upregulation. The tight choreography of
chondrocyte differentiation and osteogenesis in the perichondrium/periosteum
cedes the primary role to cartilage. It is appropriate therefore that in the
membrane bones of Ihh-/- mice, where cartilage forms
secondarily, osteogenesis is normal
(St-Jacques et al., 1999
).
Owing to the likely periosteal origin of secondary chondrocytes, one may
expect their genesis to challenge the established bifurcation between
Cbfa1-specified osteoblasts and Sox9-specified chondrocytes.
The regulation of these pivotal genes has not previously been examined in
secondary chondrogenesis, but the importance of epigenetic influences on skull
development and evolution is widely acknowledged
(Thorogood, 1993). We have
studied the evocation and function of secondary cartilage in the chick, and
found that the integration of epigenetic, and more familiar genetic, pathways
underlies its role as a signalling centre. Our study also indicates that
Sox9, like Cbfa1 and chondrocyte differentiation, can divert
pre-osteoblasts to chondrogenesis: the hypertrophic chondrocyte being the
common endpoint, reached via the functionally crucial Ihh-expressing
pre-hypertrophic chondrocyte.
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Materials and methods |
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Manipulation of the quadratojugal/quadrate joint
Fertilised wild-type chicken eggs (Ross White) were obtained from Henry
Stewart and Co., Lincolnshire, UK. Eggs were incubated at 38°C for
the stated number of days. Embryos at stage e14 were used for the ex vivo
experiments, as the joint was less liable to dislocation when manipulated. The
quadratojugal/quadrate (QJ/Q) was dissected from either side of the head and
stripped of most attendant connective tissue, except that holding the two
elements together. The Q and QJ were trimmed and then placed on a filter
(Millipore; 0.2 µM) upon a metal grid at the air/medium interface. Explants
were cultured in DMEM (Gibco) plus 15% chick serum (Sigma), at 37°C/5%
CO2. Articulation regimes involved operating the joint through its
normal movement (10 times on the hour) in order to mimic the sporadic
movements in ovo, for the specified time (following
Hall, 1967
).
Treatment with blocking antibody
Anti-hedgehog antibody, 5E1 (Ericson et
al., 1996), was obtained from the DSHB, University of Iowa, IA.
Partially purified antibody was obtained and used at a dilution of 1:10.
Cell proliferation studies
Explants were labelled in 10xBrdU diluted in culture medium (as
above), according to the manufacturer's protocol (Roche), immediately
following dissection (for 0 hour timepoint) or at the specified time
post-dissection for 90 minutes. Explants were then fixed in 4%
paraformaldehyde, processed through to wax and sectioned. BrdU incorporation
was revealed using a biotin-conjugated anti-BrdU antibody (MD-5215; Caltag,
USA). ABC kit from Vectastain (Vector Labs, Peterborough, UK) was then used,
followed by DAB (Vector Labs) for the colour reaction. Sections were
counterstained with Toluidine Blue (0.02%) for cell counting, or Alcian Blue
(prior to immunochemistry) and Chlorantine Fast Red (post-immunochemistry) to
assess differentiation. For pulse-chase experiments the QJ/Q joint was rinsed
in medium, placed on a fresh filter in fresh medium and re-incubated.
Parathyroid hormone (PTH) was used at a concentration of 10 nM.
High-density micromass culture
High-density micromass culture was carried out essentially as described by
Fang and Hall (Fang and Hall,
1996), based on the sequential digestion method for calvaria
(Wong and Cohn, 1974
). 2-3
dozen eggs were harvested to provide e13 QJs and Qs that were then dissected
free from muscle and connective tissue, and pooled in PBS. The hook,
containing the secondary chondrocytes, was removed and the shafts were
digested in 0.5% trypsin/collagenase type 1A (200 units/ml; Sigma) in PBS,
sequentially for 30 minutes, 30 minutes and 45 minutes. Between each digestion
the solution was removed and replaced. The solution containing the cells was
then spun down and rinsed in serum-containing medium. For establishing the
micromass cultures we used fractions 2 and 3. These were pooled, passed
through a cell strainer (Falcon), spun down and resuspended in 200 µl of
medium. A 10 µl drop was added to the centre of a well of a multi-well
plate, allowed to settle for 2 hours before flooding with medium (3/4 Ham's
F12+1/4 BGJb, containing 10% FBS, L-glutamine, ascorbate 50 µg/ml,
penicillin/streptomycin and ß-glycerophosphate). Proliferation was
assessed by adding BrdU for 30 minutes on the fourth day, before fixing and
processing as outlined above.
