1 Department of Craniofacial Development and Orthodontics, GKT Dental Institute,
King's College London, Floor 28, Guy's Hospital, London SE1 9RT, UK
2 Department of Craniofacial Development, GKT Dental Institute, King's College
London, Floor 28, Guy's Hospital, London SE1 9RT, UK
* Author for correspondence (e-mail: paul.sharpe{at}kcl.ac.uk)
Accepted 10 March 2004
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SUMMARY |
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Key words: Shh, Gas1, Tooth development, Diastema, Mandibular arch
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Introduction |
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Patched 1 (Ptc1; Ptch1 Mouse Genome Informatics) and smoothened
(Smo) are transmembrane proteins thought to form a receptor complex for
hedgehog ligands (Stone et al.,
1996; Marigo et al.,
1996
). The mechanism of this interaction is poorly understood
(Kalderon, 2000
;
Ingham and McMahon, 2001
;
Nybakken and Perrimon, 2002
);
however, genetic studies indicate that, in the resting state, Ptc1 inhibits
the activity of Smo, and binding of Hedgehog proteins to Ptc1 releases this
inhibition, allowing signal transduction
(Chen and Struhl, 1996
;
Quirk et al., 1997
;
Chen and Struhl, 1998
).
Hedgehog signalling is mediated principally within vertebrate cells by
Gli-family zinc-finger transcription factors
(Ruiz i Altaba, 1999
), of
which in the mouse there are three: Gli1, Gli2 and Gli3
(Hui et al., 1994
). Gli
proteins control cell fate, growth and patterning in vertebrates, having both
activating and inhibitory roles (Grindley
et al., 1997
; Lee et al.,
1997
; Platt et al.,
1997
). More recently, further novel components in the hedgehog
signalling pathway have been isolated. Hedgehog-interacting protein
(Hhip1, also known as Hip1) encodes a membrane glycoprotein
capable of binding all mammalian hedgehog proteins and attenuating the signal
(Chuang and McMahon, 1999
), and
Gas1 (growth arrest-specific gene) encodes a
glycosylphosphatidylinositol-linked membrane glycoprotein demonstrated to have
an antagonistic effect on Shh signalling in the somites
(Lee et al., 2001
).
Teeth in mammals form on the first branchial arch derivatives, the
maxillary and mandibular processes and the frontonasal process. Early tooth
development is characterised by reciprocal interactions between the oral
epithelium and the underlying neural crest-derived ectomesenchyme of the first
branchial arch (Peters and Balling,
1999; Tucker and Sharpe,
1999
; Jernvall and Thesleff,
2000
). In the mouse embryo at around embryonic day (E) 11.5,
individual thickenings in the branchial arch epithelium mark the first
morphological signs of tooth development. These thickenings undergo localised
proliferation to form an epithelial bud that, together with local
condensations of ectomesenchyme, form the tooth germ. Tooth germs progress
through a well-characterised path of differentiation, in which the
ectomesenchyme gives rise to the tooth pulp and dentine-producing
odontoblasts, and the epithelium differentiates into enamel-secreting
ameloblasts.
During the initiation process, expression of Shh is localised to
the epithelial thickenings of future teeth
(Bitgood and McMahon, 1995;
Hardcastle et al., 1998
), and
there is in vitro evidence to suggest that Shh acts as a mitogen, inducing
proliferation as these thickenings form a tooth bud
(Hardcastle et al., 1998
;
Sarkar et al., 2000
;
Cobourne et al., 2001
).
Indeed, inhibition of Shh signalling in mandibular explants from E10.5 results
in a failure of bud formation and an arrest of tooth development
(Sarkar et al., 2000
;
Cobourne et al., 2001
).
Further, conditional knockout of Shh in the developing tooth germ
from E12.5 leads to a reduction in overall size of the developing tooth bud
(Dassule et al., 2000
). Given
this localised and crucial role of Shh signalling during early odontogenesis,
it is clearly important to restrict the sites of activity along the developing
oral axis specifically to the sites of tooth development. This is well
illustrated in the developing mouse, as mice have lost their antemolar
dentition during evolution, and the incisor regions are separated from molar
regions by a diastema or non-tooth-forming edentulous region.
