The murine first molar tooth develops through a series of
morphologically distinct stages as a result of epithelial-mes-
enchymal interactions during days E9 to E18 of gestation
(for review see: Thesleff et al., 1989). These events are
characterized by, and are probably regulated by, changes in
the extracellular matrix (Thesleff et al., 1979, 1987), pro-
duction of peptide growth factors and their respective recep-
tors (Partanen and Thesleff, 1989; Cam et al., 1990; Vaah-
tokari et al., 1991), and by DNA-binding transcription
factors (MacKenzie et al., 1991a,b, 1992; Karavanova et
The epithelium, derived from the oral ectoderm, forms
the enamel organ of the first molar which secretes the
enamel matrix. The remainder of the tooth and its sup-
porting tissues are derived from the mesenchyme of the first
branchial arch (Lumsden, 1988). All of the mesenchyme of
the first arch consists of neural crest cells which migrate
from the first and second rhombomere region of the prim-
itive hindbrain (Hunt et al., 1991). By recombination exper-
iments, Lumsden (1988) showed that for tooth formation to
occur the mesenchyme must be neural crest in origin, but
that it need not be from the cranial region. Furthermore,
tooth formation can only be initiated by the oral epithelium,
which at around E10 possesses pre-patterned regions,
beneath which mesenchymal condensation occurs (Mina
and Kollar, 1987; Lumsden, 1988). During the condensa-
tion process, the mesenchyme becomes capable of induc-
ing tooth formation when cultured in combination with non-
oral epithelium (Mina and Kollar, 1987); this property of
the mesenchyme persists until at least E16 (Kollar and
The odontogenic interactions only occur at specific zones
Development 117, 461-470 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
Duplication of the msh-like homeobox gene of
Drosophila may be related to the evolution of the ver-
tebrate head. The murine homologues of this gene, msx
1 and msx 2 are expressed in the developing craniofa-
cial complex including the branchial arches, especially
in regions of epithelial-mesenchymal organogenesis
including the developing tooth.
By performing in vitro recombination experiments
using homochronic dental and non-dental epithelial and
mesenchymal tissues from E10 to E18 mouse embryos,
we have found that the maintenance of homeobox gene
expression in the tooth is dependent upon tissue inter-
actions. In homotypic recombinants, dental-type tissue
interactions occur, leading to expression of both genes
in a manner similar to that seen during in vivo devel-
opment. msx 1 is expressed exclusively in mesenchyme,
both in the dental papilla and follicle. msx 2 is expressed
in the dental epithelium and only in the mesenchyme of
the dental papilla. In heterotypic recombinants, the
dental epithelium is able to induce msx 1 expression in
non-dental mesenchyme, this potential being lost at the
bell stage. In these recombinants msx 2 was induced by
presumptive dental epithelium prior to the bud stage
but not thereafter. The expression of msx 1 and msx 2
in dental mesenchyme requires the presence of epithe-
lium until the early bell stage. However, whereas non-
dental, oral epithelium is capable of maintaining
expression of msx 1 in dental mesenchyme throughout
tooth development, induction of msx 2 was temporally
restricted suggesting regulation by a specific epithelial-
mesenchymal interaction related to the inductive events
of tooth formation.
msx 1 and msx 2, as putative transcription factors,
may play a role in regulating the expression of other
genes during tooth formation. We conclude that
expression of msx 1 in jaw mesenchyme requires a non-
specific epithelial signal, whereas msx 2 expression in
either epithelium or mesenchyme requires reciprocal
interactions between specialized dental cell populations.
