Coordination of trigeminal axon navigation and patterning with tooth

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					                                            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
              dental mesenchyme
              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:

              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) ( 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) ( 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
              by epithelial-mesenchymal
              interactions (Lumsden, 1988;
              Mina and Kollar, 1987). To
              analyze      whether      tissue
              interactions           regulate
              mesenchymal             Sema3a
              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
              mandibular         mesenchyme
              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
              mesenchymal Sema3a
              expression is present (C1-C2).
              (D1-D2) E10.5 oral epithelium

              has induced Sema3a in the
              underlying E10.5 molar area of
              mandibular mesenchyme,
              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
                                             Dental              Oral
                                           epithelium         epithelium                Tgfβ1                                 Wnt4
                                        Wnt4                                                       Condensed
                          buccal                         lingual                       Sema3A        dental     Sema3A         Sema3A
                                Msx1                    Presumptive
                                       Sema3A                dental
                                                                                             “Molar nerve”
                                                 alveolar nerve

              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
              induces Fgf3 expression in the dental mesenchyme (Kettunen           Andl, T., Reddy, S. T., Gaddapara, T. and Millar, S. E. (2002). WNT signals
              et al., 2000), which is required for Shh expression in the future      are required for the initiation of hair follicle development. Dev. Cell 2, 643-
              enamel knot (Kratochwil et al., 2002). In addition, it has been        653.
                                                                                   Bagri, A. and Tessier-Lavigne, M. (2002). Neuropilins as Semaphorin
              suggested that interactions between epithelial expressed Wnt7          receptors: in vivo functions in neuronal cell migration and axon guidance.
              and Shh determine the position of tooth initiation (Sarkar et al.,     Adv. Exp. Med. Biol. 515, 13-31.
              2000). We have found that Wnt4 maintained mesenchymal                Behar, O., Golden, J. A., Mashimo, H., Schoen, F. J. and Fishman, M. C.
              expression of Msx1 transcription factor, which is essential for        (1996). Semaphorin III is needed for normal patterning and growth of
              tooth morphogenesis (Satokata and Maas, 1994) in the early             nerves, bones and heart. Nature 383, 525-528.
                                                                                   Buchman, V. L., Sporn, M. and Davies, A. M. (1994). Role of transforming
              jaw mesenchyme, whereas Tgfβ1, the expression of which                 growth factor-beta isoforms in regulating the expression of nerve growth
              correlates with tooth morphogenesis, stimulated the                    factor and neurotrophin-3 mRNA levels in embryonic cutaneous cells at
              proliferation of the dental mesenchymal cells. Thus, besides           different stages of development. Development 120, 1621-1629.
              regulating Sema3a, Wnt4 and Tgfβ1 appear to be involved in           Cobourne, M. T. and Sharpe, P. T. (2003). Tooth and jaw: molecular
                                                                                     mechanisms of patterning in the first branchial arch. Arch. Oral Biol. 48, 1-
              the regulation of tooth formation. We propose that they may

              act as signals that mediate epithelial-mesenchymal interactions      Davies, A. M. (1988). The trigeminal system: an advantageous experimental
              and coordinate trigeminal axon growth and patterning with              model for studying neuronal development. Development 103, 175-183.
              tooth formation (Fig. 8).                                            Dickson, B. J. (2002). Molecular mechanisms of axon guidance. Science 298,
              Epithelial-mesenchymal interactions may coordinate                   Dillon, T. E., Saldanha, J., Giger, R., Verhaagen, J. and Rochlin, M. W.
                                                                                     (2004). Sema3A regulates the timing of target contact by cranial sensory
              establishment of the peripheral nerves with                            axons. J. Comp. Neurol. 470, 13-24.
              outgrowth and patterning of the mandibular process                   Erdelyi, G., Fried, K. and Hildebrand, C. (1987). Nerve growth to tooth buds
              During development of the nervous system, peripheral axons             after homotopic or heterotopic autotransplantation. Brain Res. 430, 39-47.
