1 Department of Oral Biochemistry, Sahlgrenska Academy at Göteborg
University, SE-405 30 Göteborg, Sweden
2 Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, 20
Shattuck Street, Boston, MA 02115, USA
3 Curis Inc., 45 Moulton Street, Cambridge, MA 02138, USA
4 Department of Molecular and Cellular Biology, Harvard University, 16 Divinity
Avenue, Cambridge, MA 02135, USA
* Authors for correspondence (e-mail: amcmahon{at}mcb.harvard.edu amel{at}odontologi.gu.se)
Accepted 12 August 2002
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SUMMARY |
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Key words: Sonic hedgehog, Smoothened, Patched2, mRNA subcellular localization, CyclinD1, Cell polarity, Cell size, ZO-1, Molar fusion, Tooth, Mouse
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INTRODUCTION |
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During the cytodifferentiation stage, the terminal differentiation of
odontoblasts is accomplished by their withdrawal from the cell cycle,
elongation, polarization and secretion of a predentin matrix. This, in turn,
triggers terminal differentiation of ameloblasts
(Slavkin and Bringas, 1976;
Frank and Nalbandian, 1989
).
Differentiation of the ameloblast into a highly-polarized complex secretory
cell involves considerable growth, elongation of the cytoplasm, a change in
nuclear polarity, a sequential development and change in polarity of
organelles, and the appearance of a complex cytoskeleton
(Slavkin, 1974
). During this
stage, there are other progressive changes within the enamel organ. Cells from
the SI that are adjacent to polarizing post-mitotic ameloblasts become
cuboidal in shape, except in the future enamel-free areas in rodent molars
(Cohn, 1957
;
Gaunt, 1956
;
Hay, 1961
). In addition, the
SR is invaded by blood vessels and fibroblasts emanating from the dental sac
(Lefkowitz et al., 1953
;
Hay, 1961
).
The rodent incisor is unique in its tissue organization and consists of
stem cells, differentiating cells and mature cells organized in defined
regions along its anteroposterior and labial-lingual axes. The rodent incisor
is asymmetrical, as the labial or amelogenic IDE gives rise to ameloblasts and
enamel, whereas the IDE on the lingual side does not produce enamel. The
posterior-most aspect of the incisor has been postulated to contain stem cells
which give rise to the different dental cell populations
(Smith and Warshawsky, 1975;
Harada et al., 1999
). A
posteroanterior gradient of cytodifferentiation is thus present in the rodent
incisor throughout life, with the less differentiated cells located
posteriorly and the most mature cells anteriorly. Odontoblasts differentiate
all along the epitheliomesenchymal interface of the incisor
(Cohn, 1957
; Warshavsky,
1968).
Like many organs, morphogenesis and cytodifferentiation of the tooth is
governed by sequential and reciprocal epithelialmesenchymal interactions
mediated by several soluble bioactive proteins
(Jernvall and Thesleff, 2000;
Thesleff et al., 2001
;
Thesleff and Mikkola, 2002
).
In addition, cell-matrix interactions and cell-cell junctional complexes and
cytoskeletal components have been implicated in the regulation of
histomorphogenesis and proliferation
(Thesleff et al., 1981
;
Lesot et al., 1982
;
Fausser et al., 1998
).
Sonic hedgehog (Shh), a member of the vertebrate Hedgehog
(Hh) family, encodes a secreted signaling peptide. Hedgehog signals are
received within a target tissue by the general Hedgehog receptor Patched 1
(Ptc 1 in mammals; Ptch Mouse Genome Informatics) [for review
of pathway see Ingham and McMahon (Ingham
and McMahon, 2001)]. Transduction of the signal within a
responding cell absolutely requires the activity of a second, multi-pass,
membrane protein Smoothened (Smo). Smoothened activity leads to a conserved
transcriptional response: up-regulation of Ptc1 and Gli1 in
the target tissue. Gli1 encodes a member of the Ci/Gli family of
transcriptional effectors of Hedgehog signaling.
The Hh signaling pathway is an evolutionarily well-conserved mechanism
involved in a plethora of biological processes (for reviews, see
Ingham and McMahon, 2001;
McMahon et al., 2002
).
Shh is expressed exclusively in the epithelial component of the
murine tooth from the dental lamina stage until cytodifferentiation
(Bitgood and McMahon, 1995
;
Vaahtokari et al., 1996
;
Hardcastle et al., 1998
;
Dassule et al., 2000
;
Gritli-Linde et al., 2001
). At
the cap stage, Shh is confined to the primary enamel knot
(Vaahtokari et al., 1996
;
Hardcastle et al., 1998
).
Expression spreads thereafter to the rest of the IDE laterally, the stratum
intermedium (Dassule et al.,
2000
; Gritli-Linde et al.,
2001
) and the stellate reticulum
(Gritli-Linde et al., 2001
).
At the cap stage, general transcriptional targets and effectors of Shh
signaling, including Ptc1, Gli1 and Smoothened
(Smo) are, however, expressed in both dental epithelium and
mesenchyme, but are excluded from the enamel knot
(Hardcastle et al., 1998
). In
contrast, Ptc2, while appearing to bind all mammalian Hedgehog proteins
similarly to the related Hedgehog receptor Ptc1
(Carpenter et al., 1998
), is
expressed in the enamel knot and IDE at the cap and early bell stages,
respectively (Motoyama et al.,
1998
).