Key words: odontogenesis, organ culture, in situ hybridization
Epithelial-mesenchymal interactions are required for msx 1 and msx 2
gene expression in the developing murine molar tooth
Adrian K. Jowett1,*, Seppo Vainio1, Mark W. J. Ferguson2, Paul T. Sharpe2and Irma Thesleff1
1Department of Pedodontics and Orthodontics, University of Helsinki, Mannerheimintie 172, Helsinki SF-00300, Finland
2Molecular Embryology Laboratory, Department of Cell and Structural Biology, University of Manchester, Stopford Building,
Oxford Road, Manchester M13 9PT, UK
*Present address and address for correspondence: Department of Biomedical Science, The University of Sheffield, Sheffield, S10 2TN, UK
of the epithelial-mesenchymal interface, separated by
regions of non-odontogenic epithelium (Mina and Kollar,
1987). Each of these zones codes for a single, morpholog-
ically distinct tooth. Mutations resulting in the transposi-
tion of teeth are rare (Miles and Grigson, 1990). However,
the patterning system is not flawless as missing, supple-
mental (extra teeth with indistinct morphology) supernu-
merary (additional teeth of specific morphology) and
morphologically disrupted teeth are found in humans
(Duterloo, 1991). There is only one reported case where
molar-like teeth are found anteriorly in the mouth (Kanta-
putra and Gorlin, 1992).
Homeobox genes constitute a large, highly conserved,
multigene family of developmentally regulated transcrip-
tion factors. Combinatorial expression of the Antennapedia-
like homeobox genes of mammals within mesoderm adja-
cent to the neural tube is responsible for establishing axial
regional identity of the prevertebral segments (Kessel and
Gruss, 1991), and a similar system within the neuroepithe-
lium appears to establish the identity of the caudal branchial
arches (Hunt and Krumlauf, 1991). The Antennapedia class
of homeobox genes are not expressed in rhombomeres 1
and 2, which give rise to the neural crest population of the
first branchial arch (Hunt and Krumlauf, 1991) and are thus
unlikely to regulate specification of the jaws. However, msx
1 and msx 2 (formally Hox-7 and Hox-8 respectively)
expression within the neural tube extends caudally from the
forebrain and they are expressed in the branchial arches,
including the region of the presumptive tooth germ (Hill et
al., 1989; MacKenzie et al., 1991a,b, 1992). msx 1 and msx
2 are two members of a diverged homeobox family homol-
ogous to the Drosophila muscle-segment homeobox (msh)
gene (Hill et al., 1989). In Drosophila, msh is mainly
expressed in the central nervous system and in segmented
striated muscles of the body wall. No mutations of msh are
known at present (Robert et al., 1989). In the mouse there
appear to be three distinct msh-like genes, which are found
at different loci, and are not clustered. Duplication of the
msh-like genes has been suggested to be correlated with the
emergence of the vertebrate body plan (Thorogood and
Hanken 1992; Holland, 1991).
MacKenzie et al. (1991a,b, 1992) proposed that the
initiation of tooth formation and the subsequent ability of
condensed neural crest mesenchyme to induce tooth for-
mation are related to expression of the msx 1 and msx 2
genes. They are first expressed in the early maxillary and
mandibular processes with an anteroposterior gradient
within the mesenchyme (msx 1, MacKenzie et al., 1991a)
and both epithelium and mesenchyme (msx 2, MacKenzie
et al., 1992). During subsequent odontogenesis, msx 1 is
expressed by all of the condensed dental mesenchyme.
However, msx 2 is expressed by subpopulations of both
epithelium and mesenchyme, suggesting that it may play a
role in regulating epithelial-mesenchymal interactions.
To investigate the relationship of msx 1 and msx 2
expression to epithelial-mesenchymal interactions during
odontogenesis, we performed a series of tissue recombina-
tion experiments and analyzed these by in situ hybridiz-
ation. The recombination technique has been used success-
fully to examine the expression of matrix molecules and
homeobox genes (Thesleff et al., 1990; Takahashi et al.,
1991; Vainio and Thesleff, 1992).