                                                                                   Ferguson, C. A., Tucker, A. S. and Sharpe, P. T. (2000). Temporospatial cell
              establish nerve tracts and contacts to target tissues and organs       interactions regulating mandibular and maxillary arch patterning.
              (the development of most of which is regulated by epithelial-          Development 127, 403-412.
              mesenchymal interactions) very accurately in place and time.         Fried, K., Nosrat, C., Lillesaar, C. and Hildebrand, C. (2000). Molecular
              Developmentally regulated signaling of Sema3a is shown to              signaling and pulpal nerve development. Crit. Rev. Oral Biol. Med. 11, 318-
              serve crucial functions for trigeminal axon pathfinding and             332.
                                                                                   Fristad, I., Heyeraas, K. J. and Kvinnsland, I. (1994). Nerve fibres and cells
              patterning in the mandibular process (Dillon et al., 2004;             immunoreactive to neurochemical markers in developing rat molars and
              Taniguchi et al., 1997), which grows out, undergoes patterning         supporting tissues. Arch. Oral Biol. 39, 633-646.
              and develops into teeth, various tissues and skeletal elements.      Hardcastle, Z., Mo, R., Hui, C. C. and Sharpe, P. T. (1998). The Shh
              Like the formation of the tooth, the development of the BA1            signalling pathway in tooth development: defects in Gli2 and Gli3 mutants.
                                                                                     Development 125, 2803-2811.
              is regulated by reciprocal epithelial-mesenchymal interactions,      Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J. and Ish-Horowicz,
              and epithelial signaling controls the expression of transcription      D. (1995). Expression of a Delta homologue in prospective neurons in the
              factors involved in the establishment of the polarity and pattern      chick. Nature 375, 787-790.
              of the BA1 (Cobourne and Sharpe, 2003). Furthermore,                 Jernvall, J., Kettunen, P., Karavanova, I., Martin, L. B. and Thesleff, I.
              epithelial signaling controls the expression of Nt3, which             (1994). Evidence for the role of the enamel knot as a control center in
                                                                                     mammalian tooth cusp formation: non-dividing cells express growth
              promotes trigeminal axon growth, in the maxillary process              stimulating Fgf-4 gene. Int. J. Dev. Biol. 38, 463-469.
              mesenchyme (O’Connor and Tessier-Lavigne, 1999). Our                 Kawakami, A., Kitsukawa, T., Takagi, S. and Fujisawa, H. (1996).
              results show that Sema3a expression in the E10.5-E11 early             Developmentally regulated expression of a cell surface protein, neuropilin,
              mandibular mesenchyme is dependent on the overlying                    in the mouse nervous system. J. Neurobiol. 29, 1-17.
                                                                                   Kettunen, P. and Thesleff, I. (1998). Expression and function of FGFs-4, -8,
              epithelium and that epithelial Wnt4 induces the expression of          and -9 suggest functional redundancy and repetitive use as epithelial signals
              Sema3a and maintains Msx1 when epithelial signaling controls           during tooth morphogenesis. Dev. Dyn. 211, 256-268.
              the patterning of the process (Ferguson et al., 2000) and nerve      Kettunen, P., Karavanova, I. and Thesleff, I. (1998). Responsiveness of
              branches in the mandibular process are being established               developing dental tissues to fibroblast growth factors: expression of splicing
              (Lumsden, 1982). Hence, these results suggest that tissue              alternatives of FGFR1, -2, -3, and of FGFR4; and stimulation of cell
                                                                                     proliferation by FGF-2, -4, -8, and -9. Dev. Genet. 22, 374-385.
              interactions may link the establishment and patterning of the        Kettunen, P., Laurikkala, J., Itaranta, P., Vainio, S., Itoh, N. and Thesleff,
              peripheral nerves to the outgrowth and patterning of the               I. (2000). Associations of FGF-3 and FGF-10 with signaling networks
              lower jaw. Thus, given the remarkable similarities of the              regulating tooth morphogenesis. Dev. Dyn. 219, 322-332.