We have shown previously that Shh protein produced by the enamel knot and
IDE moves many cell diameters to reach the rest of the dental epithelium and
the dental papilla, indicating that Shh has a long-range activity, consistent
with the broad expression of Shh target genes
(Gritli-Linde et al., 2001).
Together, the above observations suggest that Shh signaling may be operative
intra-epithelially as well as in mediating epithelial-mesenchymal interactions
during tooth development. Finally, we have shown that genetic removal of Shh
activity from the tooth leads to alterations in growth and morphogenesis and
results in tissue disorganization, affecting both the dental epithelium and
mesenchyme derivatives (Dassule et al.,
2000
). In this study, it was not possible to determine clearly
whether the alterations in the dental mesenchyme and its derivatives were
solely generated by lack of Shh signaling by the dental epithelium, or whether
they were secondary to the lack of proper signaling via other
bioactive molecules in the abnormal dental epithelium. Conversely,
this approach left unanswered the question of whether abnormal development of
the epithelial enamel organ in Shh mutant teeth was a result of a
loss of intra-epithelial Shh signaling, or a secondary consequence of altered
signaling by the underlying dysplastic dental mesenchyme. In order to
distinguish between these alternatives, and further define the roles of Shh in
regulating morphogenesis of the tooth, we abrogated Shh signal transduction by
genetically removing the activity of Smo from the dental epithelium and its
derivatives while maintaining Shh responsiveness in the dental mesenchyme.
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MATERIALS AND METHODS |
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Electron microscopy and histology
The tips of the mandibles containing the lower incisors from control and
mutant pups at 1 day post-partum (1 dpp) were processed for transmission
electron microscopy (TEM) as described previously
(Sun et al., 1998). Sections
for histology were prepared according to routine procedures and stained with
Hematoxylin and Eosin.
In situ hybridization
Sections from embryos and 1 dpp pups were prepared for in situ
hybridization with 35S-UTP-labeled riboprobes essentially as
described previously (Wilkinson et al.,
1987). The following probes were used: Bmp2, Bmp4 and
Bmp7 (Åberg et al.,
1997
); PDGFR
(Boström et al., 1996
);
amelin (Cerny et al.,
1996
; Fong et al.,
1998
); Shh, Ptc2, Gli1, Gli2, Gli3, Dentin Sialoprotein (DSP),
Dentin Matrix Protein 1 (DMP1) and Msx2
(Dassule et al., 2000
);
cyclin D1 (Long et al.,
2001
); Dlx3 and Dlx7
(Zhao et al., 2000
);
Ptc1 (Gritli-Linde et al.,
2001
). A 1 kb Bmp5 pro-region fragment was also used to
generate an antisense RNA probe.
Immunohistochemistry and von Kossa staining
Tissues were fixed in either 4% paraformaldehyde in 0.1 M phosphate buffer,
pH 7.4, or in Sainte Marie's solution, embedded in paraffin and processed for
immunohistochemistry as described previously
(Gritli-Linde et al., 2001).
The following antibodies were used: rabbit anti-amelin
(Fong et al., 1998
); rabbit
anti-phospho-histone H3 (Ser 10) (Cell Signaling Technology); Ab80 anti-SHH
(Bumcrot et al., 1995
);
Marti et al., 1995
); rabbit
anti-calbindin-D28K (Chemicon); rat anti-E-cadherin and rabbit anti-ZO1 (Zymed
Laboratories); rabbit anti-collagen type IV (ICN Pharmaceuticals); mouse
monoclonal anti-ß-tubulin clone 5H1, IgM fraction (BD Pharmingen).
For staining of mineralized extracellular matrix, we used a modified method
based on the von Kossa technique (Stevens
et al., 1990).
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RESULTS |
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To create a dental epithelium devoid of Smo activity, we crossed K14-Cre; Smo+/n males to Smoc/c females. As expected, approximatively 25% of newborns were the experimental genotype (K14-Cre; Smoc/Smon), hereafter referred to as `Smo mutants' for simplicity. All mutant pups were of a normal size and displayed normal external features but died within one day of birth for unknown reasons. Throughout, we compare development of teeth from K14-Cre;Smoc/Smon to those of Smo heterozygotes (Smoc/Smon), hereafter referred to as `controls'. Smoc/Smon embryos and pups were phenotypically similar to wild-type animals.
Smo ablation in the dental epithelium generates cytological
alterations within the enamel organ and disrupts normal morphogenesis of the
tooth
In order to determine the outcome of Smo ablation from the dental
epithelium, we analysed histological sections from maxillae and mandibles at 1
day post partum (dpp). As in wild-type pups, incisors and molars developed in
both jaws of Smo mutants. Ablation of Shh activity within the tooth
generates abnormally small and misshapen teeth
(Dassule et al., 2000). In
contrast, general growth of molars in Smo mutants was essentially
similar to that of control littermates
(Fig. 1A-D). Primary and
secondary enamel knots developed normally in Smo mutant molars and
expressed appropriate specific molecular markers, such as Bmp2, Bmp4, Shh,
Ptc2 and Msx2 (see below and data not shown). Despite their
normal growth, Smo mutant molars exhibited several morphological
abberrations. Parasagittal sections at 1 dpp revealed that the first and
second molars, in both the maxilla and mandible, were abnormally fused,
forming a single gigantic anlage (Fig.