MATERIALS AND METHODS
Preparation and culture of tissues
Hybrid mice (CBA×C57BL) were time-mated and the day of
locating the vaginal plug was designated as day 0. The regions of
the first mandibular molar tooth germs were removed from
embryos at days E12, E14, E16 and E18 of gestation, and the first
mandibular arch from E10 embryos. For heterotypic recombina-
tions; the second branchial arch was taken at E10, the palatal
shelves or diastema tissue at days E12 and E14, and the disto-
buccal oral mucosa at E16 and E18. The tissues were initially dis-
sected in phosphate-buffered saline (PBS) at 4°C, prior to final
preparation in calcium and magnesium-supplemented Dulbecco’s
buffered saline (DPBS).
For recombination experiments explants were incubated for 2
minutes in freshly defrosted 2.25% trypsin/0.75% pancreatin and
then kept for at least 20 minutes at room temperature in minimum
essential medium (MEM) supplemented with 10% fetal calf serum
(FCS). The epithelium and mesenchyme were teased apart, and
any follicular mesenchyme discarded. Isolated dental and non-
dental tissue fragments were then recombined on a polycarbonate
membrane (0.1 µm pore-size, Nuclepore Corp.). The recombina-
tions were cultured for 48 hours in Trowell-type (Trowell, 1954)
cultures in glutamine-supplemented MEM/10% FCS at 37°C. We
have shown previously that these culture conditions are permis-
sive for early tooth initiation at E10 (Vainio et al., 1991), and
tooth-specific cell differentiation after E16 (Thesleff et al., 1989).
Recombination experiments were performed using tissues from
E10, E12, E14, E16 and E18 embryos. These times correspond to
the stages of: induction of the mesenchyme by the epithelium,
mesenchymal condensation, organization of the enamel organ,
morphogenesis and tooth-specific cell differentiation, respectively.
The numbers of recombinations performed are detailed in Table
The tissues were fixed overnight in 4% paraformaldehyde at
4°C, prepared for wax histology and 7 µm serial sections were
dried onto silanized slides.
Preparation of probes and in situ hybridization
In situ hybridization was done as previously described (Wilkin-
A. K. Jowett and others
Table 1. Details of the number of tissue recombinations performed and analysed by in situ hybridization
The number of recombinations between oral tissues is small as the tissues were clearly negative for expression of both genes. Fig. 6 shows a subjective
assessment of the hybridization patterns.
463 msx 1 and msx 2 during tissue interactions
son and Green, 1990). Sense and anti-sense [35S]UTP-labelled
single-stranded RNA probes were synthesized by in vitro tran-
scription of linearized pSP72 vectors containing msx 1 (MacKen-
zie et al., 1991a) and msx 2 (Monaghan et al., 1991) cDNAs. All
transcripts were shortened to around 100 bases by limited alka-
line hydrolysis, purified by gel chromatography and precipitated
with ethanol. Labelling was detected by autoradiography after 10
days exposure at +10°C.
In agreement with previous work (MacKenzie et al., 1991a,
1992), we found msx 1 expression only in the mesenchyme,
and msx 2 expression in both epithelial and mesenchymal
tissues. Hybridization of the control sense probes was min-
imal by comparison with the antisense (data not shown).
In vivo expression of msx 1 and msx 2
To investigate some aspects of the in vivo expression of
msx 1 and msx 2 a limited survey of gene expression during
molar tooth development was undertaken. At E10, the first
sign of tooth development, an epithelium thickening is seen
(Fig. 1A,B). Both msx 1 and msx 2 were expressed in the
mesenchyme adjacent to, but anterior to, this thickening.
There was no expression of msx 1 in the epithelium, but
msx 2 was expressed at a low level in the epithelium from
the tip of the process into the anterior edge of the dental
epithelial thickening (Fig. 1B). There was negligible
expression of either msx 1 or msx 2 at this stage in the ante-
rior region of the second branchial arch (Fig. 1A,B).
MacKenzie et al. (1992) have shown expression in all four
arches; we attribute this difference to our mouse strain
developing faster and, at E10, being equivalent to an E11.5
MFI strain mouse.
