Development ePress online publication date 16 December 2004
Research article 323
Coordination of trigeminal axon navigation and patterning with
tooth organ formation: epithelial-mesenchymal interactions, and
epithelial Wnt4 and Tgfβ1 regulate semaphorin 3a expression in the
Päivi Kettunen1, Sigbjørn Løes1, Tomasz Furmanek1, Karianne Fjeld1, Inger Hals Kvinnsland1, Oded Behar2,
Takeshi Yagi3, Hajime Fujisawa4, Seppo Vainio5, Masahiko Taniguchi6 and Keijo Luukko1,*
Division of Anatomy and Cell Biology, Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway
Department of Experimental Medicine and Cancer Research, The Hebrew University, Jerusalem, 91120, Israel
Laboratories of Integrated Biology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita,
Osaka 565-0871, Japan
Group of Developmental Neurobiology, Division of Biological Science, Nagoya University Graduate School of Science,
Chikusa-ku, Nagoya, 464-8602, Japan
Biocenter Oulu and Department of Biochemistry, Faculties of Science and Medicine, University of Oulu, 90014, Finland
Department of Biochemistry and Molecular Biology, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku,
Tokyo, 113-0033, Japan
*Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 20 October 2004
Development 132, 323-334
Published by The Company of Biologists 2005
During development, trigeminal nerve ﬁbers navigate and epithelial Wnt4 and Tgfβ1 regulate Sema3a expression in
establish their axonal projections to the developing tooth in the dental mesenchyme. In addition, Wnt4 stimulates
a highly spatiotemporally controlled manner. By analyzing mesenchymal expression of Msx1 transcription factor,
Sema3a and its receptor Npn1 knockout mouse embryos, which is essential for tooth formation, and Tgfβ1
we found that Sema3a regulates dental trigeminal axon proliferation of the dental mesenchymal cells. Thus,
navigation and patterning, as well as the timing of the ﬁrst epithelial-mesenchymal interactions control Sema3a
mandibular molar innervation, and that the effects of expression and may coordinate axon navigation and
Sema3a appear to be mediated by Npn1 present in the patterning with tooth formation. Moreover, our results
axons. By performing tissue recombinant experiments and suggest that the odontogenic epithelium possesses the
analyzing the effects of signaling molecules, we found that instructive information to control the formation of tooth
early oral and dental epithelia, which instruct tooth nerve supply.
formation, and epithelial Wnt4 induce Sema3a expression
in the presumptive dental mesenchyme before the arrival Key words: Odontogenesis, Tissue interactions, Tooth, Axon growth,
of the ﬁrst dental nerve ﬁbers. Later, at the bud stage, Mouse
Introduction the molecular mechanisms of organ formation. The teeth
During development of the peripheral nervous system, growing develop in the oral side of the maxillary and mandibular
axons navigate and establish connections to their developing processes, and their formation is regulated by sequential and
target organs. Regulation of axon growth involves coordinated reciprocal interactions between the odontogenic epithelium
activity of diffusible and local contact-mediated attractive and and neural crest derived ectomesenchymal cells (Miletich and
repulsive guiding cues including members of for example the Sharpe, 2003; Thesleff, 2003). Signaling molecules have
netrin, Slit, ephrin and semaphorin families (Dickson, 2002; been shown to mediate inductive tissue interactions during
Tessier-Lavigne and Goodman, 1996; Varela-Echavarria and odontogenesis. In particular, early oral epithelium- and oral
Guthrie, 1997). Many of the guidance molecules show epithelium-expressed signaling molecules regulate dental
dynamic and restricted expression patterns in peripheral tissues mesenchymal expression of signaling genes and transcription
and organs that correlate with regulation of axon growth. factors that are essential for tooth formation (Miletich and
However, how the expression of guidance molecules is Sharpe, 2003; Thesleff, 2003). Trigeminal axon pathﬁnding
regulated during organ formation, and how axon navigation and nerve ﬁber patterning, in particular in the murine lower
and patterning is coordinated spatiotemporally with organ ﬁrst molar, takes place in a strictly spatiotemporally controlled
formation has remained largely unknown. manner and is tightly linked to tooth formation (Loes et al.,
The developing tooth is a useful model in which to analyze 2002; Luukko et al., 1997b; Mohamed and Atkinson, 1983).
324 Development 132 (2) Research article
The early developing tooth is innervated by nerve ﬁbers Medical Faculty of the University of Bergen under the surveillance of
originating from the sensory trigeminal ganglion. Because new the Norwegian Animal Research Authority. Production of the Sema3a
axons do not emerge from the trigeminal ganglia after E13 and Npn1 mutant mice strains and genotyping has been described
(Davies, 1988), axon navigation and their survival around the previously (Behar et al., 1996; Kitsukawa et al., 1997; Taniguchi et
embryonic tooth germ during the period of programmed cell al., 1997). The transgenic and NMRI mice were mated overnight
and the appearance of the vaginal plug was taken as day E0.5
death (E13-E18) (Davies, 1988) are essential for the proper of embryogenesis. Tissues were processed further for different
development of the sensory innervation of the dental pulp and analyses as described previously (Luukko et al., 1996; Kettunen and
periodontal ligament (Luukko et al., 1997a). The sympathetic Thesleff, 1998). Preparation of postnatal tissues for PGP9.5
innervation of the tooth develops postnatally after the onset of immunohistochemistry was performed as described previously
root formation (Fristad et al., 1994). (Fristad et al., 1994). NMRI mouse embryos were used for organ
Some light has been shed on the molecular mechanisms that culture and tissue recombination experiments. Photographs were
regulate the development of tooth nerve supply. It appears that taken with a Coolpix 4500 digital camera (Nikon Corporation, Tokyo,
this process is regulated by set of neuroregulatory molecules Japan) and ﬁgures were processed using Adobe Photoshop software
of different families (for a review, see Fried et al., 2000; (Adobe Systems, San Jose, CA, USA).