1A,B). The molars also fuse in Shh mutants; however, the
single angle in those mice were much smaller than those of Smo mutant
mice (data not shown). On parasagittal and frontal sections, the `first
molars' of Smo mutant pups displayed shallow, broad, underdeveloped
and misshapen cusps as compared to controls
(Fig. 1A-D). As in Shh
mutants (Dassule et al.,
2000
), Smo mutant molars developed close to the oral
surface, reflecting the virtual absence of a dental cord
(Fig. 1C,D). However, in
contrast to Shh mutants (Dassule
et al., 2000
), at all developmental stages the mesenchyme of the
Smo mutant molars appeared to have normal cellularity, temporospatial
patterns of odontoblast terminal differentiation and secretion of a predentin
matrix (Fig. 1C,D).
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The principal cytological differences were observed in the dental epithelium derivatives. A disorganization within the EEO was evident at the late bell stage in the principal cusps of the `first molars'. In the less-developed cusps of the `first and second molars', the EEO cytological organization was normal, similar to that of controls (Fig. 1A,B). At the tip of the principal cusps of late bell stage first molars from control 1 dpp pups, elongated polarizing, post-mitotic ameloblasts lay adjacent to a predentin matrix. The ameloblast were overlaid by the SI, which had assumed a cuboidal shape. The ODE developed into a discontinuous layer, and the coronal aspect of the SR displayed the normal metaplastic changes secondary to the initiation of its invasion by early vascular loops and fibroblasts that emanate from the dental sac, through gaps in the ODE (Fig. 1C). In the most advanced cusps of the first molar region of Smo mutant, ameloblasts were abnormally short and were overlaid by a scarce, squamous SI. The SR was also hypocellular and did not display the changes characteristic of the process of metaplasia. The absence of early vascular loops in the coronal aspect of the SR was verified by staining of blood vessel basement membrane with an anti-collagen type IV antibody (data not shown). Finally, the mutant ODE formed a continuous layer without the gaps observed in controls (Fig. 1D).
Similar cytological changes were observed, specifically on the labial side of the epithelium of Smo mutant incisors. They were, however, more dramatic, as the incisor is developmentally more advanced than the molar. At 1 dpp incisors from control pups contained, on their labial side, enamel matrix secreted by highly polarized secretory ameloblasts overlaid by cuboidal SI cells (Fig. 1E). At this stage, the ameloblasts of mutant incisors had failed to undergo polarized growth and formed a non-cohesive layer of non-polarized cuboidal cells with centrally located nuclei; the enamel matrix was absent (Fig. 1F). The SI overlying ameloblasts was sparse and exhibited a squamous morphology (Fig. 1F). In contrast to molars, the mutant incisors had a smaller diameter than the controls, which was first evident at the differentiation stage. This may reflect the differences between development of molars and incisors in rodents. In addition, the Smo mutant incisors exhibited abnormal foldings of the lateral and medial aspects of the IDE at the posterior and middle segments of the tooth, which at certain levels invaginated within the tooth (data not shown). These may also account for the slightly smaller size of the incisor (data not shown). In control incisors, during enamel secretion, a papillary layer consisting of merged SR and ODE was evident, but this structure was absent in the mutant incisors (data not shown).
In order to characterize the structural cytological alterations of Smo mutant ameloblasts and SI at the subcellular level, we examined and compared their phenotype to that of controls by transmission electron microscopy (TEM) on ultrathin sections taken from incisors at their anterior segment. In control incisors, young secretory ameloblasts were highly columnar, elongated and polarized with oval-shaped nuclei elongated along the apical-basal axis (Fig. 1G). In Smo mutant incisors, the cuboidal ameloblasts were only 15% of the apical-basal height of the secretory ameloblasts of control incisors (excluding the Tomes' process) and contained centrally located round nuclei (Fig. 1H). Furthermore, several organelles, including mitochondria, RER and Golgi were both sparse and evenly distributed in the cytoplasm, whereas in controls these organelles were abundant and showed a polarized distribution within the cell. Tomes' processes and the terminal webs had not developed in mutant ameloblasts (Fig. 1H).
Shh regulates cell-cell interactions within the EEO and is essential
for development of the cytoskeleton in ameloblasts
Junctional complexes and the cytoskeleton are important in maintaining
epithelial cell polarity and cell-cell interactions (Farquar and Palade, 1963;
Gundersen and Cook, 1999). To
assess whether defects in the EEO in Smo mutant teeth were secondary
to alterations in the development of these structures, we analyzed by
immunohistochemistry the distribution of some markers of these cellular
components.
