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Msx1 Is Required for the Induction of Patched by Sonic
Hedgehog in the Mammalian Tooth Germ
1Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana
2Department of Anatomical Sciences and Neurobiology, University of Louisville College of Medicine, Louisville, Kentucky
We have used the mouse developing tooth germ as a model system to explore the
transmission of Sonic hedgehog (Shh) signal in
the induction of Patched (Ptc). In the early developing molar tooth germ, Shh is expressed in the
dental epithelium, and the transcripts of Shh
downstream target genes Ptc and Gli1 are expressed in dental epithelium as well as adjacent
mesenchymal tissue. The homeobox gene Msx1 is
also expressed in the dental mesenchyme of the
molar tooth germ at this time. We show here that
the expression of Ptc, but not Gli1, was downregulated in the dental mesenchyme of Msx1 mutants.
In wild-type E11.0 molar tooth mesenchyme SHHsoaked beads induced the expression of Ptc and
Gli1. However, in Msx1 mutant dental mesenchyme SHH-soaked beads were able to induce
Gli1 but failed to induce Ptc expression, indicating a requirement for Msx1 in the induction of Ptc
by SHH. Moreover, we show that another signaling molecule, BMP4, was able to induce Ptc expression in wild-type dental mesenchyme, but
induced a distinct expression pattern of Ptc in
the Msx1 mutant molar mesenchyme. We conclude that in the context of the tooth germ Msx1 is
a component of the Shh signaling pathway that
leads to Ptc induction. Our results also suggest
that the precise pattern of Ptc expression in the
prospective tooth-forming region is controlled
and coordinated by at least two inductive signaling pathways. Dev Dyn 1999;215:45–53.
r 1999 Wiley-Liss, Inc.
Key words: tooth development; Sonic hedgehog;
Msx1; Ptc; Gli1; Bmp4; signaling pathway
Vertebrate organs form through sequential and reciprocal interactions between two different tissue layers,
most commonly an epithelium and a mesenchyme.
These processes involve a series of inductive and permissive tissue interactions (also known as secondary induction), which govern tissue-specific gene expression and
morphogenesis, and eventually lead to terminal cell
differentiation and organ patterning. Similar to many
other embryonic organs, murine tooth development
relies largely on such tissue interactions (reviewed in
Thesleff and Nieminen, 1996). Therefore, the murine
developing molar tooth has been employed as one of the
classical model systems for studying early inductive
interactions and their underlying molecular basis. The
first sign of mouse molar tooth morphogenesis occurs at
embryonic day 11.5 (E11.5) as a local thickening of
dental epithelium (lamina stage), which subsequently
invaginates into the subjacent mesenchyme to form
budlike structures (bud stage: E12.5–E13.5). This process is accompanied by the condensation of mesenchymal cells around the epithelial bud. The tooth bud then
progresses to the cap (E14.5) and bell stages (E16.5).
Eventually, the epithelial cells differentiate into enamelsecreting ameloblasts and the mesenchymal cells into
dentin-secreting odontoblasts, pulp, and alveolar bone
(Palmer and Lumsden, 1987).
Rapidly accumulating evidence has indicated that
peptide growth factors, such as bone morphogenetic
proteins (BMPs) and fibroblast growth factors (FGFs),
act as morphogenetic signals mediating the inductive
interactions during tooth development (Thesleff and
Sahlberg, 1996). The function of growth factors as
inductive signaling molecules in epithelial–mesenchymal interactions has been demonstrated by their ability
to substitute for one tissue in the induction of gene
expression and morphogenetic change in an adjacent
tissue (Vainio et al., 1993; Chen et al., 1996a; Neubüser
et al., 1997). For example, BMP4 can substitute for
dental epithelium to induce the expression of a number
of genes, including homeobox genes Msx1 and Msx2, an
HMG box gene Lef1 and Bmp4 itself, in dental mesenchyme (Vainio et al., 1993; Chen et al., 1996a; Kratochwil et al., 1996). The expression of growth factors in
different developmental phases and different tissue
layers may form signaling loops to achieve sequential
and reciprocal interactions (reviewed in Chen and
Grant sponsor: National Science Foundation; Grant number: IBN9796321; Grant sponsor: Tulane University (Y.P.C.); Grant sponsor:
University of Louisville/NIH Human Genetics Enhancement Program; Grant sponsor: Kentucky Spinal Cord and Head Injury Trust;
Grant sponsor: NSF (IBN-9808126) (M.S.Q.).