Luukko, 1998). However, as yet, no gene has been shown to Antibodies and immunohistochemistry
be essential for pioneer dental axon guidance or the To detect nerve ﬁbers in paraffin sections, immunohistochemistry with
establishment of early tooth innervation. The ﬁnding that the rabbit polyclonal anti-peripherin (Chemicon International, CA, USA)
developing tooth is able to promote its reinnervation when and neuropilin 1 antibodies (Kawakami et al., 1996) (1:150 and
implanted in ectopic locations (Erdelyi et al., 1987) and that 1:1000 dilution) was carried out using the Vectastain pK4001
the expression of neurotrophins and their receptors persists in kit (Vector Laboratories, Burlingame, CA) according to the
vitro, without peripheral nerve ﬁbers cultured tooth explants manufacturer’s instructions (Luukko, 1997; Fristad et al., 1994).
has suggested that the developing tooth is able to control the Negative control sections were incubated with normal rabbit serum
formation of its own innervation and that the synthesis of the instead of the primary antibody. No speciﬁc immunoreactivity was
neuroregulatory molecules is regulated locally and is an detected.
intrinsic property of the tooth germ (Luukko et al., 1996; In situ hybridization
Luukko et al., 1997a). For in situ hybridization on sections the 0.6 kb rat Gdnf, 0.1 kb rat
We have recently reported developmentally regulated Lanr, 0.9 kb mouse Ncam, 0.4 kb rat Ngf and 2.9 kb mouse Sema3a
mRNA expression of semaphorin 3a (Sema3a) in the cDNAs were used for in vitro transcription of 35S-UTP- and
developing tooth in sites that are devoid of nerve ﬁbers, digoxigenin-labeled and antisense and sense probes. Sectional and
suggesting functions in dental axon guidance and tooth whole mount in situ hybridization was performed as described
formation (Loes et al., 2001). Sema3a, a secreted repulsive previously (Luukko et al., 1996; Henrique et al., 1995). No speciﬁc
axon guidance molecule, shows broad developmentally hybridization signals were detected in tissues hybridized with control
regulated expression in the peripheral tissues and organs of the sense probes (not shown).
embryo including the ﬁrst branchial arch (BA1) and tooth Three-dimensional reconstruction
(Taniguchi et al., 1997; Wright et al., 1995). Targeted
Three-dimensional (3D) computer reconstruction of the tooth germ
inactivation of the Sema3a gene leads to abnormal was generated from 7 µm serial frontal bright- and dark-ﬁeld
fasciculation and patterning of a set of peripheral nerves, phototomicrographs (180 sections from each ﬁeld). The processing of
including the cranial trigeminal, facial and glossopharyngeal the images was done using custom scripts and programs written with
nerves, indicating the importance of Sema3a in the Java Advanced Imaging and Java 3D (Sun Microsystems, CA,
establishment of axonal trajectories (Taniguchi et al., 1997; USA) (http://java.sun.com). Three-dimensional reconstructions were
Ulupinar et al., 1999). In addition, Sema3a serves an rendered with a perspective camera view in Visualization Toolkit
organogenetic function, e.g. in bone and heart formation (Kitware, New York, USA) (http://www.kitware.com). A transparent
(Behar et al., 1996), and controls vascular morphogenesis by 3D surface of the inner dental epithelium was generated using the
inhibiting integrin function (Serini et al., 2003). In the current Marching Cubes function in Visualization Toolkit from the outlines
study, we analyzed the functions and regulation of Sema3a of the dental epithelium and the cervical loops. A hybridization signal
in the dark-ﬁeld images with an intensity over 230 was considered to
during early, crucial stages of tooth organogenesis and the represent positive Sema3a gene expression. The sections were median
formation of its trigeminal nerve supply. We found that Sema3a ﬁltered to reduce the background hybridization signal in the 3D
is an essential signal for proper tooth innervation, and that the image.
oral epithelium and dental epithelium expressed Wnt4 induce
Sema3a in the mandibular presumptive molar mesenchyme Organ and tissue culture, recombinant proteins and cell
area before the arrival of ﬁrst nerve ﬁbers. Later, during pioneer lines
dental axon navigation, Wnt4 and Tgfβ1 control Sema3a Organ and tissue cultures were performed as described earlier
expression in the dental mesenchyme. Thus, epithelial- (Kettunen et al., 1998). Explants shown are representatives of at least
mesenchymal interactions may provide a central mechanism three independent experiments. At least six explants of each
for coordination of axon navigation and patterning with the experimental setup were analyzed. Agarose (BioRad) and heparin
acrylic (Sigma) were used. Beads were incubated in recombinant
mandibular process and tooth formation. human FGF2 (100 µg/ml) and FGF4 (50 or 100 µg/ml); mouse Fgf8b
(50 or 100 µg/ml); human FGF9 (25 or 100 µg/ml); human TGFβ1
Materials and methods (10 or 100 µg/ml); mouse Shh (250 µg/ml); human BMP4 (100 µg/ml)
(R&D Systems, Minneapolis, MN); or in BSA (1 mg/ml). All bead
Preparation of tissues experiments were accompanied by positive controls to conﬁrm the
Animal use was approved by the Department of Biomedicine of the activity of the proteins used (Fig. 6, G1-I2, P1-Q2; Fig. 7I).