First, we studied the distribution of Zonula Occludens-1 (ZO-1), a member
of the membrane-associated guanylate kinase homologues (MAGUKs) and a
scaffolding protein associated with tight junctions and other cell-cell
contact sites (Tsukita et al.,
1999; Vasioukhin and Fuchs,
2001
). In control embryos at the early bell stage, ZO-1 staining
was abundant in the ODE and less so in the IDE (data not shown). By the late
bell stage, in control molars, ZO-1 protein gradually increased at the
apicolateral and basolateral domains of polarizing ameloblasts adjacent to the
first layer of predentin. At a similar stage, the ameloblasts of mutants
showed barely detectable levels of ZO-1
(Fig. 2A-D). The ODE of control
molars exhibited reduced ZO-1 immunostaining and formed a discontinuous layer
interrupted by gaps penetrated by connective tissue invading the coronal part
of the SR (Fig. 2C). In
contrast, the ODE of Smo mutants remained as a continuous layer of
cells exhibiting strong ZO-1 staining (Fig.
2D). At more advanced developmental stages, as demonstrated in
incisors, Smo mutant ameloblasts totally lacked ZO-1 staining,
whereas the ODE and SI displayed a strong cytoplasmic accumulation of the
protein, in striking contrast to control incisors
(Fig. 2E-H).
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E-cadherin, the epithelial prototype of the transmembrane core of adherens junctions, which is associated with homotypic cell-cell adhesion, also showed a dynamic distribution in polarizing ameloblasts and ODE of control molars that was similar to that of ZO-1 (Fig. 2I,K). In Smo mutant molars, some ameloblasts facing predentin had an apicolateral accumulation of E-cadherin similar to that of control ameloblasts (Fig. 2J,L), but in the less-developed cusps, immunostaining was much weaker in the Smo mutant preameloblasts than controls (Fig. 2I,J). Similarly, E-cadherin accumulated at the apicolateral and basolateral membrane domains of polarizing ameloblasts but was weak and disorganized in mutants (Fig. 2M,N). At a more anterior level, E-cadherin staining became strong in cells of the papillary layer (Fig. 2O). In Smo mutant incisors, the shrunken ameloblasts showed a weak cytoplasmic staining and the papillary layer was unrecognizable (Fig. 2P).
The polarization of both the ameloblast and odontoblast populations is characterized by the accumulation of microtubules. In control teeth, whereas ß-tubulin was present at high levels in functional odontoblasts, in polarizing ameloblasts (Fig. 2Q) and in secretory ameloblasts (data not shown), no such accumulation of ß-tubulin protein was detected in Smo mutants (Fig. 2R). In contrast, odontoblasts in the mutants displayed normal levels of ß-tubulin staining.
Thus, Shh signaling is also necessary for normal development of both the cytoskeletal organization and cell-cell junctional complexes, which are likely to play a role in establishing and maintaining ameloblast polarity and function.
Interestingly, analysis of the expression of Ptc2 and Gli1 transcripts indicated that their subcellular distribution was polarized in the developing ameloblasts. Ptc2 and Gli1 transcripts were enriched at the basal and perinuclear compartments of polarizing (see Fig. 5K below), presecretory and secretory ameloblasts closest to the Shh-expressing SI (Fig. 3A-C and see below). In contrast, transcripts of Dlx7, Bmp7, Bmp5 (data not shown), Gli2 (see below), Msx2, Ptc1 and Dlx3 (Fig. 3D-F) were either distributed uniformly throughout the ameloblast cytoplasm or enriched at its apical pole. Thus a polarized response to the SI, mediated by Shh signaling could play a role in the Shh-dependent ameloblast polarization that is deficient in Smo mutants.
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Shh responsiveness is abrogated in the epithelial enamel organ and is
conserved in the dental mesenchyme of Smo mutant teeth
In order to gain insight into the molecular basis of the cytological
alterations within the epithelial enamel organ following removal of Smo
activity, we first analysed in detail the expression of Shh and its
targets and effectors at different developmental stages of tooth development.
Shh expression and responsiveness in control and mutant molars at the
cap stage are represented in the schematics
(Fig. 4A,B). At the cap stage,
Shh was detected in the primary enamel knot in both controls and
Smo mutants (Fig.
4C,D). Ptc1 and Gli1, primary targets of Shh
signaling, were expressed in both the EEO (excluding the enamel knot) and
mesenchyme in control teeth (Fig.
4E,G). In contrast, in Smo mutants, the EEO exhibited a
dramatic loss of Ptc1 and Gli1 expressions, whereas the
mesenchyme maintained normal expression of both of these genes
(Fig. 4F,H). Expression of
Gli2 and Gli3 (data not shown), Ptc2 in the enamel
knot and Hip1 (Fig.
4I-L) was similar to controls. These data indicate that, as
expected, Shh responsiveness was absent from the EEO but maintained in the
dental mesenchyme at the cap stage.
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In late bell stage control molars, Shh was strongly expressed in the IDE, SR, SI, preameloblasts and differentiating ameloblasts (Fig. 5A). However, in mutant molars, preameloblasts adjacent to either polarized odontoblasts or to predentin, SI and SR showed barely detectable levels of Shh (arrow Fig. 5B). At this developmental stage, Ptc1 and Gli1 were expressed in both the EEO and mesenchyme, whereas Ptc2 expression was confined to the EEO in control molars (Fig. 5C,E,I). In Smo mutants, Ptc1, Gli1 and Ptc2 expressions were severely decreased in the EEO, whereas the mesenchyme showed normal Shh responsiveness (Fig. 5D,F,J). In Smo mutant molars, the expression of Hip1 in the mesenchyme was similar to that of controls (Fig. 5G,H).