MacKenzie et al. (1992) reported that msx 2 expression
in the dental papilla mesenchyme increased during the pro-
gression of the tooth from the bud to the cap stage. We
observed the appearance of strong mesenchyme hybridiz-
ation of msx 2 already at the bud stage, suggesting an induc-
tion at this stage (Fig. 1D). Expression of msx 2 by the
dental mesenchyme was observed at all later stages. Mes-
enchymal msx 2 expression was highest during the late cap
stage and was restricted to the mesenchyme condensed
around or inside the enamel organ (Fig. 2C). By contrast,
msx 1 expression in the mesenchyme was more widespread
and always included the putative follicular mesenchyme
(Fig. 2B). Within the epithelial enamel organ the enamel
knot showed marked expression of msx 2, and this was often
contiguous with a short length of the buccal external enamel
epithelium and internal enamel epithelium (Fig. 2C).
During the late cap and bell stages, we found expression
of msx 1 within all of the dental mesenchyme, and msx 2
in the dental papilla mesenchyme and epithelial enamel
organ, as previously described (MacKenzie et al., 1992).
Dental and non-dental recombinants
The patterns of expression of msx 1 and msx 2 in these
tissue recombinations are summarized in Fig. 6.
Homotypic recombinations of dental epithelium (DE) and
mesenchyme (DM) were made to confirm that under these
conditions the expression of msx 1 and msx 2 was equiva-
lent to that observed in vivo. Fig. 3 shows that after 2 days
of culture DE and DM from teeth at various stages of devel-
opment reorganized and underwent epithelial-mesenchymal
interactions. These interactions were accompanied by
Fig. 1. Expression of the msx 1 (A,C) and msx 2 (B,D) genes in
E10 and E12 mouse, bar 100 µm. (A,B) Parasagittal sections of the
branchial region of an E10 mouse embryo showing the maxillary
(x) and mandibular (d) processes of the first branchial arch. (A)
B r i g h t - field view. msx 1 shows expression in mesenchyme of the
anterior region of both mandibular and maxillary processes with a
decreasing gradient of expression away from the stomodeum.
There is also expression in the mesenchyme adjacent to the fir s t
branchial cleft (c). (B) Dark-field view of an adjacent section
showing msx 2 expression in the same regions although with a
more restricted distribution. msx 2 is also expressed in the anterior
epithelium. (A,B) The epithelial thickening indicating the origin of
the first mandibular molar (arrowed). (C,D) Coronal sections of
upper and lower bud-stage first molar tooth germs in an E12 mouse
embryo. (C) Bright-field view hybridized with the msx 1 p r o b e
indicates marked expression in the mesenchyme around the
epithelial buds. (D) Dark-field view of an adjacent section
hybridized with the msx 2 probe. Expression in the mesenchyme
and buccal external enamel epithelium of the upper tooth and in the
epithelial bud in the lower.
expression of both msx 1 and msx 2. msx 1 was seen in
mesenchyme of all recombinations. In recombinants from
E14 or earlier, it was usual to see a region of the DM which
was msx 1 positive and msx 2 negative (Fig. 3E,F), but at
E16 and E18 stages DM was positive for both msx 1 and
msx 2. The DE was negative for msx 1 in all explants. There
was no expression of msx 2 in the DE in recombinations
of E10 tissues, slight expression adjacent to the mes-
enchyme at E12 (Fig. 3C) and widespread expression in the
DE thereafter (Fig. 3F,I).
Dental mesenchyme and heterotypic epithelium
To ensure that tissues that did not express msx 1 or msx 2
in vivo behaved similarly in vitro, control homotypic
recombinants of non-dental epithelium and mesenchyme
were made. These showed no expression above background
of either gene although an intimate epithelial-mesenchymal
interface was formed (Figs 4A,B, 5A,B). When DM (i.e.
jaw mesenchyme from the presumptive area of the tooth)
from early embryos (E10 and E12) was cultured in isola-
tion, it disaggregated, whereas in explants from older
embryos the DM remained compact and often formed car-
tilage beads. msx 1 and msx 2 were not expressed in iso-
lated mesenchyme except when explanted at E18. At this
stage, the mesenchymal cells maintained expression of msx
1 (but lost expression of msx 2) during 2 days in vitro.