We have used the developing mandible as a model to investigate the relationship between downstream mediators of the Shh pathway and non-transcriptional regulation of hedgehog signalling during the initiation of odontogenesis. We find that following removal of the oral epithelium, the endogenous source of Shh, both Ptc1 and Gli1 are upregulated in the diastema of isolated mandibular mesenchyme. This upregulation was associated with Shh protein detected at a distance from the tooth-forming regions. Conversely, Gas1 was specifically downregulated in isolated diastema mesenchyme in regions corresponding to ectopic Ptc1. Electroporation of Gas1 inhibited ectopic Ptc1 expression in the diastema, suggesting that Gas1 plays a role in limiting the activity of Shh signalling along the early tooth-forming axis of the mandibular arch. Interestingly, neither ectopic Ptc1 expression nor Shh protein was detectable in the diastema of mandibular processes in the presence of epithelium. Moreover, the ability of transplanted diastema epithelium to inhibit Ptc1 in developing tooth germs, and for isolated diastema mesenchyme to express Ptc1, suggests that in the developing mandible, although Shh protein is present within non-odontogenic diastema mesenchyme, signalling activity by this protein is inhibited by the epithelium.
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Materials and methods |
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Transplant experiments
For recombination experiments, mandibular diastema or tongue epithelium was
harvested from ROSA-26 mice (exhibiting ubiquitous expression of
ß-galactosidase) (Zambrowicz et al.,
1997) and transplanted onto developing incisor tooth germs of
intact mandibular explants derived from CD-1 mice. This allows transplant
localisation following staining with X-gal
(Sanes et al., 1986
). For
transplantation of isolated diastema mesenchyme, tissue was taken from a mouse
line exhibiting ubiquitous expression of green fluorescent protein (GFP) under
the control of a ß-actin promoter (GFP-mice)
(Hadjantonakis et al., 1998
)
and transplanted into intact tongues derived from CD-1 mice. This allows
transplant visualisation under fluorescence.
In situ hybridisation
Whole-mount digoxigenin-labelled and double-labelled
(digoxigenin-fluorescein) whole-mount in situ hybridisation was carried out
according to Shamim et al. (Shamim et al.,
1998). Radioactive section in situ hybridisation using
35S-UTP radiolabelled riboprobes was performed as described by
Wilkinson (Wilkinson, 1992
).
Antisense riboprobes were generated from mouse cDNA clones that were gifts
from several laboratories: Shh
(Echelard et al., 1993
);
Ptc1 (Goodrich et al.,
1996
); Hhip1 (Chuang
and McMahon, 1999
); Gli1
(Hui et al., 1994
);
Barx1 (Tissier-Seta et al.,
1995
); Gas1 (Lee and
Fan, 2001
); Ihh
(Echelard et al., 1993
).
Immunohistochemistry
Immunohistochemistry was carried out according to Gritli-Linde et al.
(Gritli-Linde et al., 2001).
Primary antibody (Shh rabbit polyclonal IgG, Ihh rabbit polyclonal IgG; Santa
Cruz) was detected using a biotinylated monoclonal anti-rabbit IgG (Sigma) and
visualised with DAB (Vector Laboratories). Slides were counterstained with
Haematoxylin (Vector Laboratories).
Electroporation
The full-length sequence of Gas1 (Chen-Ming Fan, Carnegie
Institution of Washington) was cloned into the expression vector pcDNA3. The
avian expression vector pcAß-IRES-mGFP
(McLarren et al., 2003)
expresses a myristylated EGFP whose fluorescence can be detected through thick
tissue. pcAß-IRES-mGFP was co-electroporated with pcDNA3-Gas1 to
visualise targeting efficiency. A DNA solution containing 3 µg/µl of
pcDNA3-Gas1, pcAß-IRES-mGFP and Fast Green (Sigma; 1/10,000) was injected
into the diastema mesenchyme using a micropipette. Two tungsten electrodes
(0.1 mm) were inserted into the mesenchyme surrounding the diastema area. DNA
was then transferred into the cells using an Electro-Square-PoratorTM ECM
830 (Genetronics), applying 2-3 sets of 5 pulses: 50 V/50 ms duration, 100 ms
intervals. A control experiment was performed using 3 µg/µl of
pcAß-IRES-mGFP to assess any effect of electroporation or GFP expression
on Ptc1 expression. All explants were cultured for 24 hours before
further processing.