Immunohistochemistry for Shh protein showed staining in the EEO and mesenchyme as well as in the basement membrane and predentin matrix in molars from control pups (Fig. 5K). The mutant molars (Fig. 5L) exhibited normal Shh immunostaining in the dental mesenchyme and SR; however, immunostaining was dramatically reduced in preameloblasts adjacent either to polarized odontoblasts or to a thin layer of predentin, as well as in the SI overlying these cells.
As in molars, alterations of Shh expression and responsiveness were also found in the mutant incisors. In the middle segment of control incisors at 1 dpp, Shh was strongly expressed in the IDE, SI, preameloblasts and differentiating ameloblasts on the labial side of the tooth (Fig. 5M). In contrast, in Smo mutants, Shh expression was severely decreased in preameloblasts adjacent either to polarized odontoblasts or to predentin matrix (Fig. 5N). The SI continued to express Shh for only a short time before expression was decreased in this cell layer too (Fig. 5N). At this stage, Ptc1 (data not shown) and Gli1 (Fig. 5O) were expressed in both the mesenchyme and labial epithelium of control incisors. In contrast, expression of both genes (Fig. 5P and data not shown) was severely decreased in the epithelium but not in the mesenchyme of Smo mutant incisors. In the anterior segment of control incisors, Shh expression declined in polarizing ameloblasts (arrowhead in Fig. 5Q), dramatically decreased in presecretory ameloblasts (arrow in Fig. 5Q), and was totally abrogated in secretory ameloblasts (data not shown). The SI continued to express Shh (Fig. 5Q), even at a more advanced secretory stage (data not shown). In mutant incisors, Shh expression was dramatically decreased (Fig. 5R). In control incisors, polarizing, presecretory and young secretory ameloblasts and the SI, expressed Gli3 (data not shown), Ptc1, Ptc2, Gli1 and Gli2; however, with the exception of Gli3 (data not shown), expression of all these genes was decreased to background levels in these cell populations in Smo mutants (Fig. 5S-Z).
These data demonstrate that removal of Smo activity from the dental epithelium affects Shh responsiveness within all components of the EEO without altering Shh signaling in the dental mesenchyme. Furthermore, the results provide evidence that in the developing tooth, with the exception of the enamel knot, Ptc2 transcription is Shh dependent, suggesting that Ptc2, like Ptc1, is a target of Shh signaling, and implicating Ptc2 in the regulation of Shh signaling activity within the EEO.
Shh signaling within the EEO is necessary for regulating cellular
proliferation
The Smo mutant molars were small, misshapen and had shallow cusps
and the incisors were also abnormally small. The enamel knots have been
suggested to control cusp morphogenesis in molars. However, primary and
secondary enamel knot development in Smo mutant molars appeared
normal at both the morphological and molecular level. Cell proliferation
profiles have been suggested to govern tooth morphogenesis and
cytodifferentiation (Ruch,
1990). To address whether cell proliferation, altered by
Smo loss-of-function, might contribute to the morphological
alterations we observed, we first examined the distribution of phosphorylated
histone H3 (PH-H3), a marker of mitosis. In control molars and incisors,
preameloblasts and the overlying SI facing polarized odontoblasts showed PH-H3
staining. PH-H3 staining was lost thereafter in ameloblasts adjacent to
predentin matrix (Fig. 6A and
data not shown). In contrast, in Smo mutant molars and incisors,
PH-H3 staining was lost at an earlier stage, in preameloblasts and SI facing
polarized odontoblasts, before predentin secretion
(Fig. 6B and data not
shown).
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Cyclin D1 is a G1 cyclin, which has recently been shown to be
transcriptionally induced by Shh and Indian hedgehog
(Kenney and Rowitch, 2000;
Long et al., 2001
). We found
that expression of cyclin D1 was similar in control and Smo
mutant molars from the cap stage until the early bell stage (data not shown).
In the EEO of control molars at 1 dpp, cyclin D1 was expressed in the
IDE, SI and preameloblasts adjacent to polarized odontoblasts. Expression was
thereafter abrogated in post-mitotic ameloblasts adjacent to predentin matrix,
whereas the SI continued to express cyclin D1
(Fig. 6C). In Smo
mutant molars, however, cyclin D1 transcripts were lost from
preameloblasts and the adjacent SI in young cusps that contained polarized
odontoblasts but before predentin secretion by these cells
(Fig. 6D). As expected, no
alterations in PH-H3 staining and cyclin D1 expression were found in
the dental mesenchyme (Fig.
6A-D). Thus, it is likely that mutant preameloblasts and cells of
the SI exit the cell cycle prematurely, and that Shh may promote proliferation
within the dental epithelium at least in part through the transcriptional
regulation of cyclin D1.