*Correspondence to: Dr. YiPing Chen, Department of Cell and
Molecular Biology, Tulane University, New Orleans, LA 70118. E-mail:
Received 30 November 1998; Accepted: 5 February 1999
Fig. 1. (A) Front view of the mouse face. (B)
Oral view of a mandible at E11. The region (mandible) used for this study is outlined with dashed
lines in Figure A. fb, forebrain; I, incisor; M, molar;
mb, midbrain; md, mandible; mx, maxillary arch; np,
nasal pit; T, tongue.
Fig. 2. Expression of Shh , Ptc, and Msx1 in the early mouse
developing tooth germ. Whole-mount in situ hybridization showed the
expression of Shh (A, D, J), Ptc (B, E, K), and Msx1 (C, F, L) in the incisor
(white arrows in panels A and B) and molar (black arrows in panels D, E, F,
J, K, and L) teeth from E11.0 to E12.5 (oral view). Section in situ
hybridization indicated that Shh transcripts are localized to the dental
epithelium (G) at E11.5 (sagittal section), while Ptc and Msx1 transcripts
are found in dental mesenchyme (H, I) at E11.5 (cross section). Note the
weak expression of Ptc in the dental epithelium (panel H). de, dental
epithelium; dm, dental mesenchyme; m, molar primordium; ma, mandibular arch; mx, maxillary arch; T, tongue. Scale bar: A–F, J–L ⫽ 500 µm;
G–H ⫽ 50 µm; I ⫽ 25 µm.
Fig. 3. Expression of Ptc but not Gli1 is altered in the Msx1 mutant
dental mesenchyme. (A–F) Oral view of mandibles. (G–J) Cross section
through molar teeth. Marked reduction of Ptc expression was observed in
the Msx1 mutant dental mesenchyme from E11.5 to 13.5 (panels A, D,
and G). Insert in panel A shows that Ptc expression at E11.5 is preserved
in epithelium but is downregulated in mesenchyme of the Msx1 mutant
molar tooth. Gli1 expression was preserved in the Msx1 mutant tooth
germ from E11.5 to 13.5 (panels C, F, and J), as compared to the wild-type
Gli1 expression at the same stages (panels B, E, and I). Panel H shows
expression of Ptc in an E13.5 wild-type molar tooth germ. e, epithelial bud.
Scale bar: A–F ⫽ 500 µm; G–J ⫽ 50 µm.
Maas, 1998). It has been suggested that homeobox
containing genes participate in epithelial–mesenchymal interactions by regulating the expression of inductive signaling molecules (Chen and Maas, 1998). This is
exemplified by the fact that Msx1 controls Bmp4 expression in the dental mesenchyme (Chen et al., 1996a).
Mice lacking Msx1 exhibit an arrest of tooth development at the bud stage, which is accompanied by the
downregulation of several genes, including Bmp4, Fgf3,
Dlx2, Lef1 and syndecan-1 (Satokata and Maas, 1994;
Chen et al., 1996a; Bei and Maas, 1998).
Recent studies have revealed the importance of vertebrate homologues of the Drosophila segment polarity
gene hedgehog (hh) in the control of organogenesis
(reviewed in Hammerschmidt et al., 1997). Several
members of the vertebrate hh gene family that encode
secreted signaling molecules have been identified.
Among them, Sonic hedgehog (Shh) is best characterized. Shh is expressed in tissues with inductive and
polarizing activities in several vertebrate organs (Hammerschmidt et al., 1997), including Hensen’s node
(Levin et al., 1995, Chen et al., 1996b), limb (Riddle et
al., 1993), neural tube (Echelard et al., 1993; Roelink et
al., 1994), gut (Roberts et al., 1995), hair (Bitgood and
McMahon, 1995), lung (Bellusci et al., 1997) and tooth
(Bitgood and McMahon, 1995; Koyama et al., 1996;
Vaahtokari et al., 1996; Hardcastle et al., 1998). Similar
to HH action in Drosophila, SHH exerts its short- and
long-range effects by activating downstream gene expression (reviewed in Johnson and Tabin, 1995). The
Ptc gene, originally identified as a Drosophila segment
polarity gene (Hooper and Scott, 1989), encodes a
transmembrane protein which serves as a SHH receptor and plays a key role as a negative regulator in the
SHH signaling pathway (Ingham et al., 1991; Marigo et
al., 1996a; Stone et al., 1996). In addition, Ptc is also
one of the general downstream targets of SHH signal.