              334       Development 132 (2)                                                                                                                  Research article

              Kispert, A., Vainio, S. and McMahon, A. P. (1998). Wnt-4 is a mesenchymal              interactions between FGF and BMP signaling pathways: a mechanism for
                signal for epithelial transformation of metanephric mesenchyme in the                positioning the sites of tooth formation. Cell 90, 247-255.
                developing kidney. Development 125, 4225-4234.                                     Nosrat, C. A., Fried, K., Ebendal, T. and Olson, L. (1998). NGF, BDNF, NT3,
              Kitsukawa, T., Shimizu, M., Sanbo, M., Hirata, T., Taniguchi, M., Bekku,               NT4 and GDNF in tooth development. Eur. J. Oral Sci. 106, 94-99.
                Y., Yagi, T. and Fujisawa, H. (1997). Neuropilin-semaphorin III/D-mediated         O’Connor, R. and Tessier-Lavigne, M. (1999). Identification of maxillary
                chemorepulsive signals play a crucial role in peripheral nerve projection in         factor, a maxillary process-derived chemoattractant for developing trigeminal
                mice. Neuron 19, 995-1005.                                                           sensory axons. Neuron 24, 165-178.
              Kratochwil, K., Galceran, J., Tontsch, S., Roth, W. and Grosschedl, R.               Obara, N. and Takeda, M. (1993). Expression of neural cell adhesion molecule
                (2002). FGF4, a direct target of LEF1 and Wnt signaling, can rescue the arrest       (NCAM) during the first molar development in the mouse. Anat. Embryol.
                of tooth organogenesis in Lef1(–/–) mice. Genes Dev. 16, 3173-3185.                  187, 209-219.
              Kvinnsland, I. H., Luukko, K., Fristad, I., Kettunen, P., Jackson, D. L.,            Patapoutian, A., Backus, C., Kispert, A. and Reichardt, L. F. (1999).
                Fjeld, K., von Bartheld, C. S. and Byers, M. R. (2004). Glial cell line-             Regulation of neurotrophin-3 expression by epithelial-mesenchymal
                derived neurotrophic factor (GDNF) from adult rat tooth serves a distinct            interactions: the role of Wnt factors. Science 283, 1180-1183.
                population of large-sized trigeminal neurons. Eur. J. Neurosci. 19, 2089-2098.     Peters, H., Neubuser, A., Kratochwil, K. and Balling, R. (1998). Pax9-
              Lillesaar, C. and Fried, K. (2004). Neurites from trigeminal ganglion explants         deficient mice lack pharyngeal pouch derivatives and teeth and exhibit
                grown in vitro are repelled or attracted by tooth-related tissues depending on       craniofacial and limb abnormalities. Genes Dev. 12, 2735-2747.
                developmental stage. Neuroscience 125, 149-161.                                    Qian, X. B. and Naftel, J. P. (1996). Effects of neonatal exposure to anti-nerve
              Loes, S., Kettunen, P., Kvinnsland, I. H., Taniguchi, M., Fujisawa, H. and             growth factor on the number and size distribution of trigeminal neurones
                Luukko, K. (2001). Expression of class 3 semaphorins and neuropilin                  projecting to the molar dental pulp in rats. Arch. Oral Biol. 41, 359-367.
                receptors in the developing mouse tooth. Mech. Dev. 101, 191-194.                  Rochlin, M. W. and Farbman, A. I. (1998). Trigeminal ganglion axons are
              Loes, S., Kettunen, P., Kvinnsland, H. and Luukko, K. (2002). Mouse                    repelled by their presumptive targets. J. Neurosci. 18, 6840-6852.
                rudimentary diastema tooth primordia are devoid of peripheral nerve fibers.         Sarkar, L. and Sharpe, P. T. (1999). Expression of Wnt signalling pathway
                Anat. Embryol. 205, 187-191.                                                         genes during tooth development. Mech. Dev. 85, 197-200.