To assess whether the premature withdrawal from the cell cycle of mutant
ameloblasts was associated with their differentiation, we examined the
expression of amelin and its protein product as well as calbindin
D28K immunostaining. These markers are expressed in differentiating and in
mature ameloblasts (Elms and Taylor,
1987; Fong et al.,
1998
). In control molars and incisors, amelin mRNA and
protein were present in the dental papilla, preodontoblasts and polarizing
odontoblasts (Fig. 6E,I and
data not shown). Thereafter, amelin mRNA and protein appeared in
differentiating ameloblasts and was strongly increased and maintained in
ameloblasts (Fig. 6K and data
not shown). In contrast, in mutant molars and incisors, amelin
transcripts and protein were already detected in preameloblasts that were
adjacent to either preodontoblasts or polarizing odontoblasts, prior to
predentin secretion (Fig. 6F,J
and data not shown), and remained strong at later stages
(Fig. 6L and data not shown).
Immunohistochemistry with anti-calbindin D28K antibody showed staining in
preameloblasts that are either adjacent to polarized odontoblasts or a thin
layer of predentin (Fig. 6G),
whereas in Smo mutants molars calbindin staining was present at a
developmentally earlier stage, in preameloblasts adjacent either to
preodontoblasts or polarized odontoblasts
(Fig. 6H). These data suggest
that mutant ameloblasts `mature' precociously and express genetic markers of
post-mitotic ameloblasts, without undergoing the cytological changes
characteristic of the ameloblast proper.
Smo ablation leads to down-regulation of transcripts for DSP
and Dlx7 in ameloblasts without affecting other genetic markers in
odontoblasts and presecretory ameloblasts
As Shh signaling appears to be intact in the dental mesenchyme and its
derivatives, we expected that these would not be affected in Smo
mutants. However, the altered differentiation and responsiveness of
ameloblasts in Smo mutants might have indirectly affected mesenchymal
components. To address this issue, we examined the expression of a number of
genetic markers, that are known to be initiated or upregulated upon ameloblast
or odontoblast differentiation, including PDGFR, DSP,
DMP1, Dlx3, Dlx7, Msx2, Bmp2, Bmp4, Bmp5, and Bmp7
(Fig. 7 and data not
shown).
|
Odontoblasts differentiated normally on schedule and secreted normal
predentin/dentin matrix in Smo mutant teeth. This is supported by the
presence of normally polarized odontoblasts and normal mineralization of
dentin matrix, as shown by von Kossa staining
(Fig. 7A,B) and odontoblast
expression of PDGFR, DMP1, Bmp2, Dlx7 and DSP
(Fig. 7C-P and data not shown).
In contrast, cells of the ameloblastic lineage showed temporospatial
alterations of the expressions of Dlx7, DSP, Bmp4, Bmp5 and
Bmp7. In Smo mutant incisors and molars, expression of
Dlx7 in differentiating ameloblasts was considerably less than in
controls (Fig. 7G-J). These
data suggest a possible regulation of Dlx7 by Shh during ameloblast
differentiation. It is noteworthy that in the dental epithelium of incisors,
Dlx7 expression is confined to the amelogenic labial epithelium
(Fig. 7G). As shown in control
incisors and molars, in addition to its expression in odontoblasts,
DSP is known to be transiently expressed in preameloblasts adjacent
to the first layer of predentin (Fig.
7K,M) and then lost in secretory ameloblasts
(Fig. 7O). However, in
Smo mutant incisors and molars
(Fig. 7L,N) DSP
transcripts were lost precociously from ameloblasts
(Fig. 7L,N). This was not
secondary to cell death in mutant ameloblasts, as these cells continued to
express other late genetic markers, such as Msx2 and amelin
(see Fig. 6P and data not
shown) at very high levels in the anterior portion of the incisor. These data
provide further evidence for the premature differentiation of Smo
mutant ameloblasts. Finally, while in control incisors, Bmp4, Bmp5
and Bmp7 (Fig. 7Q and
data not shown) were expressed in secretory ameloblasts, expressions of these
genes were severely down-regulated in mutant ameloblasts
(Fig. 7R and data not shown).
However, at an earlier stage in both incisors and molars from Smo
mutants, preameloblasts and ameloblasts expressed Bmp4 and
Bmp5 as in controls (Fig.
7S,T and data not shown).
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DISCUSSION |
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Epithelial Shh signaling regulates cell proliferation and cell
differentiation in epithelial compartments of the tooth
Loss of Smo activity in the dental epithelium led to the expected
abrogation of Shh responsiveness within the EEO, whereas Shh signaling was
maintained in the dental mesenchyme, which developed normally at all stages up
to 1 dpp. Smo mutant molars were fused and displayed alterations in
cusp morphogenesis. In Smo mutant teeth, preameloblasts and cells of
the SI withdrew from the cell cycle prior to predentin secretion, as evidenced
by loss of cyclin D1 mRNA and PH-H3 protein, and exhibited molecular
features of more mature cells. These data suggest a direct requirement for Shh
activity in regulating proliferation within the dental epithelium. Rather than
exiting the cell-cycle coincident with secretion of the first layer of
predentin matrix (Ruch, 1987;
Ruch, 1990
), in the absence of
Shh signal transduction, preameloblast and cells of the SI withdraw before
matrix accumulation. Thus, Shh may exert a direct mitogenic effect on these
cells, at least in part, by promoting cyclin D1 transcription to
control the G1/S transition. After exiting the cell cycle,
preameloblasts underwent premature differentiation. Thus, maintaining these
cells in a proliferative state may be one mechanism by which the pace and
position of ameloblast differentiation is controlled in the IEE.