Regulation of Sema3a by tissue interactions 325
The Wnt4 and Wnt6 cell lines have been described previously had diverged into the buccal and shorter lingual branches
(Kettunen et al., 2000). Quantitative comparison of Wnt protein levels (Fig. 1A3,B3). At the cap stage (E14.5), nerve ﬁbers extended
was not performed, but an induction of kidney tubule formation by to the buccal side of the tooth germ and were located in the
Wnt4- and Wnt6-producing cells in E11 metanephric mesenchyme area between the Sema3a-expressing mesenchymal dental
explants was used as a positive control in each experiment to conﬁrm follicle and forming alveolar bone (Fig. 1A4,B4). Later
the activity of the Wnt proteins. Wnt4 and Wnt6 cell clusters, which
were approximately same size had similar tubulogenetic effects on
during the bell stage (E16.5 and E18.5) nerve ﬁbers were
E11 metanephric mesenchyme (Fig. 7G,H) (Kispert et al., 1998). No located in the mesenchymal dental follicle target ﬁeld
effects were observed in control NIH3T3 cells. between Sema3a-expressing outer dental epithelium and
alveolar bone, as well as in the base of the dental papilla
Cell proliferation assay mesenchyme between the epithelial cervical loops, which
The effect of exogenous proteins on dental mesenchymal cell also expressed transcripts as shown for the E18 tooth (Fig.
proliferation was analyzed as described earlier (Kettunen et al., 1998). 1A5,B5). Thus, Sema3a is expressed in sites that harbor the
The explants were labeled for 1.5 hours with 10 mM BrdU (Sigma) mesenchymal dental axon pathway and target ﬁeld around the
after 24 hours’ culture. The incorporated BrdU was detected by developing tooth.
indirect immunoperoxidase method with monoclonal antibody against
BrdU (Sigma, CA) and the biotinylated goat
anti-mouse secondary antibody (Jackson
ImmunoResearch Laboratories, West Grove, PA,
USA) in wholemounts and tissue sections.
Sema3a is absent from the
mesenchymal dental axon pathway
and dental follicle target ﬁeld
To address possible neuronal functions of
Sema3a we ﬁrst compared its mRNA
expression with the localization of nerve
ﬁbers in the embryonic mouse mandibular
ﬁrst molar tooth germ using peripherin
antibodies from the serial paraffin sections.
Sema3a was ﬁrst observed in the presumptive
dental mesenchyme between the thickened
dental epithelium and buccal nerve at E11.5
(Fig. 1A1,B1). At the early bud stage (E12.5)
Sema3a expression was observed in the
condensing dental and deep jaw mesenchyme
(Fig. 1A2) adjacent to the trigeminal nerve
branch, the ‘molar nerve’, which had
extended from the inferior alveolar nerve next
to the tooth germ (Fig. 1B2). At the bud stage,
Sema3a continued in the condensed dental
mesenchyme under which the molar nerve
Fig. 1. Sema3a regulates the timing of tooth
innervation and dental axon guidance and
patterning. Expression of Sema3a in the embryonic
mandibular molar tooth germ (A1-A5) at the
epithelial thickening (E11.5) (A1), early bud
(E12.5) (A2), bud (E13.5) (A3), cap (E14.5) (A4)
and bell (E18.5) (A5) stages compared with the
localization of nerve ﬁbers in corresponding stages
of wild-type (Sema3a+/+) (B1-B5) and Sema3a null
mouse embryos (Sema3a–/–) (C1-C5) using
peripherin antibodies. (A1-A5) The ‘molar nerve’
is indicated by arrows in B2. b, developing alveolar
bone; cdm, condensing dental mesenchyme; cm,
condensed dental mesenchyme; de, dental
epithelium; dp, dental papilla mesenchyme; df,
dental follicle; tn, trigeminal inferior alveolar
nerve; pm, presumptive dental mesenchyme.
Arrowheads mark nerve ﬁbers. Scale bars: 100 µm.
326 Development 132 (2) Research article
Fig. 2. Npn1 mediates Sema3a signaling in dental axons.
Frontal sections of E13.5 trigeminal ganglia (A,B) and
tooth germs (C,D). Bright- (A1,B1) and dark-ﬁeld
(A2,B2) images. (A1-B2) A prominent Npn1 expression
is seen in the trigeminal ganglion, while no speciﬁc
expression of Npn2 is observed. (C) Npn1
immunoreactivity is seen in the dental nerve ﬁbers
(arrows) next to the condensed dental mesenchyme (dm)
and in the trigeminal superior alveolar nerve trunk (sn).
(D) E12.5 Npn1–/– heads analyzed by peripherin
antibodies show that ectopic nerve ﬁbers are prematurely
present next to the dental epithelium and in the area of
the condensing dental mesenchyme (arrows). Trigeminal
inferior (in) and superior alveolar nerve trunks are
abnormally defasciculated. Scale bars: 100 µm.
dental axons, we ﬁrst analyzed its mRNA expression
in trigeminal sensory ganglion. In situ hybridization
revealed a prominent Npn1 expression in the
ganglion cells during E12.5-E14.5, i.e. during the
period when the dental axons are navigating to and
around the tooth germ as shown for E13.5 ganglion,
while hardly any neuropilin 2 (Bagri and Tessier-
Lavigne, 2002) expression was seen (Fig. 2A1-B2).
Immunohistochemical analysis from the E12.5-
E13.5 head sections using Npn1 antibodies showed
that dental axons expressed the protein (Fig. 2C),
which is in agreement with the recent report showing
Npn1 in dental axons at E15 (Lillesaar and Fried,
Sema3a regulates timing and pattering of tooth 2004). To investigate the in vivo roles of Npn1 in tooth, we
innervation analyzed Npn1 mutant mouse embryos (Kitsukawa et al.,
To investigate whether Sema3a regulates dental axon guidance, 1997). Apparently owing to the defects in the cardiovascular
we studied the localization of nerve ﬁbers in tooth of the system, the embryos die at about E12.5 when the molar tooth
Sema3a mutant mouse embryos (Taniguchi et al., 1997). germ is at the early bud stage. No defects in tooth formation
Ectopic nerve ﬁbers were found prematurely in the in the E11.5-E12.5 mutant embryos were detected compared
mesenchyme next to the dental epithelium at the epithelial to corresponding wild-type embryos. Immunohistochemical
thickening (E11.5) and early bud stage (E12.5) (Fig. 1C1,C2). analysis of 12.5 Npn1–/– embryos revealed that nerve ﬁbers
At the bud stage (E13.5), axons were ectopically present in the were prematurely present next to the dental epithelium, and
condensed dental mesenchyme, and some had reached the some were also ectopically localized in the condensing dental
epithelial bud (Fig. 1C3). Although many nerve ﬁbers showed mesenchyme area (Fig. 2D). This phenotype resembled the
largely proper localization in the mesenchymal dental follicle defects observed in corresponding stages of Sema3a–/–
target ﬁeld around the cap and bell stage tooth germs (E14.5, embryos but appeared to be less severe.