Ectopic Shh expression leads to ectopic Ptc expression
in several vertebrate developing organs, including neural tube (Goodrich et al., 1996; Marigo and Tabin,
1996), limb (Marigo et al., 1996b), and lung (Bellusci et
al., 1997).
It has also been demonstrated that Gli1, one of the
three vertebrate Gli gene family members that are
orthologous to the Drosophila cubitus interruptus (ci)
gene, may function as a component of the Shh signaling
pathway (Orenic et al., 1990; Hui et al., 1994). In the
Drosophila wing disc, ci functions downstream of hh to
activate Ptc expression (Alexandre et al., 1996). The
involvement of ci in the regulation of Ptc by HH is
conserved in vertebrates. For example, ectopic Shh
expression in the chick limb and mouse lung results in
upregulation of Ptc and Gli1 transcription (Marigo et
al., 1996b, 1996c; Bellusci et al., 1997; Grindley et al.,
1997). This upregulation of Gli1 by SHH appears to
mediate the ability of SHH to upregulate Ptc expression, since misexpression of Gli1 also induces ectopic
Ptc expression (Marigo et al., 1996c). Additional evidence for this idea comes from the observation that the
expression of Ptc and Ptch2, a second mouse Patched
gene, is downregulated in mutant mice lacking Gli2
(Motoyama et al., 1998).
Although the important role of Shh in induction and
patterning processes in vertebrate organogenesis has
been well established by loss-of-function and misexpression studies (Chiang et al., 1996; reviewed in Hammerschmidt et al., 1997), components of the SHH signal
transduction pathway and the general target genes of
SHH signaling remain partly unknown. Here we present evidence that Shh, which is normally expressed in
mouse dental epithelium, can induce expression of Ptc
and Gli1 in mouse dental mesenchyme. In the dental
mesenchyme of Msx1 mutant mice, Ptc, but not Gli1
expression, is markedly downregulated at the stages
that precede overt morphologic differences in the Msx1
mutant tooth germ. This result suggests a requirement
of Msx1 for Ptc expression in dental memesenchyme.
Consistent with this hypothesis, we further demonstrate that induction of Ptc, but not Gli1, in dental
mesenchyme by SHH requires functional MSX1 protein, implicating MSX1 as a component of the Shh
signaling pathway that leads to the induction of Ptc
expression. Finally we show that BMP4 is able to
induce Ptc expression even in the absence of Msx1,
indicating the involvement of different signaling pathways in the regulation of Ptc expression in the developing mouse molar tooth germ.
Shh and Ptc Are Expressed in the Early
Tooth Germ
In order to explore the Shh signaling pathway in the
mouse tooth germ, we began with an analysis of Shh
and Ptc expression in the early developing tooth. Whole
mandibular arches from E11.0 to E13.5 mouse embryos
were used for in situ hybridization (Fig. 1). At E11.0,
Shh transcripts were first detected weakly in the
incisor-forming region (Fig. 2A), while Ptc expression
was found in the incisor as well as molar-forming
regions (Fig. 2B). At E11.5, Shh expression becomes
stronger in the incisors and starts to appear in the
molar tooth germ (Fig. 2D). Shh expression is restricted
to the dental epithelium and remains there until E13.5
(Fig. 2G,J, and data not shown). At E14.5, Shh transcripts are localized to the enamel knot of the dental
epithelium (Koyama et al., 1996; Vaahtokari et al.,
1996) (data not shown). Ptc transcripts were detected
relatively weakly in dental epithelium but strongly in
adjacent dental mesenchyme from E11.5 to E13.5 (Figs.
2E,H,K and 3H). Similar results on the expression
pattern of Shh and Ptc in mouse tooth germ have
recently been reported by Dassule and McMahon (1998)
and Hardcastle et al. (1998). The expression pattern of
Ptc always overlaps with, but is broader than, that of
Shh in the tooth-forming regions at the same stages.
Thus, similar to other Shh signaling centers (Goodrich
et al., 1996), Ptc is also expressed in cells close to
Shh-expressing cells in the developing tooth germ. In
the dental mesenchyme, Ptc expression coincides with
that of Msx1 temporally and spatially (Fig. 2C,F,I,L;
MacKenzie et al., 1991), suggesting a potential correlation between the expression of these two genes during
early tooth development.