Alternatively, Shh may regulate cell proliferation and cell differentiation
independently. In this regard it is noteworthy that a second member of this
family, Ihh, coordinates proliferation and differentiation of chondrocytes by
distinct mechanisms during morphogenesis of the endochondral skeleton
(reviewed by McMahon et al.,
2002
).
A key role of hedgehog members as mitogens has been shown in several
developmental settings (for a review, see
McMahon et al., 2002). A
direct hedgehog input controlling cell proliferation has been best
characterized in the cerebellum (Dahmane
and Ruiz i Altaba, 1999
;
Wallace, 1999
;
Wechsler-Reya and Scott, 1999
;
Kenney and Rowitch, 2000
) and
cartilage (Long et al., 2001
),
as well as in the optic lamina and eye in Drosophila
(Huang and Kunes, 1996
;
Huang and Kunes, 1998
;
Duman-Scheel et al., 2002
).
Recent evidence indicates that hedgehog proteins are likely to exert their
mitogenic activity through regulation of G1 and G1/S
cyclins. In the cerebellum, Shh stimulates granule cell progenitor (GCP)
proliferation by promoting D-type cyclins
(Kenney and Rowitch, 2000
). In
the developing cartilage, Ihh's mitogenic activity also promotes cyclin
D1 transcription (Long et al.,
2001
). Finally, recent studies in Drosophila established
a link between hedgehog and D-type and E-type cyclins in controlling cell
proliferation and growth (Duman-Scheel et
al., 2002
). Together, these data indicate that transcriptional
induction of D-type cyclins is a likely common and conserved mechanism by
which hedgehog proteins promote cell proliferation. In addition, studies in
transfected cells have shown that Shh ligand disrupts an interaction between
Ptc1 and cyclin B1 at the G2/M transition, thereby allowing the
activation of M-phase-promoting factor and cell cycle progression
(Barnes et al., 2001
). It would
be of interest to determine whether these events also occur in normal cells
that are responsive to Shh.
During the late bell stage, both the proliferating preameloblasts and the cells of the SI strongly express Shh, and both respond to Shh signaling as judged by expression of key targets such as Ptc1 and Gli1. Whether Shh operates in an autocrine or paracrine manner within and between these cell layers, respectively, is unclear at present. One possible way to examine this issue would be by removal of Shh activity specifically from the SI.
What makes preameloblasts exit the cell cycle? Our analyses show that both the proliferating preameloblasts and the newly differentiated ameloblasts both express high levels of Shh and are Shh responsive. This raises the question of what normally triggers exit from the cell cycle in ameloblasts. The same situation is found in the developing cerebellum, where GCPs migrate towards Purkinje neurons, the source of Shh, and thus become exposed to higher concentrations of the protein. However, GCPs stop dividing by the time they reach the internal part of the external germinal layer, although they remain highly responsive to Shh. It is unlikely that termination of preameloblast proliferation is due to reduced exposure or responsiveness to Shh. This may be the result of conversion of a proliferative response into a differentiative one or to the activation of antagonizing signals.
Smo mutant first and second molars were fused and developed into a
single anlage. It is noteworthy that molar fusions can occur in both animals
and humans (Sofaer and Shaw,
1971; Turell and Zmener,
1999
), but their etiology is unknown. In addition, Smo
mutant molars had abnormally shaped, small and shallow cusps. Signaling from
the enamel knots has been shown to pattern cuspidogenesis in molars
(Thesleff et al., 2001
). In
Smo mutants molars, the primary and secondary enamels knots developed
normally and expressed several genetic markers of these structures. We
therefore propose that cusp dysmorphogenesis in Smo mutant molars may
be secondary to the alterations in cell proliferation profiles within the
EEO.
Shh as a modulator of epithelial-epithelial interactions necessary
for ameloblast and SI cytodifferentiation
Smo mutant ameloblasts failed to assume the morphological features
of differentiating cells, and the SI remained squamous in nature. At this
developmental stage in Smo mutant teeth, odontoblasts developed and
differentiated normally, and predentin/dentin matrices were secreted on
schedule. These data indicate that Shh activity within the dental epithelium
is necessary for proper cytodifferentiation of preameloblasts and cells of the
SI.
In the mouse incisor, Shh and Dlx7 were expressed exclusively in the labial amelogenic epithelium, and Dlx7 expression was down-regulated in Smo mutant teeth. Transcripts for Ptc2 and Gli1 showed a polarized localization at the basal and perinuclear pole of polarizing ameloblasts, which is adjacent to the SI.