E16, E18.5), disoriented nerve ﬁbers were present next to the
outer dental epithelium and in the dental papilla mesenchyme Sema3a expression in the postnatal tooth
(Fig. 1C4-C5). No defects were observed in tooth formation in The ﬁrst trigeminal nerve ﬁbers from the dental follicle
the studied Sema3a–/– embryos (Fig. 1C1-C5) or in embryos of penetrate the dental papilla (future dental pulp) of the mouse
another Sema3a mutant mouse strain (Behar et al., 1996), mandibular ﬁrst molar at about three days postnatally (P3)
which suffers from non-neuronal defects and dies after birth when the shape of the tooth crown is largely ready, crown
(not shown). Thus, Sema3a regulates the timing of tooth calciﬁcation has started, and the formation of the mesial and
innervation as well as dental axon navigation and patterning distal roots, which attach the tooth to the alveolar bone, starts
but is not needed for early tooth organ formation. Moreover, (Mohamed and Atkinson, 1983). Closer observation of E18
Sema3a is the ﬁrst signaling molecule shown to be necessary teeth revealed that Sema3a was expressed in the middle part of
for the development of tooth innervation. the base of the dental papilla mesenchyme, while the mesial
and distal regions were mostly devoid of transcripts. In situ
Npn1 mutant mice show defects in axon navigation hybridization analysis from the sagittal sections and 3D-
The receptor neuropilin 1 (Npn1) mediates Sema3a signaling reconstruction of PN1 tooth conﬁrmed Sema3a expression in
by forming receptor complexes with plexins (Bagri and the area of the future pulp ﬂoor and mesenchymal cells
Tessier-Lavigne, 2002). Experimental and genetic analyses adjacent to epithelial cervical loops (Fig. 3A1,A2,B). Later, at
have provided evidence that Npn1 mediates in vivo effects of P4, Sema3a was expressed in preodontoblasts next to the
Sema3a on trigeminal axons during their pathﬁnding epithelial cells of the pulp ﬂoor as well as next to the epithelial
(Kitsukawa et al., 1997; Rochlin and Farbman, 1998). To root sheaths, which form the mesial and distal roots of the
analyze whether Npn1 is a signaling receptor for Sema3a in molar (Fig. 2C1-C2). Thus, Sema3a-free areas mark the sites
Regulation of Sema3a by tissue interactions 327
Fig. 3. Expression of Sema3a in 1- (PN1) and 4-day postnatal (PN4) ﬁrst (I), second (II) and third (III) lower molar tooth germs. Bright-
(A1,C1) and dark-ﬁeld (A2,C2) images of sagittal sections as well as (B) three-dimensional reconstruction of Sema3a (in red) expression in
PN1 ﬁrst molar tooth germ shown from below. Sema3a is absent from the sites of the secondary anterior (mesial) and posterior (distal)
secondary apical foramina. The inner dental epithelium (gray) is 30% transparent to better visualize Sema3a expression in the dental papilla
around the secondary apical foramina. Three-dimensional reconstruction was rendered using a perspective camera view. Scale bars: 200 µm.
White arrows indicate Sema3a expression in dark-ﬁeld images. a, developing alveolar bone; cl, cervical loop; d, dentin; e, enamel; dp, dental
papilla; ode, outer dental epithelium; p, developing pulp ﬂoor; sr, stellate reticulum.
of the secondary apical foramina through which the sensory Qian and Naftel, 1996), is expressed in the mesenchymal axon
and sympathetic nerve ﬁbers enter the dental pulp. However, pathway, and, later, together with glial cell line-derived
most probably owing to the fact that ectopic nerve ﬁbers were neurotrophic factor (Gdnf), in the dental follicle target area
present in the dental papilla of Sema3a mutant mice already around the tooth germ (Luukko et al., 1997a; Luukko et al.,
during embryonic development (see Fig. 1C1-C5), we were not 1997c; Mitsiadis et al., 1992; Nosrat et al., 1998). Similarly,
able to observe obvious defects in timing or patterning of the neural cell adhesion molecule (Ncam) and low-affinity
dental papilla innervation, or in tooth formation in Sema3a neurotrophin receptor (Lanr), which may sequester
mutant molars at P5 when the ﬁrst nerve ﬁbers are normally neurotrophins, are present in the dental follicle target ﬁeld
present in the dental pulp from tissue sections using PGP9.5 during axon navigation (Luukko et al., 1996; Mitsiadis et al.,
antibodies (not shown). 1992; Obara and Takeda, 1993). In addition, mRNAs for netrin
3 (Net3) are expressed in the mesenchymal axon pathway and
Ngf, Gdnf, Lanr, Ncam and Net3 expression is not dental follicle region and may therefore be involved in the
altered in Sema3a mutant tooth regulation of dental axon growth (Loes et al., 2003). In situ
The ﬁnding that some of the nerve ﬁbers showed normal hybridization analysis of the late bud/early stage tooth germs
localization within the tooth target ﬁeld in Sema3a–/– mice, in (E14–) revealed no differences between the expression patterns
particular at the later morphogenetic cap and bell stages, of Ngf, Gdnf, Lanr, Ncam and Net3 in Sema3a–/– and wild-type
suggested that other neuroregulatory molecules might guide mice (Fig. 4A1-J2).