Msx1 Is Required for Ptc But Not Gli1
Expression in Dental Mesenchyme
Observations that the expression pattern of Msx1
and Ptc overlaps in the molar dental mesenchyme
prompted us to test the hypothesis that Msx1 and Ptc
reside within the same signaling pathway in the early
tooth development. We examined Ptc expression in the
Msx1 mutant tooth germs of E11.5 to E13.5 embryos by
in situ hybridization. At E11.5, weak signals of Ptc
expression were detected in the incisor and molar tooth
germ of the Msx1 mutant mandible by whole-mount in
situ hybridization (Fig. 3A; cf. Fig. 2E). Sectioning of
the samples revealed that Ptc expression occurred only
in the dental epithelium but was downregulated from
the adjacent mesenchyme (insert in Fig. 3A; cf. Fig.
2H). At E12.5, Ptc expression was almost undetectable
in the Msx1 deficient tooth germ (Fig. 3D), as compared
with Ptc expression in the wild-type tooth germ at the
same stage (Fig. 2K). At E13.5, Ptc expression remained undetectable in the dental mesenchyme but
was weakly detected in the dental epithelium of Msx1
mutants (Fig. 3G; cf. wild-type expression in Fig. 3H).
However, Ptc expression was not affected in the developing tongue where Msx1 is not expressed (Fig. 3A,D),
indicating the specificity of the reduction of Ptc expression in the developing tooth germ of Msx1 mutants.
Since SHH is an inducer of Ptc expression, this downregulation of Ptc expression could result from altered
Shh expression in the Msx1 mutant dental epithelium.
However, we have found that Shh expression was
preserved in the dental epithelium of E11.5 Msx1
mutants (Zhang et al., unpublished observation). Based
on these results, we conclude that Ptc is a downstream
gene from Msx1.
It was previously reported that Gli1 may function as
a component of Shh signal transduction machinery,
residing downstream of Shh but upstream to Ptc (Marigo
et al., 1996b, 1996c; Grindley et al., 1997). In addition,
Gli1 expression was detected in both dental epithelium
and dental mesenchyme at E14.5, coinciding with Ptc
expression pattern (Hui et al., 1994) (data not shown).
We therefore asked whether Gli1 also resides downstream from Msx1 in the early mouse developing tooth
germ. In situ hybridization was performed to examine
Gli1 expression in wild-type and Msx1 mutant tooth
germ from E11.5 to E13.5. Gli1 expression was detected
in the wild-type incisor and molar tooth germs of all
stages examined (Fig. 3B,E,I). The transcripts were
localized to dental epithelium and mesenchyme (Fig.
3I), as recently reported (Dassule and McMahon, 1998;
Hardcastle et al., 1998). The wild-type expression pattern of Gli1 in the early developing tooth germ mirrors
that of Ptc. However, by contrast, Gli1 expression in the
Msx1 mutant tooth germ is mainly preserved (Fig.
3C,F,J). Thus, Msx1 is clearly required for the expression of Ptc, but not Gli1, in mouse dental mesenchyme.
TABLE 1. Induction of Ptc and Gli1 Expression
in Wild-Type and Msx1 Mutant Mandibles
nd, not done.
*Biased expression; see text for details.
to Ptc expression induced by SHH beads in the wildtype molar mesenchyme (Fig. 4A). These results indicate that Msx1 is required for the induction of Ptc by
Shh in the mouse tooth germ. By contrast, SHH beads
were able to induce Gli1 expression in the Msx1 mutant
dental mesenchyme (Fig. 4D and Table 1). Thus, SHH
can induce Gli1 expression in the absence of Msx1.
Msx1 Is Required for Induction of Ptc But Not
Gli1 by SHH in Dental Mesenchyme
BMP4 Induces a Distinct Pattern of Ptc
Expression in Wild-type and Msx1
Mutant Dental Mesenchyme
Since SHH protein can induce Ptc and Gli1 expression in mandibular mesenchyme (Dassule and McMahon, 1998; Hardcastle et al., 1998), and since Ptc
expression is downregulated in the Msx1 mutant dental
mesenchyme, we asked whether the Shh induction of
Ptc expression is mediated by Msx1. We first analyzed
induction of Ptc and Gli1 expression by SHH in wildtype dental mesenchyme. Bacterially expressed recombinant SHH protein was prepared and purified. Whole
mandibles isolated from wild-type E11.0 and E11.5
embryos were subjected to microsurgical separation of
epithelium from mesenchyme after enzyme treatment.