The events leading to ameloblast cytodifferentiation are not clear. Early
tissue recombination studies using dental and nondental tissues have shown
that ameloblast cytodifferentiation requires functional odontoblasts
(Kollar and Baird, 1970;
Ruch et al., 1973
), and that
acellular dental matrices can promote ameloblast cytodifferentiation as well
(Karcher-Djuricic et al.,
1985
). In those studies (and J. V. Ruch, personal communication),
the development of an EEO (and not only a monolayer of IDE) from non-dental
and dental epithelia was shown to be a prerequisite for induction of
ameloblast cytodifferentiation by the mesenchymal components. Thus, there is
additional need for an epithelial endogenous control, possibly involving
interactions between the SI and preameloblasts. However, the different
developmental fates of the labial amelogenic and lingual non-amelogenic IDE of
the incisor have been shown to be governed by an endogenous epithelial
regulation, independent of odontoblasts/predentin/dentin
(Amar et al., 1986
;
Amar et al., 1989
;
Ruch, 1990
). The role of the
SI is unknown. This cell layer has been suggested to play a role in ameloblast
differentiation based on morphological differences in the relationships
between ameloblasts and the SI in the amelogenic zones as compared to the
non-amelogenic zones of rodent teeth
(Wakita and Hinrichsen, 1980
;
Nakamura et al., 1991
). The SI
has also been suggested to give rise to cells of the SR
(Hunt and Paynter, 1963
). The
polarized Shh responsiveness in ameloblasts suggests that Shh emanating from
the SI may play a role in ameloblast cytodifferentiation. However, Shh is
expressed in both the SI and the differentiating ameloblasts.
Together, these data suggest that although the dental mesenchyme is necessary for ameloblast cytodifferentiation, it is not sufficient, and autocrine and/or paracrine epithelial-epithelial interactions within the EEO play an important role. Thus, our Smo conditional allele provides a molecular basis for understanding the early experimental studies, and suggests that Shh may be an endogenous epithelial factor regulating ameloblast cytodifferentiation. The asymmetrical expression of Shh and Dlx7 in the incisor and the downregulation of Dlx7 expression in mutant ameloblasts raise the question of whether Shh is, indeed, involved in the determination of the different developmental fates between the labial and lingual dental epithelia through the regulation of Dlx7 expression.
Shh regulates growth and polarization of epithelial dental cells
The preameloblast is a unique epithelial cell, as upon differentiation into
a secretory ameloblast it reverses its polarity: the pole of the cell that was
originally basal (towards the basement membrane) becomes structurally and
functionally apical (Frank and Nalbandian,
1967). In addition, differentiation of the ameloblast is
accompanied by a significant increase in size and by an extensive development
of cytoplamic organelles (Frank and
Nalbandian, 1967
). At this time, cells of the SI also increase in
size (Wakita and Hinrichsen,
1980
).
At the differentiation stage, despite the presence of predentin/dentin matrices, Smo mutant ameloblasts and cells of the SI failed to grow in size, remained unpolarized, and were characterized by a paucity of organelles. In spite of this, mutant ameloblasts expressed several genetic markers of differentiated cells. As in polarizing ameloblasts, Ptc2 and Gli1 transcripts were enriched in the basal and perinuclear compartment of control presecretory and secretory ameloblasts. By this time, Shh production was barely detectable in these cells, whereas the overlying SI continued to produce Shh. These data suggest that ameloblast growth and polarization and the development of a cuboidal SI are Shh dependent. Hence, Shh may have anabolic activities regulating organelle and membrane biosynthesis. In addition, the phenotype of Smo mutant ameloblasts suggests that control of differentiation may be uncoupled from regulation of growth and polarization.
Smo mutant ameloblasts failed to show the typical polarized and
localized distribution of E-cadherin and ZO-1 and lacked ß-tubulin
accumulation. Polarized epithelial cells interact with each other through
specialized junctional complexes (Farquhar
and Palade, 1963). Junctional complexes and microtubules are
involved not only in maintaining epithelial cell polarity and partitioning the
plasma membrane into apical and basolateral domains, but also in integrating
mechanical and signaling pathways
(Kirkpatrick and Peifer, 1995
;
Barth et al., 1997
;
Gundersen and Cook, 1999
;
Tsukita et al., 1999
;
Vasioukhin and Fuchs, 2001
;
Jamora and Fuchs, 2002
).
Previous TEM and immunological studies have shown that during the
cytodifferentiation stage, numerous junctional complexes are established
within the ameloblast and SI layers as well as between these two cell layers
(Wakita and Hinrichsen, 1980
;
Fausser et al., 1998
).
Together, these data reveal roles for Shh signaling in controlling epithelial
cell size and polarity.
The intracellular localization of Ptc2 and Gli1
transcripts to the basal and perinuclear compartments of presecretory and
secretory ameloblasts is an intriguing finding. This suggests that signaling
from the SI to ameloblasts may be linked to the polarized distribution of
these RNAs, potentially providing an efficient mechanism for the targeting of
their protein products to the appropriate subcellular compartments. Epithelial
polarity is established and maintained through specific subcellular
trafficking of proteins to different subcellular compartments or membranes
(Ikonen and Simons, 1998;
Mostov et al., 2000
). Protein
trafficking and cell polarity involve not only the directed movement of
specialized vesicles after protein synthesis
(Ikonen and Simons, 1998
), but
also occur via the subcellular localization of transcripts (RNA sorting) prior
to translation (Palacios and St. Johnston,
2001
). Why Ptc2 and Gli1 transcripts show this
distribution in the ameloblast cytoplasm, but Ptc1 and Gli2
transcripts do not, is not clear.
In conclusion, the expression patterns of Shh signaling components and the tooth phenotype of Smo mutant mice provide evidence for the presence of subtle and yet exquisite Shh signaling within the dental epithelium. Tooth development is orchestrated by Shh-dependent epithelial-mesenchymal and epithelial-epithelial interactions.
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ACKNOWLEDGMENTS |
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