In recombinations of DM with non-dental epithelium
(OE), msx 1 expression was maintained in the mesenchyme
near to the epithelial-mesenchymal interface at stages E10
to E18 (Fig. 4D). This expression was usually restricted to
several cell layers. In these heterotypic recombinations, msx
2 expression was never seen in the mesenchyme, and only
occasionally in OE (at E12, data not shown).
Dental epithelium and heterotypic mesenchyme
Until the bell stage (i.e. E10 to E16), DE induced expression
of msx 1 in the non-dental mesenchyme (Fig. 5D,G,I). This
induction occurred through much of the mesenchyme and
a gradient of expression could be discerned in large pieces
of mesenchyme (Fig. 5D,I). From the density of the silver
grains, it appeared, subjectively, that the inductive ability
of the DE decreased with increasing age (compare Figs 5G
and I). At E18 induction did not occur. It was only at E10
that DE (i.e. first arch epithelium) was able to induce
expression of msx 2 in non-dental mesenchyme (Fig. 5E).
msx 2 expression was not seen in the DE of any of these
Expression studies of msx 1 and msx 2 in vivo confirmed
the previous results of MacKenzie et al. (1992) with the
exception that we observed marked mesenchymal
expression of msx 2 at the late bud and early cap stages.
The transition of expression into the mesenchyme appears
to occur around the time when the mesenchyme becomes
competent to induce odontogenesis (Mina and Kollar,
1989). msx 1 was expressed in both the papillary and fol-
licular mesenchyme in a pattern similar to that for the cell
surface-proteoglycan syndecan (Thesleff et al., 1988;
Vainio et al., 1991). The lack of msx 2 expression by the
follicular cells clearly reflects the diverging differentiation
pathways of the two cell populations. This pattern of
expression is similar to that of int-2, where transcripts are
restricted to the dental papilla (Thesleff et al., 1990 and
unpublished data), but the reverse of IGFII where tran-
scripts are restricted to the dental follicle and around the
dental papilla (Ferguson et al., 1992). Follicular tissue dif-
ferentiates into the progenitor cells of the cementum, peri-
odontal ligament and alveolar bone after the late bell stage
(Palmer and Lumsden, 1987).
A. K. Jowett and others
Fig. 2. (A-C) Coronal sections of an E14 late cap stage molar. Bar 100 µm. (A) Bright-field view. (B) msx 1 expression in both the dental
papilla and dental follicle mesenchyme, no expression in the epithelium. (C) Expression of msx 2 in the dental papilla mesenchyme, in the
epithelial enamel knot (e) and adjacent internal and external enamel epithelia (arrowed).