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Results |
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Ectopic expression of Ptc1 is restricted to diastema mesenchyme
In the absence of epithelium, localised upregulation of Ptc1 and
Gli1 was restricted to the proximal regions of the mandibular axis,
in either the molar or diastema-forming regions of mesenchyme. Following
epithelial removal, isolated mesenchymal explants undergo considerable change
from their original shape during a 24 hour culture period, and, for this
reason, direct comparison with normal explants was inconclusive in
definitively establishing the region of ectopic expression. The exact location
of ectopic Ptc1 was therefore identified using molecular markers. The
experiments were repeated and expression of Barx1 was analysed in
conjunction with Ptc1. Barx1 is a homeobox-containing gene known to
be restricted specifically to molar-forming mandibular arch mesenchyme
(Tissier-Seta et al., 1995);
at E11.5 this expression is fixed, even in the absence of epithelium
(Fig. 2A)
(Ferguson et al., 2000
).
Double-labelling of mesenchymal explants with both Ptc1 and
Barx1 confirmed that ectopic Ptc1 expression extended more
distally than the molar-restricted Barx1 domain
(Fig. 2B; arrow). This
suggested that ectopic Ptc1 was present in the diastema, but did not
exclude the possibility that it extended throughout both regions. However,
analysis of adjacent sections using radio-labelled in situ hybridisation
demonstrated that the Ptc1 and Barx1-expressing regions were
distinct along the proximodistal axis (Fig.
2C,D). Together, these findings suggested that Ptc1
upregulation was occurring in isolated diastema mesenchyme after 24 hours of
culture, in a region distinct from the proximal molar-forming mesenchyme.
|
The deposition of Meckel's cartilage in the middle of the first branchial
arch has been shown to be under control of the epithelium. The epithelium
inhibits chondrogenesis; if it is removed, large amorphous masses of cartilage
are found instead of a narrow rod (Kollar
and Mina, 1991). The possibility therefore existed that ectopic
Ihh signalling from such regions might be responsible for the observed
Ptc1 and Gli1 induction seen in isolated mandibular
mesenchyme. The morphology of Meckel's cartilage was investigated at E11.5, in
both the presence and absence of epithelium, using Pro
1(II)-lacZ
transgenic mice (Zhou et al.,
1995
). Although Meckel's cartilage was found to extend further
distally in mandibles cultured for 24 hours in the absence of epithelium, no
ectopic masses of cartilage were detected
(Fig. 2I,J; n=8).
Therefore it was unlikely that the consistent, bilateral and symmetrical
upregulation of Ptc1 and Gli1 observed in the diastema
region was due to Ihh signalling from ectopic cartilage. This was further
confirmed by an absence of Ihh transcription in Meckel's cartilage
during these early stages of mandibular development
(Fig. 2K,L).
Shh protein distribution in the mandibular process
The indirect evidence to suggest a source of Shh being responsible for
inducing Ptc1 and Gli1 transcription in isolated mandibular
diastema mesenchyme led to an attempt at localising the distribution of Shh
protein within the mandibular axis during early tooth development. At E11.5
during initiation, Shh was detected throughout the epithelial thickenings of
the future teeth but was absent from the underlying mesenchyme. Interestingly,
this protein generally had a wider distribution in the epithelium than that of
Shh transcripts (Fig.
3A,B). During the early bud stage, Shh was detectable in an
expanded region of bud epithelium and also in mesenchymal cells situated
around the bud tip; however, Shh transcription was again more
localised, detected only in a small group of epithelial cells situated at the
tip of the tooth bud (Fig.
3C,D). By E13.5 at the late bud stage, Shh protein distribution
had increased, being strongly detected in the outer regions of bud epithelium
and in the surrounding mesenchyme, whereas Shh expression remained
localised to the epithelial cells situated at the tip of the tooth bud
(Fig. 3E,F). By E14.5 at the
early cap stage, Shh protein was present within the enamel knot epithelium,
internal enamel epithelium and mesenchyme of the dental papilla
(Fig. 3G). At this stage,
Shh transcripts remained highly localised to the epithelial cells of
the enamel knot (Fig. 3H).
Therefore, during the very earliest stages of odontogenesis, Shh protein is
able to move beyond Shh-expressing cells within the odontogenic
epithelium to cells within the mesenchymal component that express downstream
mediators of Shh signalling. Thus, early tooth germs appear to be a viable
source of Shh protein, detectable within adjacent mesenchyme.
|
Inhibitory properties of diastema epithelium
The observation that Shh protein was only active and detectable in the
diastema of isolated mandibular mesenchyme suggested that Shh activity was
being inhibited in the presence of epithelium. If this was the case, then
isolated diastema epithelium might be expected to downregulate endogenous
Shh-induced Ptc1 expression if transplanted onto presumptive tooth
germs. Isolating diastema epithelium from E11.5 mandibular processes and
transplanting it unilaterally over early incisor tooth buds tested this.