dental axons and partially rescue the tooth innervation
phenotype in Sema3a mutant embryos. To study this, we Epithelial-mesenchymal interactions regulate
analyzed mRNA expression of molecules implicated in dental Sema3a expression
axon guidance and patterning. Nerve growth factor (Ngf), The observation that Sema3a mRNAs appeared in the
which is essential for tooth innervation (Matsuo et al., 2001; presumptive dental mesenchyme at E11.5 under the thickened
328 Development 132 (2) Research article
dental epithelium before the
arrival of the pioneer dental
nerve ﬁbers suggested that
Sema3a expression in tooth is
not controlled by growing
axons but by a local
mechanism. Tissue separation
and recombination studies
have demonstrated that tooth
formation is regulated
interactions (Lumsden, 1988;
Mina and Kollar, 1987). To
analyze whether tissue
expression, we ﬁrst separated
epithelial and mesenchymal
tissue components from
E10.5 and E11.5 mandibular
processes, as well as E11 and
E12 molar tooth germs,
and cultured the isolated
mesenchymes in a Trowell-
type culture for 24 hours.
The explants were serially
sectioned and analyzed by
in situ hybridization. The
removal of the epithelium
resulted in a loss of almost if
not all Sema3a expression in
the E10.5 mandibles, while in
the intact cultured mandibles
Sema3a expression persisted
(Fig. 5B1-C2). In E11
explants, some Sema3a
transcripts persisted in the
proximal deep aboral part,
whereas little if any
endogenous Sema3a was
found in the presumptive
molar mesenchyme area (Fig.
5F1,F2), as also conﬁrmed
by culturing isolated
E11 presumptive molar
mesenchyme explants alone
Fig. 4. Ngf, Lanr, Gdnf, Ncam and Net3 expression is not altered in the Sema3a–/– tooth. Bright- and dark-
(not shown). At E12, ﬁeld images of frontal sections of the E14- late bud/early cap stage wild-type (A1-A2,C1-C2,E1-E2,G1-
however, more endogenous G2,I1-I2) and Sema3a mutant (B1-B2,D1-D2,F1-F2,H1-H2,J1-J2) upper and lower ﬁrst molar tooth
expression of Sema3a germs. Abbreviations: de, dental epithelium; dp, dental papilla. Scale bar: 100 µm.
persisted in the isolated
cultured dental mesenchymes
(Fig. 5J1,J2), while transcripts were not observed in the E12 dental epithelia, onto the proximal presumptive molar area of the
dental epithelia (not shown). Thus, Sema3a expression in the E10.5 mandibular mesenchyme, which is devoid of Sema3a
E10.5 mandibular mesenchymes and E11 presumptive dental (Fig. 5A1,A2). In situ hybridization revealed Sema3a expression
mesenchyme is dependent on epithelial signaling. in the mesenchyme adjacent to E10.5 oral and E11 dental
To speciﬁcally address whether Sema3a expression in the epithelium after 24 hours and 2 days culture respectively
oral side of the E11.5 proximal jaw mesenchyme containing (Fig. 5D1,D2,G1,G2). Similarly, a prominent expression was
the presumptive dental mesenchyme area is induced by the observed in the mesenchyme next to the E12 dental epithelium,
overlying epithelium, we placed E10.5 oral epithelium, which which had reached the bud stage after 3 days culture (Fig.
includes presumptive dental epithelium as well as E11 and E12 5L1,L2). In addition, in homochronic E11 and E12 tooth
Regulation of Sema3a by tissue interactions 329
Fig. 5. Epithelial-mesenchymal
interactions regulate Sema3a
expression in the mandibular
and dental mesenchyme.
Expression of Sema3a in the
E10.5 head (A1,A2) and E11.5
(E1,E2) and E12.5 (I1-I2)
mandibular molar tooth germs,
as well as in cultured
mandibular and dental
mesenchymes and tissue
recombinants analyzed by in
situ hybridization from frontal
sections after 24 hours (B1-
2 days (G1-G2) and 3 days (L1-
L2) of culture. Bright- and
dark-ﬁeld images. (B1-B2) No
speciﬁc Sema3a expression is
seen in the E10.5 mandibular
mesenchyme cultured alone
without epithelium (compare
also images in Fig. 5J1-L2
cultured with BSA-soaked
beads and Wnt6 and NIH3T3
cells), while in the cultured
intact mandible a prominent
expression is present (C1-C2).
(D1-D2) E10.5 oral epithelium
has induced Sema3a in the
underlying E10.5 molar area of
which is devoid of Sema3a (arrows in A1). (F1,F2) In cultured E11 mandibular mesenchyme, Sema3a transcripts are largely absent from the
presumptive molar area (arrow), whereas the remaining Sema3a is seen in the deep aboral mesenchyme. (G1-G2,L1-L2) Sema3a induction is
observed next to the E11 and E12 dental epithelia, which were placed onto the proximal, molar region of E10.5 mandibular mesenchyme. (H1-
K2) A prominent Sema3a expression is seen in the E11 jaw and E12 dental mesenchyme under the E11 and E12 dental epithelia, respectively,
while some endogenous Sema3a is observed in the dental mesenchyme explant. The expression patterns of Sema3a in these homochronic
recombinant explants correlate with the Sema3a expression in in vivo developed teeth (compare E1,E2 with I1,I2). de, dental epithelium; dm,
dental mesenchyme; m, mandibular mesenchyme; Md, mandibular process, Mx, maxillary process; oe, oral epithelium. Scale bars: 100 µm
(200 µm for D2).