465msx 1 and msx 2 during tissue interactions
Homotypic recombinations show a normal pattern
Progressive commitment of the dental papilla mesenchyme
is highlighted by the homotypic recombinants. In recombi-
nants of E10 and E12 tissue, the DE was often small com-
pared to the DM. This led to a bare region of DM which
did not express the homeobox genes. At E18, even isolated
mesenchyme continued to express msx 1 showing that by
this stage the cells were independent of epithelial mainte-
nance, although requiring the presence of DE to express
msx 2. At E14, when isolated DM did not express either
msx 1 or msx 2, the size of the DE was often adequate to
cover the DM. All of the mesenchyme expressed msx 1,
but msx 2 was only expressed in regions of the DM that
were enclosed by an enamel organ-like structure. This
implies that the inductive events for msx 1 and msx 2 are
different, with msx 2 expression only occurring over part
of the DE/DM interface. This effect is probably due to the
interaction of relatively uncommitted dental mesenchyme
with different aspects of the cap stage epithelial enamel
organ. The internal enamel epithelium was shown by Ruch
et al. (1982) to be able to stimulate the dental papilla cells
to proliferate, whereas the external enamel epithelium could
not. This effect was believed to be mediated by the spe-
cialized composition of their basement membranes. As the
dental papilla of late cap and early bell staged teeth can
Fig. 3. Homotypic dental recombinants. Bar 100 µm. (A-C) Homotypic recombination of E12 DE and DM. (A) Bright-field view
showing the formation of an epithelial bud. (B) msx 1 transcripts throughout the mesenchyme. (C) Dark-field view of A showing that msx
2 expression is restricted to that part of the epithelial bud adjacent to the mesenchyme. The mesenchyme is negative. (D-F) Homotypic
recombination of E14 DE and DM. (D) Bright-field view showing the formation of early bell stage-like structures. (E) msx 1 transcripts
present in all of the dentally derived mesenchyme both inside and outside the enamel organ, by contrast to msx 2 (F) which is found only
in the enclosed dental papilla mesenchyme (p). All of the enamel organ is positive for msx 2 and negative for msx 1. (G-I) Homotypic
recombination of E18 DE and DM (m). (G) The epithelium adjacent to the mesenchyme is polarized. (H) The mesenchyme is positive for
msx 1. There is no expression within the epithelium. (I) msx 2 is expressed throughout the mesenchyme and epithelium with higher levels
found in the presumptive ameloblast layers.
form both papillary and follicular tissues (Palmer and
Lumsden, 1987), we suggest that the DM adapts to the
nature of epithelium adjacent to it in the recombination and
expresses msx 2 accordingly.
Heterotypic recombinants show stage-specific
As in vivo, the selected non-dental tissues did not clearly
express either of the genes when recombined and cultured.
We observed that both isolated DM and DE became dis-
rupted in vitro and did not express the genes except at E18.
Such dedifferentiation has been described in both oral
(Smith and Hall, 1990) and limb (Coelho et al., 1991) sys-
tems. As non-dental branchial-arch epithelium was capable
of maintaining the expression of msx 1 within the previ-
ously msx 1-expressing DM, we believe that its effect is
permissive and that some epithelial signal is required prior
to final dental commitment. The absence of msx 2
expression in these recombinations suggests that there are
differences in the specificity of epithelial-mesenchymal
interactions. Other experiments confirm the presence of
n o n - s p e c i fic epithelial-mesenchymal interactions. For
example, it is clear that epithelium is important for the
integrity of immature mesenchyme by maintaining the rate
of tissue-specific proliferation (Minkoff, 1991). Addition-
ally, in both in vivo and in vitro experiments the distrib-
ution of expression of syndecan around the developing
tooth (Vainio et al., 1991) is very similar to that of msx 1.
Furthermore, like msx 1, its expression is induced in dental
mesenchyme by non-dental epithelium (Vainio et al.,
1 9 8 9 ) .
Although the dental mesenchyme has been reported to
be able to induce tooth formation in heterotypic epithelium
(Kollar and Baird, 1969; Mina and Kollar, 1987) we did
not see formation of morphologically distinct dental struc-
tures, and only once found msx 2 expression in the non-
dental epithelium (at E12, data not shown). The previous
experiments in which such inductive events were described
utilized reimplantation of the recombinants into host ani-
mals. It is possible that the improved nutritional conditions
were permissive for reorganization and that such reorgani-
zation cannot occur during 2 or 3 days in vitro.