Infection with retroviruses was achieved by adding filtered conditioned
medium at the flooding stage. Alkaline phosphatase activity was detected using
Napthol AS MX-PO4 (Sigma) and Red Violet (Sigma), and mineralised
matrix was detected using 2.5% silver nitrate enhanced by carbonate
formaldehyde (von Kossa). The RCAS retroviral construct used in this study,
RCAS-Ihh, has been previously described
(Vortkamp et al., 1996);
RCAS-Gfp was constructed as described by Church et al.
(Church et al., 2002
).
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Results |
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We further characterised the secondary chondrocytes by examining the
expression of Col2, an early chondrocyte matrix constituent;
Ihh, expressed by pre-hypertrophic chondrocytes
(Vortkamp et al., 1996);
Col10, a marker for terminally differentiated chondrocytes
(Devlin et al., 1988
); and
Frzb, expressed by condensing mesenchyme and early chondrocytes
(Ladher et al., 2000
).
Col2 was expressed in a domain coincident with that of Alcian Blue staining (Fig. 2A,B); in this example, both in anterior and posterior SC domains of the tip of the QJ, but not in the germinal region. Col10 expression on an adjacent section (Fig. 2C) revealed that although the domain of Col10 was contained within that of Col2, it had been rapidly upregulated by the secondary chondrocytes. This apparent rapid hypertrophy was borne out by the co-expression of Ihh (Fig. 2E) and Col10 (Fig. 2F), and was consistent with the enlarged appearance of the secondary chondrocytes. Additionally, a negative regulator of chondrocyte differentiation, Pthrp, was undetectable at e11 and at e14 (data not shown). As secondary chondrocytes are believed to arise from the germinal region, we examined the expression of known perichondrial/periosteal markers to see whether changes in the expression of these genes presaged or reflected the formation of SCs. We found that Bmp7 was expressed in the periosteum of the membrane bones (Fig. 2H), including around the SCs. Gdf5 expression was not detected in the germinal region (Fig. 2I), even after SC formation, nor was it expressed in the remaining periosteum, but it could be detected in the quadrate perichondrium. We also looked at further markers of chondrocytes, for example, the pre-chondrogenic marker Frzb (Fig. 2L), which was expressed coincident with Sox9 upregulation (Fig. 2K) and in advance of Alcian Blue staining. For markers of definitive osteoblasts and late stage hypertrophic chondrocytes we also compared the expression of osteocalcin (ocn) and osteopontin (opn) in the quadratojugal. Neither gene was expressed in SCs up to e13 (Fig. 2M,N,O; data not shown). In the membrane bone, opn was expressed by the osteocytes of the trabecular bone (Fig. 2N), whereas ocn was expressed earlier, in the osteoblasts adjacent to the periosteum (Fig. 2O). Opn was upregulated in SCs at e14 (Fig. 2P,Q), coinciding with the erosion of secondary cartilage by chondroclasts. By contrast, ocn expression remained osteoblast specific (Fig. 2R).
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To explore the role of cell division in differentiation, we assessed the expression of Col2 and Col10 in explanted QJ/Q joints labelled with BrdU, on adjacent sections. As at e11 and e13 (see Fig. 2B,C; data not shown), Col2 expression (Fig. 4H) slightly preceded that of Col10 (Fig. 4H), although BrdU labelling within the Col2-expressing area was rare (Fig. 4G). When we compared this to the situation after a 24 hour chase (Fig. 4I, BrdU labelling at 0 hours), we found that the Col2 and Col10 expression domains at the germinal region/SC boundary were now coincident (Fig. 4J). Also, cell doublets could now be observed within the Col10 expression domain (Fig. 4I). This observation was consistent with a cell committing to the chondrocyte lineage, dividing once, and then upregulating Col10 (n=3). The rapid hypertrophy indicated by this result, led us to assess whether this was an intrinsic property of SCs, or was related to the absence of Pthrp expression. We attempted to delay hypertrophy by adding PTH peptide to cultured QJ/Q explants. We found no significant difference between PTH-treated and untreated explants when we compared Col2 with Col10 expression on adjacent sections after 24 hours culture (n=4; data not shown).
Ihh signalling during secondary chondrogenesis
The proliferation rate in the germinal region was almost three times that
of the periosteum in other regions, indicating that the germinal region was
subject to influences unique to this location. In order to assess whether
hedgehog (Hh) signalling from SCs was responsible, we analysed the expression
of Ptc2 in relation to Ihh. As an inducible transcriptional
target, Ptc2 is a sensitive marker for receipt of Hh signal
(Pearse et al., 2001;
Hartmann and Tabin, 2001
). At
e11, although Ihh was readily detectable
(Fig. 2E,
Fig. 5B), Ptc2
expression was at the limit of detection
(Fig. 5C). However, at e12 and
e14 Ptc2 expression was evident within the germinal layer
(Fig. 5F,I), adjacent to the
Ihh domain in the SCs (Fig.