The whole mandibular mesenchyme was applied to
organ culture with prospective tongue side facing up.
Removal of the epithelium resulted in complete loss of
Ptc expression in E11.0 mandibular explants after 24
hours in culture, indicating a requirement of epithelial
signals to maintain Ptc expression at this stage. By
contrast, endogenous Ptc expression is retained in
E11.5 mandibular mesenchyme without epithelium after 24 hours in culture. Therefore, E11.0 mandibles
were used for the induction assay. SHH-soaked beads
were placed on top of the right side molar region, while
BSA-soaked beads were placed on the left side molar
region as controls. After 24 hours in culture, samples
were assayed for gene expression by whole-mount in
situ hybridization. The results, summarized in Table 1,
demonstrated that beads soaked with SHH were able to
induce expression of Ptc (Fig. 4A) and Gli1 (Fig. 4C) in
the cells around the implanted beads in the dental
mesenchyme. Whole mandibular mesenchyme from
E11.0 Msx1 mutant embryos was applied to similar
bead implantation culture before analysis of Ptc and
Gli1 expression. Very weak or no induction of Ptc
expression by SHH was observed in the Msx1 mutant
molar mesenchyme (Fig. 4B and Table 1), as compared
It has been demonstrated that BMP4 is a strong
inducer of Msx1 expression in dental mesenchyme
(Vainio et al., 1993). Bmp4 expression is found in dental
epithelium at the initiation stage and then shifts to the
dental mesenchyme shortly afterward (Vainio et al.,
1993). Based on the observations that Msx1 is required
for Ptc expression, we asked whether BMP4 can also
induce Ptc expression in dental mesenchyme; if so,
whether this induction requires Msx1. To address this
question, BMP4-soaked beads were implanted to the
molar region of mandibular mesenchyme from E11.0
wild-type and Msx1 mutant embryos. Samples were
analyzed for Ptc expression after 24 hours in culture.
BMP4 did indeed induce Ptc expression (Table 1). The
results demonstrated that in the wild-type, unlike
Msx1, which is induced in the mesenchymal cells
around the BMP4 beads (Fig. 5A), Ptc expression was
induced by BMP4 with a strong bias on the lingual and
mesial sites of molar mesenchyme (Fig. 5B). Of the17
samples assayed, 15 exhibited a biased induction, while
the other 2 samples showed induction in cells around
the implanted BMP4 beads. These results indicate the
presence of a distinct signaling pathway for the induction of Ptc expression in dental mesenchyme. This idea
is supported by the observations that neither SHH
induced Bmp4 expression nor BMP4 induced Shh expression in the dental mesenchyme (data not shown).
Similar observations were also reported recently (Dassule and McMahon, 1998). However, although BMP4
could also induce Ptc expression in Msx1 mutant dental
mesenchyme, the induction is seen in cells surrounding
the beads in all samples examined (Fig. 5C; Table 1).
The results indicate a requirement of Msx1 for the
biased induction of Ptc by BMP4 in molar mesenchyme.
BMP2, a BMP4 closely related signaling molecule,
which is also expressed in the dental epithelium during
Fig. 4. Induction of Ptc but not Gli1 by SHH in
molar mesenchyme requires Msx1. (A) Strong induction of Ptc by SHH-soaked bead is evident in the
right side molar mesenchyme of an E11.0 wild-type
mandible, as compared with no induction of Ptc by
BSA bead placed on the left side molar mesenchyme. (B) SHH-soaked bead (blue bead on the
right side) failed to induce PtcMsx1 mutant dental
mesenchyme. White bead soaked with BSA placed
on the left side served as a control. The sample was
overdeveloped for color reaction to detect residual
expression. Endogenous Ptc expression in the
tongue is seen. (C) SHH bead (blue bead on the
right side) induced Gli1 expression in an E11.0
wild-type molar mesenchyme, BSA-soaked bead
(white bead on the left side) served as a control. (D)
SHH beads were able to induce Gli1 expression
(right side) in an E11.0 Msx1 mutant molar mesenchyme. BSA-soaked bead was placed on the left
side as a control. Scale bar ⫽ 500 µm.