Control transplants were performed using tongue epithelium, and, in both
cases, donor epithelium was taken from ROSA-26 mice to demonstrate
localisation of the transplants (Fig.
4A,B). After 24 hours, Ptc1 expression was examined and
found to be normal in the incisor regions exposed to tongue epithelium
(Fig. 5A-C; n=5). By
contrast, Ptc1 was downregulated in incisor regions exposed
unilaterally to diastema epithelium (Fig.
5D-F; n=4). Diastema epithelium thus had the ability to
inhibit endogenous Shh protein activity within these tooth germs. At E11.5,
expression of Shh remained normal in isolated incisor regions exposed
to tongue epithelium (Fig. 5G;
arrowhead, compare with normal expression arrowed; n=5). However,
this expression was lost following the transplantation of diastema epithelium
(Fig. 5H; arrowhead, compare
with normal expression arrowed; n=5). This suggested that Shh
signalling in the developing tooth germ might be required to maintain
Shh transcription during initiation; a finding confirmed by the
ability of 5E1-soaked beads to inhibit Shh transcription at E11.5
(Fig. 5I; n=7). This
provided evidence of an autoregulatory loop, where Shh signalling was required
to maintain Shh transcription in the dental epithelium during the
initiation of tooth development.
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Therefore, diastema epithelium was capable of blocking the activity, either directly or indirectly, of Shh protein normally present, but undetectable, within the underlying mesenchyme; in the absence of epithelium, this protein was both detectable and capable of inducing Ptc1. Furthermore, it was active pre-existing Shh protein that was able to achieve this because no transcription of Shh was ever detected in regions of diastema mesenchyme.
Regulation of Shh activity by Gas1
It is clear that the epithelium plays an important role in restricting the
activity of Shh along the early mandibular axis. Recently, Gas1 has been
demonstrated to have an antagonistic effect on Shh signalling in the somites
(Lee et al., 2001). We
therefore analysed the expression of Gas1 in the murine first
branchial arch. At E11.5, Gas1 was noticeably absent from epithelium
and mesenchyme of the developing incisor and molar regions, but strongly
expressed in the non-odontogenic regions of mandibular arch mesenchyme,
including the diastema (Fig.
6A,B; arrowed). This expression pattern was consistent with Gas1
acting as an additional inhibitor of Shh signalling in non-odontogenic
mesenchyme. Interestingly, further analysis of E11.5 isolated mandibular
mesenchyme demonstrated a localised and progressive downregulation of
Gas1 in the diastema region between the developing incisor and molar
regions between 18-24 hours of culture. However, Gas1 expression was
maintained in the peripheral non-odontogenic mesenchyme throughout this time
course, indicating differential regulation of Gas1 transcription by
the epithelium in odontogenic and non-odontogenic regions of the mandibular
arch (Fig. 6C-F). Section in
situ hybridisation confirmed that Gas1 downregulation corresponded to
the diastema regions of ectopic Ptc1 expression. The progressive
Ptc1 upregulation occurred between 18-24 hours, in contrast,
Gas1 was progressively downregulated in this region over the same
time-course (Fig. 6G-L).
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Discussion |
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In this study, the expression domains of downstream Shh targets were
investigated in the mandibular arch following removal of the source of
Shh transcription, namely the oral epithelium. Removal of the
epithelium from E11.5 mandibular explants resulted in ectopic expression of
Ptc1 and Gli1 in isolated regions of mesenchyme after 24
hours. By contrast, Hhip1 was downregulated over the same time
period. Subtle differences in regulation of Shh target gene expression have
previously been reported in the first branchial arch derivatives, where
Msx1 is required for Shh to induce Ptc1, but not
Gli1 in dental mesenchyme (Zhang
et al., 1999). Shh also interacts with Prx genes
(Ten Berge et al., 2001
) and
Tbx1 (Garg et al.,
2001
) in the first arch. Together, these data imply that
interactions of genes both upstream and downstream of Shh are controlled and
coordinated by several inductive signalling pathways.