The signaling between the epithelium and mesenchyme
is likely to be partly mediated by diffusible peptide growth
factors. This has been speculated to be the case for the
induction of syndecan expression in early dental mes-
enchyme by the presumptive dental epithelium (Vainio and
Thesleff, 1992). Most probably there are many factors
involved. Some of these may mediate non-tissue-specific
interactions (resulting in expression of, for example, msx 1
and syndecan), whilst others signal tissue-specific fate,
inducing the expression of molecules restricted to deter-
mined cell lineages (for example; msx 2 and Egr-1; Kara-
vanova et al., 1992; and reviewed by Hall and Ekanayake,
The range over which non-dental epithelium could acti-
vate msx 1 in mesenchyme seemed to be less than the dis-
tance for DE in both homotypic DE/DM and in heterotypic
DE/OM recombinations. This could represent an effect due
to different combinations (and/or concentrations) of diffus-
ing inducers interacting with the extracellular environment.
Furthermore, msx 1 expression in the mesenchyme appeared
to be greater when E12 DE was used than when E16 DE
A. K. Jowett and others
Fig. 4. Recombination of dental mesenchyme with non-dental epithelium. Bar 50 µm. See diagrams for types of tissue. (A,B) Negative
control homotypic recombination of E14 palatal epithelium (OE) and mesenchyme (OM). (A) Bright-field view. (B) No expression of
msx 1 in either epithelium or mesenchyme. No expression of msx 2 (not shown). (C,D) Heterotypic recombination of E14 OE (e) and DM
(m). (C) Bright-field view. (D) Expression of msx 1 is restricted to the DM adjacent to the two pieces of epithelium. msx 2 was not
expressed in this recombinant (not shown).
467 msx 1 and msx 2 during tissue interactions
was used. A loss of inductive ability of the dental epithe-
lium was detailed by Mina and Kollar (1987). Although the
DE retained an ability to induce msx 1 expression, the abil-
ity to induce msx 2 was only found at E10. As expression
of msx 2 seems to be a more specific marker for dental
tissues than does msx 1, this finding is in agreement with
Mina and Kollar’s (1987) report that the DE loses its
instructive potential at the bud stage.
Fig. 5. Recombination of dental epithelium with non-dental mesenchyme. Bar 100 µm. See diagrams for types of tissue. (A,B) Negative
control homotypic recombination of E12 diastema-region OM and OE. (A) Bright-field view. (B) No expression of msx 1, or msx 2 (not
shown). (C-E) Heterotypic recombination of E10 2ndarch mesenchyme (OM) with 1starch epithelium (DE) (e). (C) Bright-field view. (D)
msx 1 and (E) msx 2 are expressed in the mesenchyme around the DE. (F,G) Heterotypic recombination of E12 OM and DE. (F) Bright-
field view. (G) msx 1 is expressed at a high level in the OM. No expression of msx 2 (not shown). (H,I) Heterotypic recombination of E15
OM and DE. (H) Bright-field view. (I) msx 1 is expressed in the OM but hybridization is weaker than in Fig. 5G, showing how the
inductive potential of the DE may decrease with the age of the DE.
In the hindbrain, expression of the Antennapedia-like home-
obox genes first occurs in the neuroepithelium and is main-
tained in the neural crest cells as they migrate into the
caudal branchial arches. The neural crest cells are then able
to induce expression of the same homeobox genes in the
overlying ectoderm (Hunt et al., 1991). As msx 1 and msx
2 are expressed in the neural folds and adjacent crest cells,
it is possible that a similar mechanism is responsible for
the expression of msh-like genes in the mesenchyme and
ectoderm of the first branchial arch and facial processes,
creating the pattern of expression observed by MacKenzie
et al. (1991a, 1992) at E9.