Fig. 5. Induction of Ptc by BMP4 in molar
mesenchyme. Blue beads on the right side of
mandible are protein-soaked beads, while
white beads on the left side are BSA controls.
(A) BMP4-soaked bead could induced Msx1
expression in molar mesenchyme of an E11.0
wild-type mandible. Cells around the beads
showed response to BMP4 induction. No induction is seen on the left side where a BSA
bead was implanted. (B) In contrast to Msx1
expression, Ptc could only be induced by
BMP4 bead in the cells localized on the lingual
site of molar mesenchyme of an E11.0 wildtype mandible. BSA bead failed to induce Ptc
expression on the left side molar mesenchyme. (C) BMP4 could induce Ptc expression in cells around the bead in an E11.0 Msx1
mutant molar mesenchyme. (D) BMP2-soaked
bead did not induce Ptc expression in an
E11.0 wild-type mandibular mesenchyme.
Scale bar ⫽ 500 µm.
early tooth morphogenesis (Turecková et al., 1995;
Dassule and McMahon, 1998; Thesleff and Pispa, 1998),
failed to induce Ptc expression when BMP2-soaked
beads were implanted in E11.0 wild-type molar region
(Fig. 4D and Table 1), as reported recently (Dussale and
McMahon, 1998). This induction of Ptc expression in
the molar mesenchyme is thus unique to BMP4.
Msx1 Is a Component of Shh Signaling Pathway
A number of downstream target genes of the Shh
signal, including Ptc, have been described in vertebrates in the past few years. Msx1, which coexpresses
with Ptc in dental mesenchyme, is required for expression of Ptc in the dental mesenchyme. The specific
reduction of Ptc expression in the Msx1 mutant dental
mesenchyme establishes that Msx1 is genetically epistatic to Ptc. The requirement of Msx1 for SHH to
induce Ptc expression implicates Msx1 as a key component of SHH signaling pathway. However, whether Ptc
is a direct downstream gene regulated by Msx1 remains
unclear. These results further indicate that Msx1 may
not only be involved in epithelial–mesenchymal interactions by controlling the expression of inductive signals
in the dental mesenchyme (Chen et al., 1996a; Bei and
Maas, 1998), but may also participate in pattern formation by controlling Ptc expression.
Our results have also demonstrated that expression
of Gli1, a known downstream target of Shh and a
component of Shh signaling transduction machinery, is
preserved in the Msx1 mutant dental mesenchyme
where Ptc expression is eliminated. Furthermore, SHH
can induce Gli1 expression in the absence of Msx1. Gli1
may be either upstream of or parallel with Msx1 in the
Shh signaling pathway. However, based on the fact that
SHH-soaked beads failed to induce Msx1 expression in
E11.0 wild-type dental mesenchyme (data not shown),
we suggest that Gli1 is more likely to act in parallel
with Msx1. It has been suggested that regulation of Ptc
by Gli1 may be direct, since GLI consensus binding
sites are found in the promoter region of the Drosophila
Ptc gene (Alexandre et al., 1996). Deletion of these
consensus binding sites in the Ptc promoter results in
failure of promoter activity in response to HH signal. In
the mouse dental mesenchyme, however, Gli1 alone
apparently may not directly regulate the expression of
Ptc induced by SHH signal. It is possible that the Msx1
gene product interacts with GLI1 protein to activate Ptc
expression. This hypothesis warrants further experiments. Since Ptc serves as a general downstream target
as well as a receptor for SHH, one question that is how
SHH can induce Gli1 expression in the Msx1 mutant
dental mesenchyme in the absence of Ptc. The explanation could be that there exist residual Ptc transcripts
that are below the sensitivity of in situ hybridization
detection. Alternatively, other receptors for SHH, such
as Ptch2, which is known to be present in the mouse
tooth germ (Motoyama et al., 1998), may present in the
Msx1 mutant tooth germ and mediate SHH signaling.