              Loes, S., Luukko, K., Hals Kvinnsland, I., Salminen, M. and Kettunen, P.             Sarkar, L., Cobourne, M., Naylor, S., Smalley, M., Dale, T. and Sharpe, P.
                (2003). Developmentally regulated expression of Netrin-1 and -3 in the               T. (2000). Wnt/Shh interactions regulate ectodermal boundary formation
                embryonic mouse molar tooth germ. Dev. Dyn. 227, 573-577.                            during mammalian tooth development. Proc. Natl. Acad. Sci. USA 97, 4520-
              Lumsden, A. G. (1982). The developing innervation of the lower jaw and its             4524.
                relation to the formation of tooth germs in mouse. In TEETH; Form, Function        Satokata, I. and Maas, R. (1994). Msx1 deficient mice exhibit cleft palate and
                and Evolution, Vol. 62, pp. 32-43. New York, NY: Columbia University Press.          abnormalities of craniofacial and tooth development. Nat. Genet. 6, 348-356.
              Lumsden, A. G. (1988). Spatial organization of the epithelium and the role of        Serini, G., Valdembri, D., Zanivan, S., Morterra, G., Burkhardt, C.,
                neural crest cells in the initiation of the mammalian tooth germ. Development        Caccavari, F., Zammataro, L., Primo, L., Tamagnone, L., Logan, M. et
                103, 155-169.                                                                        al. (2003). Class 3 semaphorins control vascular morphogenesis by inhibiting
              Luukko, K. (1997). Immunohistochemical localization of nerve fibres during              integrin function. Nature 424, 391-397.

                development of embryonic rat molar using peripherin and protein gene               Taniguchi, M., Yuasa, S., Fujisawa, H., Naruse, I., Saga, S., Mishina, M. and
                product 9.5 antibodies. Arch. Oral Biol. 42, 189-195.                                Yagi, T. (1997). Disruption of semaphorin III/D gene causes severe
              Luukko, K. (1998). Neuronal cells and neurotrophins in odontogenesis. Eur. J.          abnormality in peripheral nerve projection. Neuron 19, 519-530.
                Oral Sci. 106, 80-93.                                                              Tessier-Lavigne, M. and Goodman, C. S. (1996). The molecular biology of
              Luukko, K., Moshnyakov, M., Sainio, K., Saarma, M., Sariola, H. and                    axon guidance. Science 274, 1123-1133.
                Thesleff, I. (1996). Expression of neurotrophin receptors during rat tooth         Thesleff, I. (2003). Epithelial-mesenchymal signalling regulating tooth
                development is developmentally regulated, independent of innervation, and            morphogenesis. J. Cell Sci. 116, 1647-1648.
                suggests functions in the regulation of morphogenesis and innervation. Dev.        Trumpp, A., Depew, M. J., Rubenstein, J. L., Bishop, J. M. and Martin, G.
                Dyn. 206, 87-99.                                                                     R. (1999). Cre-mediated gene inactivation demonstrates that FGF8 is required
              Luukko, K., Arumae, U., Karavanov, A., Moshnyakov, M., Sainio, K.,                     for cell survival and patterning of the first branchial arch. Genes Dev. 13, 3136-
                Sariola, H., Saarma, M. and Thesleff, I. (1997a). Neurotrophin mRNA                  3148.
                expression in the developing tooth suggests multiple roles in innervation and      Tucker, A. S., Matthews, K. L. and Sharpe, P. T. (1998). Transformation of
                organogenesis. Dev. Dyn. 210, 117-129.                                               tooth type induced by inhibition of BMP signaling. Science 282, 1136-1138.