recombinants, which were cultured for 24 hours, Sema3a Fgf8 mRNAs are expressed in the E10.5 proximal oral
expression in the mesenchyme correlated to the expression in epithelium and later in the early dental epithelium (Kettunen
in vivo developed teeth (compare Fig. 5E1,E2,I1-I2,H1- and Thesleff, 1998) and are essential for the outgrowth and
H2,K1,K2). Taken together, our results indicate that patterning of the BA1 and for tooth formation (Trumpp et al.,
odontogenic epithelium induces Sema3a into the presumptive 1999). As reported earlier (Neubuser et al., 1997), Fgf8
dental mesenchyme, and later the dental epithelium continues to induced a prominent Pax9 expression in the presumptive molar
control Sema3a expression domains in the dental mesenchyme. area of E10 mandibular processes, whereas no Sema3a
induction was observed around the Fgf8 beads (Fig. 6A1-B2).
Epithelial Wnt4 and Tgfβ1 induces mesenchymal Similarly, beads soaked in Bmp4, Fgf9 and Shh, mRNAs of
Sema3a expression which are expressed in the presumptive and early dental
Early oral and dental epithelium expressed signals in particular epithelium (Hardcastle et al., 1998; Kettunen and Thesleff,
members of the ﬁbroblast growth factor, bone morphogenetic 1998; Vainio et al., 1993), had no effect on Sema3a expression
protein, Wnt and Hedgehog families control gene expression in the molar area of E10.5 or E12.5 mandibular mesenchymes
in the underlying mesenchyme (Miletich and Sharpe, 2003; as shown for Fgf9 at E12.5 (Fig. 6N1,N2; not shown).
Thesleff, 2003). To identify the epithelial signal(s) that Furthermore, protein-soaked beads for the enamel knot
induce(s) and regulate(s) Sema3a expression, we applied expressed Fgf4 (Jernvall et al., 1994) did not have effects on
protein-soaked beads and protein-producing cells of different Sema3a expression in the molar region of E12 mandibular
members of these families to the mandibular and dental mesenchyme explants (not shown).
mesenchyme explants and cultured them for 24 hours The ﬁnding that the E10.5 oral epithelium was able to induce
(Kettunen and Thesleff, 1998). The explants were serially Sema3a suggested that the expression of the putative signaling
sectioned and analyzed for Sema3a expression by in situ molecule(s), which induce(s) the initial mesenchymal Sema3a
hybridization. expression, is or are not limited to the presumptive dental
330 Development 132 (2) Research article
Fig. 6. Wnt4 and Tgfβ1 induce Sema3a in the mandibular and dental mesenchymes. Bright- and dark-ﬁeld images. (A1-B2) Fgf8 induces Pax9
in the presumptive molar mesenchyme at E10, while no effects on Sema3a expression are observed. (C1-E2) A prominent Sema3a expression is
seen around the Wnt4-producing cells in the E10.5 and E11 mandibular mesenchyme and E12 dental mesenchyme explants. (F1,F2)
Endogenous expression of Sema3a in E12.5 dental mesenchyme explants. (G1,G2) Tgfβ1-releasing bead (arrow indicates the site where the
bead was located) stimulates Sema3a expression in the dental mesenchyme area of E12.5 lower jaw mesenchyme explants. (J1-K2) Wnt4 cells
stimulate Msx1 in the proximal presumptive molar mesenchyme at E10.5, while no expression is observed around the NIH3T3 cells. (L1-P2)
No Sema3a stimulation is seen in the mesenchyme surrounding the Wnt6-producing or control NIH3T3 cells or around the Fgf9- or BSA-
soaked beads. Wnt6 cells show some endogenous Sema3a hybridization signal. (H1-I2,Q1-Q2) Bmp4, Fgf4 and Fgf9 induce the expression of
target genes in control explants. Scale bars: 100 µm. c, cell cluster.
epithelium. The Wnts form a large family of conserved presumptive molar mesenchyme area of E10.5 and E11.5 lower
secreted signaling molecules that regulate neuronal and non- jaws as well as onto isolated E12.5 dental mesenchyme. Wnt4-
neuronal development. Several Wnts are expressed in the lower producing cells upregulated Sema3a expression in the adjacent
jaw and dental epithelia (Sarkar and Sharpe, 1999). In mesenchymal cells at all stages studied (Fig. 6, C1-E2), while
developing limb, epithelial Wnt4 induces neurotrophin 3 (Nt3) cells producing Wnt6, mRNAs of which are present in the oral
expression in the adjacent mesenchyme (Patapoutian et al., epithelium (Sarkar and Sharpe, 1999), showed no effect on
1999), the development of which is dependent on epithelial mesenchymal Sema3a expression at E10.5 or E11.5 (Fig.
mesenchymal interactions. Moreover, Wnt4 and Wnt5 regulate 6J1,J2 and not shown). mRNAs for Tgfβ1 appear in the
commissural axon guidance (Lyuksyutova et al., 2003; epithelial dental bud at E12.5 (not shown), and later at the cap
Yoshikawa et al., 2003). To investigate whether Wnt factors stage they also appear in the dental mesenchyme (Vaahtokari
control Sema3a, we placed clusters of Wnt4-producing cells, et al., 1991). Tgfβ1 regulates Ngf and Nt3 mRNA levels in the
mRNAs of which are present in the oral and dental epithelium epithelial and mesenchymal cells of the maxillary process in
during E10.5-E14.5 (Sarkar and Sharpe, 1999), onto the culture (Buchman et al., 1994). When Tgfβ1-soaked beads
Regulation of Sema3a by tissue interactions 331
al., 1993). We found that Wnt4-producing cells were able to
stimulate endogenous Msx1 in E10 mandibular mesenchyme
explants, whereas no Msx1 was observed around the control
NIH3T3 cell clusters (Fig. 6H1-I2). No effects on Pax9
expression in E10.5 presumptive dental mesenchyme around
the cells were observed (not shown).