BMP4 Signaling Represents a Distinct Pathway
for Ptc Induction
Among the inductive factors in the early dental
epithelium postulated to be involved in the initiation of
tooth morphogenesis, BMP4 was the first identified
signal mediating the inductive interactions between
epithelium and mesenchyme (Vainio et al., 1993). The
function of BMP4 has been demonstrated by its ability
to mimic the effect of presumptive dental epithelium in
induction of a number of genes in dental mesenchyme
(Vainio et al., 1993; Chen et al., 1996a). We show here
that BMP4 also induces Ptc expression in dental mesenchyme. Ptc expression induced by BMP4 is limited to
the lingual and mesial sites of molar mesenchyme. The
specificity of BMP4 in Ptc induction in dental mesenchyme is further supported by the fact that BMP2 failed
to induce Ptc expression in the similar condition (Dussale and McMahon, 1998; this study). Although BMP2
shares with BMP4 95% amino acid sequence identity
and many functions, including gene induction in tooth
germ (Vainio et al., 1993; Chen et al., 1996a; Thesleff
and Pispa, 1998), these results provide evidence for the
distinct function of BMP2 and BMP4 in early tooth
A genetic model for the regulation of Ptc expression in
the early tooth germ is thus proposed (Fig. 6). In this
model, epithelial SHH induces Gli1 expression in the
mesenchyme. Gli1 may activate Ptc expression by
interacting with the product of Msx1, which is induced
in the mesenchyme by the epithelially derived BMP4
(Vainio et al. 1993; Tucker et al., 1998). Mesenchymal
BMP4, whose expression requires Msx1 and provides a
positive feedback signal for maintenance of Msx1 expression (Chen et al., 1996a), can also induce Ptc expression. Epithelial BMP4 seems unlikely to be able to
induce Ptc expression in the mesenchyme, since Bmp4
expression is preserved in the dental epithelium of
Msx1 mutant tooth germ where Ptc expression is
downregulated (Chen et al., 1996a; this study).
Fig. 6. A model of genetic pathway regulating Ptc expression in the
early tooth germ. Epithelial BMP4 induces in the mesenchyme the
expression of Msx1 which is required for the mesenchymal Bmp4
expression. Epithelially derived SHH may induce Ptc expression through
the induction of Gli1. Msx1 participates in this induction of Ptc by SHH
probably by interacting with Gli1. Meanwhile, mesenchymally expressed
Bmp4 can also regulate Ptc expression. Ptc expression is thus regulated
by at least two distinct pathways in the early mouse tooth germ. See text
for details.
Interestingly, BMP4 can induce Ptc expression in the
absence of Msx1, but the asymmetric induction of Ptc by
BMP4 obviously requires functional MSX1. Expression
of Msx1 in dental mesenchyme may precondition cells
in response to BMP4. Our results show that mesenchymal cells at different sites of molar tooth germ respond
differentially to different inductive factors in terms of
Ptc expression. These observations suggest that the
prospective dental mesenchymal cells at the initiation
stage have not only been specified in general to odontogenesis (Neubüser et al., 1997), but also behave differently according to their positions in the tooth germ. The
neural crest derived cells that give rise to prospective
dental mesenchyme may have been imprinted with
different positional codes at the beginning of tooth
morphogenesis. Msx1 expression, like that of Dlx genes,
could be among these positional codes (Sharpe, 1995;
Weiss et al., 1995; Thomas et al., 1997).
Based on the results presented above, we conclude
that Msx1 is a component in the Shh-Ptc pathway,
required for the induction of Ptc expression by SHH. In
the Bmp4-Ptc pathway, Msx1 may play a role in restricting the induction of Ptc by BMP4 to certain mesenchymal cells. Ptc expression pattern is thus regulated and
coordinated by at least two distinct inductive signaling
pathways in which Msx1 plays different roles in the
early mouse dental mesenchyme.
Embryo Collection and Genotyping
Mouse embryos used for in situ hybridization and
tissue recombination were collected from matings of
CD-1 mice. Msx1 mutant embryos were collected from
Msx1⫹/⫺ females crossed with an Msx1⫹/⫺ male. The
day of vaginal plug discovery was designated as embryonic day 0.5 (E0.5). The genotype of Msx1 mutant
embryos was determined by PCR using genomic DNA
isolated from extraembryonic membranes, as previously described (Chen et al., 1996a).
In Situ Hybridization
Probes. All riboprobes used in this study were labeled with digoxygenin-UTP. The 0.65-kb Shh probe
was made from a HindIII linearized template using T3
RNA polymerase (Bitgood and McMahon, 1995). The
2.25-kb Patched probe was transcribed with T7 RNA
polymerase from a BglII linearized template covering
the whole coding region (from Dr. M. Scott). The Gli1
probe was transcribed using T3 polymerase from a
1.7-kb template linearized by NotI (Hui et al., 1994).