Definitive identification of the regions of ectopic Ptc1 and
Gli1 expression in isolated mandibular mesenchyme was not
straightforward; the mandibular process undergoes a considerable shape change
during culture without the integrity of an intact epithelium, isolated
mesenchyme lacks histologically reproducible landmarks and there are few
molecular markers for specific regions of mesenchyme devoid of epithelium. The
use of double-labelled whole-mount and section in situ hybridisation with a
specific molar-marker, Barx1, identified these bilaterally
symmetrical ectopic regions as distal to the presumptive molar regions,
corresponding to the diastema. Barx1 is expressed in the molar and
proximal-most regions of mandibular arch mesenchyme, and these expression
domains are both established and fixed by E11.5
(Ferguson et al., 2000). The
regions of ectopic expression were clearly more distal to the
Barx1-expressing zone; however, the possibility of any degree of
overlap existing between these two regions could not be definitively
excluded.
Evidence existed to suggest that Shh signalling was responsible for the
ectopic gene expression seen in isolated diastema. Firstly, Ptc1 and
Gli1 are only known to be targets of Shh in the first branchial arch
(Hardcastle et al., 1998;
Dassule and McMahon, 1998
).
Secondly, upregulation of these genes was blocked in the presence of 5E1
antibody, a known inhibitor of Shh
(Ericson et al., 1996
). As
Shh transcripts were never detected in isolated mandibular arch
mesenchyme at any time point following the loss of epithelium, this implied
that there must be an active source of Shh within the mesenchyme, capable of
inducing both Ptc1 and Gli1. Serial sections of E11.5
mandibular explant cultures, both with and without epithelium, were
investigated using immunohistochemistry to detect the presence of any ectopic
Shh protein. No Shh was detected in the diastema of the mandibular process in
the presence of epithelium; however, examination of isolated mandibular
mesenchyme revealed localised Shh accumulation in the diastema regions after
approximately 18 hours of culture. These regions increased in immunoreactivity
over the next 6 hours.
What is the source of the Shh detected in diastema mesenchyme? The absence
of Shh transcription suggests this protein must originate from other
known regions of Shh production at these stages. In the mandibular arch, the
most obvious sources would be the developing molar and incisor tooth germs
that border the diastema. In support of this, the accumulation of Shh protein
in both the epithelium and condensing ectomesenchyme of early tooth buds, at a
distance from regions of transcription, suggested that the tooth germs could
act as a source of Shh along the mandibular axis. However, both Ptc1
and Hhip1, two members of the pathway that are known to bind Shh,
show high-level transcription in the odontogenic mesenchyme surrounding the
tooth germs (Hardcastle et al.,
1998; Cobourne and Sharpe,
2002
). This point not withstanding, Shh has been demonstrated to
diffuse further than areas where Ptc1 is expressed at high levels
(Gritli-Linde et al., 2001
),
and it would appear that in the mandibular process Shh is able to move beyond
these fields of sequestration into the diastema. Interestingly, rudimentary
tooth primordia present in the vole maxillary diastema have been demonstrated
to express Shh prior to their apoptotic removal
(Keränen et al., 1998
),
and transient Shh expression has also been shown at E11.5 in mouse
diastema epithelium (Dassule and McMahon,
1998
). In this study, no specific expression of Shh was
detected in the diastema epithelium of the mouse mandible. However, if
Shh transcription does occur in the diastema epithelium at earlier
stages, albeit transiently and at low levels, theoretically this would provide
an additional potential source of the Shh protein demonstrated in the
underlying mesenchyme of the diastema.