5E,H). Ptc2 was not expressed in the periosteum
surrounding the membrane bone, and we were unable to detect Ptc1
expression at either e11 or e14 (data not shown).
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The rapid shift in cell-type differentiation, dependent upon the presence or absence of mechanical stimulation, indicated that the germinal region was extremely plastic, with the fate decision being a late event in germinal region expansion. To determine whether this was the case, we labelled e11 explanted QJ/Qs at 0 hours followed by a 48 hour chase (n=3). The resultant BrdU-labelling of both chondrocytes and osteoblasts (Fig. 8G) indicated that precursor cells capable of giving rise to osteoblasts, as well as cells committed to giving rise to chondrocytes, had both been proliferating under the influence of mechanical stimulation (Fig. 8H).
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Discussion |
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It is well known that immature chondrocytes of the growth plate gradually
acquire the range of markers outlined above, but start from a population of
precursors expressing Sox9. Misexpression of Cbfa1 in
immature, Sox9-expressing chondrocytes has shown that it is
sufficient to promote hypertrophy and exit from the cell cycle
(Enomoto et al., 2000;
Takeda et al., 2001
). What our
study of SCs shows is that the reverse is also true: mechanical evocation of
Sox9 in ovo, in a population expressing Cbfa1, leads to the
same array of gene targets becoming upregulated. Consequently, we have shown
that the key motivator of chondrogenesis on the one hand (Sox9) and
osteogenesis on the other (Cbfa1) appear to affect the same outcome
(pre-hypertrophic chondrocytes) when co-expressed, irrespective of their
primacy. Consequently, a bifurcating pathway of endochondral ossification and
secondary chondrogenesis can be described in which early lineage determination
from a mesenchymal precursor, arguably governed by Sox9 and Cbfa1 activity,
much later rejoins with the differentiation of the hypertrophic chondrocyte
(Fig. 9).
|
Exit from the cell cycle follows commitment to the chondrogenic
lineage
The transition from perichondrium to periosteum during endochondral
ossification follows exit from the cell cycle and pre-hypertrophy in the
underlying chondrocytes. This process of chondrocyte maturation is
meticulously regulated and deficiency of any of a number of genes that affect
proliferation or cell cycle control causes its perturbation, for example,
Pthrp (Karaplis et al.,
1994), Pthrp-r
(Lanske et al., 1996
),
Ihh (St-Jacques et al.,
1999
), p57 (Zhang et
al., 1997
) and Cyclin D1
(Beier et al., 2001
). When we
consider secondary chondrogenesis, the periosteum (germinal region) is the
source of the chondrocytes as well as the responding tissue. However, the
chondrocytes formed rapidly exit the cell cycle and undergo hypertrophy. Thus,
commitment to the chondrocyte lineage appears not to be accompanied by the
formation of a self-renewing proliferative layer; this is in contrast to the
growth plate of the long bone, where chondrocyte precursors are maintained
independent of movement of the joint. This conclusion is also supported by in
ovo experiments that show that, following paralysis of the embryo,
proliferating secondary chondrocyte-committed cells are not found
(Hall, 1979
).
This achieves one end - rapid hypertrophy - but necessitates continued
mechanical articulation of the joint to re-commit precursors to the
chondrocyte lineage (Murray and Smiles,
1965; Hall, 1967
;
Hall, 1968
;
Hall, 1979
) (this paper). Our
finding that Col10 expression expanded to coincide with the
Col2 domain after 24 hours in culture indicated that commitment,
differentiation and exit from the cell cycle are intimately correlated in SCs.
Thus the initial spur to chondrogenesis - compression and upregulation of
Sox9 (Takahashi et al.,
1998
) - appears to be followed by a single round of division and
hypertrophy.
Ihh drives proliferation of the germinal region
The mouse chondrodysplasias mentioned above result in either delayed or
accelerated hypertrophy. These changes then feed into ossification via
Ihh signalling, resulting in reduced or increased bone formation,
respectively. As in endochondral ossification, accelerated exit from the cell
cycle in SCs is accompanied by Ihh upregulation, which then signals
to the germinal region but not to the chondrocytes. As a result, cycling cells
in the germinal region express Ptc2, but, as they become
chondrocytes, the cells exit the cell cycle, downregulate Ptc2 and
upregulate Ihh. Hence, the cells progressed from Hh-signal receiving
to Hh-signal producing at the germinal region/SC boundary, superficially
resembling an extreme form of the growth plate of the Pthrp-r
knockout mouse (Lanske et al.,
1996).