The 1.0-kb Bmp4 and 0.8-kb Msx1 probes were generated as described previously (Hill et al., 1989; Jones et
al., 1991). Probe size and yield were checked by electrophoresis on a 1.5 % agarose gel with an RNA standard.
Whole-mount in situ hybridization. Samples were
fixed with ‘‘M’’ buffer (3.7% formaldehyde, 100 mM
MOPS, pH 7.4, 2 mM EGDA, 1 mM MgSO4) at 4°C
overnight, followed by bleaching with 10% H2O2 in ‘‘M’’
buffer at room temperature. Whole-mount in situ hybridization was performed as described previously (Chen et
al., 1996b). Briefly, all probes were hydrolyzed to similar size before use. Hybridization and washing temperatures were set at 60°C. Proteinase K concentrations
and digestion time varied, depending on the sample
size and targeting tissue. Signals were visualized with
NBT/BCIP (Boehringer Mannheim, Indianapolis, IN).
Samples were washed with PBS and then refixed with
‘‘M’’ buffer for 30 minutes before photography and
Section in situ hybridization. Tissues were fixed
in 4% paraformaldehyde/phosphate buffered saline
(PBS) overnight and dehydrated with ethanol before
embedding in paraffin wax. Sections of 8 µm were cut
and used for nonradioactive in situ hybridization.
Briefly, about 1 µg/ml of each probe was used. Hybridization and washing temperatures were set according to
the size of probes. Maleic acid buffer and blocking
reagent (from Boehringer Mannheim) were included in
blocking and antibody washing steps. Signals were
developed with BM purple alkaline phosphatase substrate. Sections were counterstained briefly with 1%
Safranin and then mounted with Permount.
Preparation of Protein-Soaked Beads
BMP4 protein was obtained from Genetics Institute
(Cambridge, MA). Recombinant SHH protein was prepared from a Shh-expression vector containing sequences encoding six histidine residues upstream to the
mouse Shh-coding sequences (amino acids 25–198)
(from Dr. A. McMahon). The protein was induced and
purified according to the method described previously
(Marti et al., 1995). The inductive activity of SHH
protein was examined and confirmed by its ability to
induce digit duplication of chick wing buds after SHHsoaked beads (1 mg/ml) were implanted to the anterior
margin of a host wing bud as well as its ability to induce
cPix2 expression in an early chick embryo (St. Amand
et al., 1998). Affi-Gel blue agarose beads (Bio-Rad) were
washed with PBS before incubating with BMP4 protein
(60 mg/ml), BMP2 (100 mg/ml) and SHH protein (1
mg/ml), respectively, at 37°C for 30 minutes. Control
beads (white heparin beads) were soaked with similar
concentrations of BSA under same conditions. All protein-soaked beads were stored at 4°C and used within 1
Bead Implantation and Organ Culture
To separate the epithelium from the whole mandibular arch, E11.0 mandibles were incubated with 2.25%
trypsin/0.75% pancreatin on ice for 10 minutes, followed by incubation in PBS/horse serum on ice for 20
minutes. Mandibular epithelium was then separated
microsurgically. Freshly separated mesenchymal tissues were placed on Millipore filters (pore size, 0.1 µM)
supported by metal grids. The whole mandibular mesenchyme was oriented with tongue side facing up.
Beads were placed on the top the molar tooth regions of
the whole mandibular mesenchyme, with SHH or
BMP4-soaked beads placed on the right-side molar,
while BSA beads on the left-side molar regions. All
explants were cultured in Dulbecco’s minimal essential
medium (DMEM) with 10% FCS at 37°C for 24 hours.
Samples were fixed with ‘‘M’’ buffer and processed for
whole-mount in situ hybridization.
We thank Drs. Andrew McMahon (Harvard) and
Matthew Scott (Stanford) for plasmids, Anthony Celeste (Genetics Institute, Inc., Cambridge, MA) for
BMP2 and BMP4 recombinant proteins, Richard Maas
(Harvard), in whose laboratory this study was originally initiated, for Msx1 mutant mice, and Ken Muneoka and Carol Burdsal and members of their laboratories for sharing laboratory resources and help during
the initiation phase of the Chen laboratory. Y.P.C.
thanks Dr. Ken Muneoka for his encouragement.
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