The failure to detect Shh protein in diastema mesenchyme in the presence of
epithelium implies that this protein is either present, but being masked (and
therefore non-functional), in whole explants, or that Shh can rapidly
accumulate in these regions following epithelial removal. Certainly, loss of
epithelium results in downregulation of Ptc1 and Hhip1 in
odontogenic mesenchyme surrounding early tooth germs and, as the products of
these genes normally sequester Shh, this downregulation might facilitate
movement of protein into the diastema. However, diastema mesenchyme
transplanted into a tongue host immediately following epithelial removal was
able to express Ptc1 but not Shh, suggesting that Shh was
normally present, but undetectable in the presence of epithelium. It should be
noted that tongue epithelium does contain small sources of Shh within
the gustatory papillae from E12.5 (Hall et
al., 1999); however, this expression is highly localised and would
not be responsible for the high-level Ptc1 expression seen in
experimental transplants. Together with the detection of ectopic Ptc1
and Gli1 in the diastema of mandibular arch mesenchyme, and the
association of this expression with Shh protein, these data indicate the
presence of a Shh-inhibitory mechanism in diastema mesenchyme that is
responsible for repressing Shh activity. This inhibition would be reliant upon
the epithelium, confirmed by the ability of diastema epithelium to inhibit the
activity of Shh when transplanted over whole incisor tooth germs. However, the
transplantation of diastema epithelium onto early incisors at E11.5 was also
demonstrated to inhibit Shh transcription, which could explain the
downregulation of Ptc1 expression seen in diastema transplants
carried out on early bud stage incisor tooth germs. But this capability of
diastema epithelium to downregulate Shh transcription might not
entirely account for the dramatic downregulation of Ptc1 seen in
these transplanted tooth germs. Shh protein was readily detectable in the
epithelium and mesenchyme at the tip of these developing teeth, and this
protein might be expected to continue inducing Ptc1 expression in the
absence of Shh transcription over the period of culture.
Ptc1 expression was dramatically downregulated in the
diastema-transplanted tooth germs, suggesting that the diastema epithelium is
able to mask the activity of pre-existing Shh protein.
A key question is the mechanism of action of any putative Shh inhibitor in
the diastema of the mandibular arch, but clearly this process requires an
intact epithelium. Several members of the Bmp family of signalling peptides
are expressed in the diastema epithelium during these early stages of tooth
development (Åberg et al.,
1997), and Bmp4 is involved in negatively regulating Shh
in the mouse tooth germ (Zhang et al.,
2000
). However, recombinant Bmp4 protein was unable to repress
Ptc1 transcription in isolated diastema mesenchyme (data not shown).
More recently Gas1 has been demonstrated to have an antagonistic effect on Shh
signalling in the somites: overexpression of Gas1 in pre-somitic
mesoderm inhibits the Shh-induced markers Ptc1 and Pax1
(Lee et al., 2001
). Gas1 forms
a unique and distinct physical complex with the active signalling form of Shh
through binding contributions made by the carbohydrate moiety and polypeptide
chain (Lee et al., 2001
).
Gas1 is expressed in a variety of embryonic tissues in a
complementary, but partially overlapping, domain to Ptc1 and it has
been suggested that it may act as an additional inhibitor of Shh in regions
where Ptc1 is not upregulated
(Lee and Fan, 2001
). However,
the exact mechanism whereby Gas1 affects Shh function is not fully
understood. It has been postulated that it might act via a direct physical
interaction with the receptor complex, through sequestration of the signalling
protein itself, or even as a new receptor for Shh
(Mullor and Ruiz i Altaba,
2002
).
Gas1 was strongly expressed in the mandibular process during the
early stages of tooth development, in the peripheral regions of odontogenic
mesenchyme and throughout the diastema. Importantly, however, Gas1
was downregulated in the Ptc1-expressing diastema regions of isolated
mandibular mesenchyme. This downregulation began after around 18 hours of
culture and coincided with Ptc1 upregulation. Interestingly,
Gas1 expression remained in the incisor and molar regions devoid of
epithelium throughout the 24-hour period of culture. Clearly, the epithelial
dependence of Gas1 differs in the tooth-forming and non-tooth-forming
(diastema) regions of the mandibular axis. The overexpression of Gas1
into isolated diastema led to downregulation of ectopic Ptc1,
suggesting a possible mechanism, consistent with the expression domains of
Gas1, for the observed restriction of Shh activity in isolated
mandibular mesenchyme (Fig. 7).
This model proposes that Shh protein in the diastema is masked because it is
in a complex with Gas1 or another unidentified protein. Clearly, an area of
further investigation would be the use of immunoprecipitation experiments to
demonstrate a physical relationship between Shh and Gas1 in whole diastema
regions. What is also not understood is how loss of the overlying epithelium
only results in downregulation of Gas1 in the diastema and not the
tooth-forming regions. These observations raise the possibility that
epithelial induction of Gas1 is compartmentalised along the oral
axis. In the somite, Gas1 expression is known to be induced by
several members of the Wnt family of signalling molecules
(Lee et al., 2001), and,
although several Wnt genes do demonstrate regionally restricted expression in
first arch epithelium, none have currently been identified whose expression is
restricted to the diastema (Sarkar and
Sharpe, 1999
).
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ACKNOWLEDGMENTS |
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