Tgfβ1 stimulates dental mesenchymal cell
Because Tgfβ1 is prominently expressed in the highly
proliferative cells in the cervical loops and dental papilla
mesenchyme during the cap stage (Vaahtokari et al., 1991)
when dental axons are growing around the tooth germ, we
analyzed the effects of Tgfβ1 on dental cell proliferation.
Tgfβ1-soaked agarose beads were applied onto the molar area
of isolated dental mesenchyme at E12 when Tgfβ1 is expressed
in the epithelial bud but not in the underlying mesenchyme.
The explants were cultured for 24 hours, the last 1.5 hours with
5-bromo-2-deoxyuridine (BrdU). Whole-mount and sectional
immunohistochemical analysis showed that E12 dental
mesenchymal cells around Tgfβ1 and positive control Fgf2
Fig. 7. Tgfβ1 stimulates proliferation (Kettunen et al., 1998)-releasing beads had incorporated BrdU
of the dental mesenchymal cells as markedly (Fig. 7A-D). This mimicked the effects of E12 dental
detected by BrdU incorporation from epithelia, which also stimulated the proliferation of adjacent
tissue sections (A,B) and whole dental mesenchymal cells in homochronic recombinants (Fig.
mounts (C). The E12 mandibular 7C,D). No elevated BrdU incorporation was seen in explants
mesenchyme or dental recombinant cultured with BSA-soaked beads (Fig. 7E). Thus, besides
explants were cultured for 24 hours controlling the establishment of tooth innervation, Tgfβ1 may
with protein-soaked agarose beads. Increased cell proliferation is
regulate tooth morphogenesis by stimulating dental cell
seen in the mesenchymal cells next to the Tgfβ1 (A-C) and control
Fgf2 (D)-releasing beads, as well as cells next to the dental proliferation.
epithelium (E12), which also shows BrdU incorporation. Near the
control BSA (1 mg/ml) bead (E), hardly any BrdU-positive cells are Discussion
seen. B shows proliferating cells in A at a higher magniﬁcation.
Arrows indicate the ﬁrst molar mesenchyme region where the beads Sema3a regulates timing of tooth innervation and
were located. (G,H) Induction of tubulogenesis in isolated E11 dental axon navigation and patterning
metanephric mesenchyme by Wnt4- and Wnt6-expressing cells after Teeth are essential for survival, and the presence of a sensory
5 days in culture. NIH3T3 control cells do not support survival and innervation is of great importance for their function and
differentiation of the mesenchyme. (I) Stimulation of Gli1 expression protection. To investigate the regulatory mechanisms of
in E11 mandibular mesenchyme by Shh-containing beads. b, bead; c,
peripheral axon guidance and patterning, we studied the
cell cluster; e, dental epithelium; m, dental mesenchyme. Scale bars:
200 µm in A,E,I; in C, 100 µm for C,D; 50 µm in B; in H, 500 µm functions and regulation of Sema3a in the formation of the
for C,F,H. mouse lower molar, which is tightly linked with the
development and patterning of the mandibular process.
Analysis of Sema3a knockout mice revealed that Sema3a is
were placed onto the molar area of E12.5 mandibular jaw an essential signal for the establishment of early tooth
mesenchyme, a prominent Sema3a expression was observed in innervation, though not for its formation, and that its effects
the surrounding cells (Fig. 6G1,G2). No effects on Sema3a was appear to be mediated by the Npn1 receptor expressed in the
observed in the mesenchyme cultured with control NIH3T3 dental axons. That the nerve ﬁbers prematurely innervate the
cells or beads soaked in BSA (1 mg/ml) from E10 to E12.5 Sema3a–/– tooth already at the epithelial thickening stage
(Fig. 6K1-M2; not shown). indicates that Sema3a, by forming exclusion areas in the jaw
and presumptive dental mesenchyme, regulates the timing of
Wnt4 stimulates Msx1 expression in the jaw tooth innervation by apparently by preventing ingrowth of
mesenchyme other trigeminal nerve ﬁbers such as buccal nerve. At the early
Because Wnt signaling is essential for tooth formation (Andl bud stage, Sema3a signaling appears to regulate the formation
et al., 2002; van Genderen et al., 1994), we investigated of the single ‘molar’ nerve and channels the growth of pioneer
whether Wnt4 is involved in odontogenesis by analyzing its dental axons to the restricted mesenchymal pathway towards
effects on the expression of Msx1 and Pax9 transcription the tooth. During subsequent morphogenetic bud, cap and bell
factors. Msx1 and Pax9 are necessary for tooth morphogenesis stages, Sema3a restricts axon growth into the mesenchymal
beyond the bud stage and their expression in the early target ﬁeld around the tooth and prevents their ingrowth to the
mandibular mesenchyme is induced by and dependent on condensed dental mesenchyme and the dental papilla. The
epithelial signaling (Ferguson et al., 2000; Neubuser et al., absence of Sema3a from the sites of the developing secondary
1997; Peters et al., 1998; Satokata and Maas, 1994; Vainio et apical foramina during E18-PN4 suggests that Sema3a
332 Development 132 (2) Research article
exclusion areas in the base of the dental papilla do not dental epithelium controls Sema3a in the dental mesenchyme.
determine the timing of nerve ﬁber penetration to the dental Thus, these results suggest that local epithelial-mesenchymal
papilla but are involved in regulation of the sites through which interactions control Sema3a expression and the establishment
the nerve ﬁbers are able to enter, i.e. through the forming root of tooth innervation. As tooth formation has been shown to be
canals. controlled by interactions between epithelial and mesenchymal
We also noticed that many nerve ﬁbers showed largely tissues (Lumsden, 1988; Mina and Kollar, 1987), tissue
normal localization within the tooth target ﬁeld in Sema3a–/– interactions may therefore provide a mechanism to coordinate
embryos, and that errors in dental axon patterning became axon navigation and patterning spatiotemporally with tooth
increasingly corrected as tooth morphogenesis proceeded, formation.