The threefold enhanced proliferation evident in the germinal region around
the SCs was quenched by blocking Ihh function with the 5E1 antibody
(Ericson et al., 1996;
Wu et al., 2001
). This finding
was consistent with the demonstrated role of Ihh signalling to immature
chondrocytes to drive proliferation (Long
et al., 2001
), but was transposed to the germinal region. Given
the pivotal roles of Ihh in chondrogenesis and osteogenesis of the long bones,
it might also be expected to affect differentiation of the germinal cells.
However, we did not find precocious or delayed expression of the osteogenic
marker osteocalcin in 5E1 treated samples (data not shown). It was
also apparent from the osteocalcin expression data at 24 and 48 hours
that Ihh alone, in the absence of mechanical stimulation, was not sufficient
to cause chondrogenic differentiation in germinal region descendants. This is
consistent with the fact that Ihh is expressed only after SCs have
formed, excluding it from any role in the initiation of SCs themselves. Thus
the principal, and perhaps sole, function of Ihh signalling in secondary
chondrogenesis is to drive proliferation of the germinal region and thereby
increase indirectly recruitment to the chondrogenic fate. In turn, exit from
the cell cycle appears to be a prerequisite for the synthesis of Ihh.
The mitogenic effect of Ihh in micromass culture was not directly
comparable with the role of Ihh in SC genesis owing to the different fates of
the responding cells. However, it was consistent with the conclusion above.
Our in vitro micromass data additionally demonstrate that Ihh promotes
osteogenesis, as measured by nodule formation and deposition of mineralised
matrix. This conclusion is comparable to that from the phenotype of the
Ihh-/- mice, that Ihh is necessary for
osteogenesis in cartilage-replacement bones
(St-Jacques et al., 1999). Our
findings are significant not only for understanding secondary cartilage but
also because they extend the similarities between periostea of membrane bones
and long bones. This implies that it is ontogenetic differences that separate
these periostea, not genetic ones
(St-Jacques et al., 1999
).
Mechano-transduction regulates gene expression and cell fate in the
germinal region
One of the most striking features of the SCs described is that their
formation is dependent upon articulation of the QJ/Q joint. In ovo this is
achieved by muscular contraction that opens and closes the beak, which can be
mimicked in explant by the manual operation of the joint
(Hall, 1968) (this paper).
Whereas evocation of chondrogenesis has certainly been described, we found
that the discreet nature of the germinal region as proliferative centre
allowed us to explore the short-term response to mechanical articulation. The
ability of mechanical articulation to prevent the default osteogenic pathway,
as determined by ocn upregulation, indicates a direct role for
mechanical stimulation in the regulation of gene expression. Ocn is
upregulated by osteoblasts as they exit the cell cycle
(Aubin and Liu, 1996
), and in
the absence of mechanical stimulation the germinal cells follow this route.
Thus, the cells of the germinal region that give rise to secondary
chondrocytes would normally exit the cell cycle at this juncture, but the
acquisition of chondrogenic fate diverts them - a limited form of
bi-potentiality (Fang and Hall,
1996
). The fact that no self-renewing chondrocyte-committed
precursors appear to be established, as evidenced by gene expression and BrdU
labelling (see also Hall,
1979
), accentuates this as a defining feature of secondary
chondrogenesis in the chick.
Conclusion
The unusual differentiation undergone by SCs, and their transient nature,
alludes to a role for SCs that is not structural, but is rather as a growth
and signalling centre; our study demonstrates that a molecular basis for this
function is Ihh production. The clear inference is that the pre-hypertrophic
chondrocyte is of such utility as a source of extracellular signals for bone
morphogenesis that, even when no cartilage scaffold exists, pre-hypertrophic
chondrocytes are evoked. The finding that Ihh stimulates proliferation of the
germinal region highlights the reciprocity between epigenetic and genetic
pathways that characterise the development of the cranial tissues, and that
underpin their evolution. Such a scenario resonates with other phenomena, such
as fracture repair, where a short-lived, Ihh-synthesising soft callus
forms from the periosteum under the influence of movement
(Vortkamp et al., 1998;
Ferguson et al., 1999
).
Conversely, the common characteristics that we have found between endochondral
and secondary chondrogenesis indicate that the subordinate role of bone to
cartilage [and other epigenetic influences
(Herring, 1993
)] is a
skeleton-wide phenomenon. Moreover, although requiring confirmation in
mammals, our findings provide additional mechanistic evidence in support of
Scott's view of cartilage as a pacemaker of cranial bone growth
(Scott, 1954
).
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ACKNOWLEDGMENTS |
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