              Luukko, K., Sainio, K., Sariola, H., Saarma, M. and Thesleff, I. (1997b).            Ulupinar, E., Datwani, A., Behar, O., Fujisawa, H. and Erzurumlu, R.
                Localization of nerve cells in the developing rat tooth. J. Dent. Res. 76, 1350-     (1999). Role of semaphorin III in the developing rodent trigeminal system.
                1356.                                                                                Mol. Cell. Neurosci. 13, 281-292.
              Luukko, K., Suvanto, P., Saarma, M. and Thesleff, I. (1997c). Expression of          Vaahtokari, A., Vainio, S. and Thesleff, I. (1991). Associations between
                GDNF and its receptors in developing tooth is developmentally regulated and          transforming growth factor beta 1 RNA expression and epithelial-
                suggests multiple roles in innervation and organogenesis. Dev. Dyn. 210, 463-        mesenchymal interactions during tooth morphogenesis. Development 113,
                471.                                                                                 985-994.
              Lyuksyutova, A. I., Lu, C. C., Milanesio, N., King, L. A., Guo, N., Wang, Y.,        Vainio, S., Karavanova, I., Jowett, A. and Thesleff, I. (1993). Identification
                Nathans, J., Tessier-Lavigne, M. and Zou, Y. (2003). Anterior-posterior              of BMP-4 as a signal mediating secondary induction between epithelial and
                guidance of commissural axons by Wnt-frizzled signaling. Science 302, 1984-          mesenchymal tissues during early tooth development. Cell 75, 45-58.
                1988.                                                                              van Genderen, C., Okamura, R. M., Farinas, I., Quo, R. G., Parslow, T. G.,
              Matsuo, S., Ichikawa, H., Henderson, T. A., Silos-Santiago, I., Barbacid, M.,          Bruhn, L. and Grosschedl, R. (1994). Development of several organs that
                Arends, J. J. and Jacquin, M. F. (2001). trkA modulation of developing               require inductive epithelial-mesenchymal interactions is impaired in LEF-1-
                somatosensory neurons in oro-facial tissues: tooth pulp fibers are absent in          deficient mice. Genes Dev. 8, 2691-2703.
                trkA knockout mice. Neuroscience 105, 747-760.                                     Varela-Echavarria, A. and Guthrie, S. (1997). Molecules making waves in
              Miletich, I. and Sharpe, P. T. (2003). Normal and abnormal dental                      axon guidance. Genes Dev. 11, 545-557.
                development. Hum. Mol. Genet. 12, R69-R73.                                         White, F. A. and Behar, O. (2000). The development and subsequent
              Mina, M. and Kollar, E. J. (1987). The induction of odontogenesis in non-              elimination of aberrant peripheral axon projections in Semaphorin3A null
                dental mesenchyme combined with early murine mandibular arch epithelium.             mutant mice. Dev. Biol. 225, 79-86.
                Arch. Oral Biol. 32, 123-127.                                                      Wright, D. E., White, F. A., Gerfen, R. W., Silos-Santiago, I. and Snider, W.
              Mitsiadis, T. A., Dicou, E., Joffre, A. and Magloire, H. (1992).                       D. (1995). The guidance molecule semaphorin III is expressed in regions of
                Immunohistochemical localization of nerve growth factor (NGF) and NGF                spinal cord and periphery avoided by growing sensory axons. J. Comp. Neurol.
                receptor (NGF-R) in the developing first molar tooth of the rat. Differentiation      361, 321-333.
                49, 47-61.                                                                         Yoshikawa, S., McKinnon, R. D., Kokel, M. and Thomas, J. B. (2003). Wnt-
              Mohamed, S. S. and Atkinson, M. E. (1983). A histological study of the                 mediated axon guidance via the Drosophila Derailed receptor. Nature 422,
                innervation of developing mouse teeth. J. Anat. 136, 735-749.                        583-588.
              Neubuser, A., Peters, H., Balling, R. and Martin, G. R. (1997). Antagonistic

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