which is in line with the corrections of other sensory axon Earlier tissue recombination studies have shown that E10-
projections in Sema3a–/– mice (White and Behar, 2000). We E11 mouse oral and dental epithelium possesses instructive
found that mRNA expression of Ngf, Gdnf, Lanr, Ncam and information to control tooth formation as well as the potential
Net3 was not affected in the dental follicle target area of the to determinate tooth type (Lumsden, 1988; Mina and Kollar,
Sema3a–/– teeth. This indicates that their expression is not 1987; Tucker et al., 1998). Our tissue recombination
regulated or dependent on Sema3a, and that they appear to experiments showing that the E10.5 oral and E11 dental
partially rescue tooth innervation phenotype in Sema3a–/– epithelia induce mesenchymal Sema3a expression indicate
mice. Thus, these results provide genetic evidence for the that, besides the odontogenic information, the presumptive
model that axon guidance and establishment of tooth nerve dental epithelium also possesses the instructive information to
supply involves redundant and independent signaling of control the formation of early tooth nerve supply and possible
neuroregulatory genes of different families. tooth-speciﬁc sensory innervation that is distinct, in some
aspects, from the adjacent cutaneous sensory system (Fried et
Epithelial-mesenchymal interactions regulate the al., 2000; Kvinnsland et al., 2004). Furthermore, because the
establishment of tooth nerve supply formation of teeth of all types is controlled by the epithelial-
The ﬁnding that precisely regulated expression domains of mesenchymal interactions, the interactions also appear to
Sema3a are crucial to the timing of tooth innervation as well provide a rationale for the fact that the timing and pattern of
as dental axon guidance and patterning led us to use Sema3a tooth innervation in different teeth and species correlate better
as a marker gene for analysis of the basal regulatory to the developmental stage of the individual tooth than the
mechanisms behind the establishment of tooth nerve supply. chronological age of the animal.
By performing tissue recombination experiments, we found
that the oral epithelium is necessary for Sema3a expression in Epithelial-mesenchymal interactions mediated by
the E10.5 and E11 mandibular mesenchyme, and that E10.5 Wnt4 and Tgfβ1 may coordinate trigeminal axon
oral epithelium as well as E11 and E12 dental epithelia are able navigation and patterning with tooth formation
to induce Sema3a expression in the presumptive molar Early oral and dental epithelium expressed signaling molecules
mesenchyme area of the lower jaw lacking Sema3a. have been implicated in the mediation of organogenetic tissue
Furthermore, we showed that later at E12 when the ﬁrst dental interactions. We found that oral and dental epithelium
axons are about to or are navigating to the developing tooth, expressed Wnt4 induces Sema3a in the mandibular
Initiation Early bud stage Bud stage
E11.5 E12.5 E13.5
epithelium epithelium Tgfβ1 Wnt4
buccal lingual Sema3A dental Sema3A Sema3A
Fig. 8. Schematic model for coordination of early tooth organogenesis and establishment of nerve supply by epithelial-mesenchymal
interactions. The Sema3a exclusion areas (in red) regulate timing of tooth innervation and the innervation pattern. Prior to the histological onset
of tooth formation (E10.5), the odontogenic oral epithelium, which instructs tooth formation and also possesses information to control tooth-
speciﬁc nerve supply, induces (mediated by Wnt4) Sema3a in the presumptive dental mesenchyme. During subsequent morphogenesis
epithelial signaling and Wnt4 and Tgfβ1 continue to control Sema3a expression domains in the dental mesenchyme target area. Wnt4 and
Tgfβ1 contribute to the regulation of tooth morphogenesis by maintaining Msx1 (the effect of Wnt4 on Msx1 expression at E11.5 is
hypothetical) and stimulating dental mesenchymal cell proliferation, respectively. The trigeminal molar nerve located in the mesenchymal axon
pathway and tooth target ﬁelds are indicated in black.
Regulation of Sema3a by tissue interactions 333
presumptive dental mesenchyme and that, later, Wnt4 and developmental processes, it is tempting to propose that
Tgfβ1, which are present in the early bud stage dental epithelial-mesenchymal interactions may provide an important
epithelium during dental axon growth, stimulate Sema3a mechanism for coordinating tissue and organ formation, and
expression in the dental mesenchyme. Thus, Wnt4 and Tgfβ1 establishment of the peripheral nerve supply.
may act as in vivo epithelial signals that control mesenchymal
Sema3a expression. Of particular interest is the observation We thank Dr M. Saarma for the Ngf, Gdnf and Lanr cDNAs; Dr
that Wnt signaling has been shown to be essential for tooth M. Tessier-Lavigne for the Net3 cDNA; Dr M. P. Scott for the
formation, as evidenced by the ﬁnding that overexpression of Patched1 cDNA; and Dr M. Takeda for the Ncam cDNA. We also
thank Ms Kjellﬁd Haukanes, Ms Helen Olsen and Ms Anne Nyhaug
the Wnt inhibitor Dickkopf1 in the BA1 ectoderm and targeted for their technical assistance. This study was supported by the
inactivation of Lef1 transcription factor (which is needed for Norwegian Cancer Society, the L. Meltzer’s foundation, the
Fgf4 expression in the primary enamel knot signaling center) University of Bergen and the Research Council of Norway.
in transgenic mice result in arrest of tooth formation prior to
the bud and cap stages, respectively (Andl et al., 2002; van
Genderen et al., 1994; Kratochwil et al., 2002). Epithelial Fgf4 References
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