Expression of the msh-like genes is found in the ver-
tebrate eye (Monaghan et al., 1991), heart and limb (Robert
et al., 1991; Suzuki et al., 1991). Within these tissues, which
develop by epithelial-mesenchymal interactions, msx 1 and
msx 2 often show complementary expression in the tissues,
suggesting that they may be involved in regulating inter-
actions. The concept of two phases of msh-like homeobox
gene expression is most clear in the chicken limb. In the
early limb bud (H&H stage 17), the chicken homologue of
msx 1 (GHox-7) is expressed throughout the mesoderm and
GHox-8 expressed most intensely anteriorly (Coelho et al.,
1991). This expression is independent of epithelial-mes-
enchymal interactions. However, later in development
(H&H stage 20 onwards), expression of both genes requires
tissue interactions involving the specialized apical ectoder-
mal ridge epithelium (Coelho et al., 1991; Robert et al.,
1991). This repeating expression pattern during epithelial-
mesenchymal organogenesis has also been described by
Suzuki et al. (1991). Indeed, in Aves, it now appears clear
that the second stage of expression of GHox-7, in chicken
limb mesoderm (Coelho et al., 1991), and Quox-7, in quail
branchial arch mesenchyme, are dependent upon the pres-
ence of competent epithelium. Coelho et al. (1991) further
speculate that expression of GHox-8 by the epithelium is
essential for normal mesoderm homeobox gene activation.
From our data it also appears that localized expression of
the msx 1 and msx 2 genes in the developing tooth is reg-
ulated by local epithelial-mesenchymal interactions.
Interestingly, members of the recently identified Dlx
homeobox gene family (homologous to Drosophila distal-
less gene) show a similar pattern of distribution to the msh-
like genes in both facial and limb tissues (Dollé et al.,
1992). Like the msh-like genes, the Dlx homeobox genes
are present only as single copies in insects, but amplified
in vertebrates. Gaunt (1991) suggests that duplication of
Hox families may be related to the evolution of new sub-
sets of tissues and organs.
A. K. Jowett and others
msx 1 msx 2
Fig. 6. Diagrammatic summary of msx 1 and msx 2 expression in dental and non-dental tissue recombinants. The four possible
permutations (OM/OE, OM/DE, DM/OE, DM/DE) are shown as idealized recombinations. Epithelium is shown in yellow and
mesenchyme in blue, the dental components are shown with a more dense colour. Specific hybridization of msx 1 and msx 2 (by
comparing the antisense and sense probes) is indicated by red and green, respectively. For example, at E16, in a recombination of non-
dental epithelium (pale yellow) with dental mesenchyme (mid blue), msx 1 (red) was expressed by the mesenchyme adjacent to the
469msx 1 and msx 2 during tissue interactions
Expression of msx 2 by the dental mesenchyme appears to
be indicative of a dental phenotype. This is suggested by
its pattern of in vivo localization and its appearance in tissue
recombinants. Early dental epithelium is capable of induc-
ing msx 2 in mesenchyme, which then gains some induc-
tive potential. Expression of msx 1 by the dental mes-
enchyme also requires epithelium. Until the late cap stage,
the dental epithelium can induce msx 1, but not msx 2,
expression in non-dental mesenchyme. We conclude that
expression of msx 1 in mesenchyme can be induced by a
relatively non-specific epithelial signal, whereas msx 2
expression in both epithelium and mesenchyme requires
reciprocal interactions between specialized dental cell pop-
The similarity of expression patterns of msx 1 and msx
2 in murine dental tissues (our data), the eye (Monaghan
et al., 1991) and in chick limb (Coelho et al., 1991; Robert
et al., 1991) tissues, and the absence of clustering (cf. Antp-
like genes; Gaunt, 1991), leads us to suggest that msx 1 and
msx 2 are involved in epithelial-mesenchymal interaction-
controlled organogenesis, rather than positional specifica-
tion, and that msx 2 is expressed by those cells possessing
the organizing potential.
We thank Dr R. Hill of the MRC Human Cytogenetics Unit,
Edinburgh for the msx 2 probe. The skillful technical assistance
of Ms Merja Mäkinen and Ms Riikka Santalahti is gratefully
acknowledged. This work was supported by grants from The
Finnish Academy, the Sigrid Juselius Foundation and by NIH
grant DE09399. A. K. J. was a Royal Society European Exchange
Fellow funded by The Finnish Academy.
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(Accepted 19 October 1992)
A. K. Jowett and others