Teeth are vital for survival for most animals, and, therefore, the presence of a sensory nerve supply, which originates from the trigeminal ganglion, is of great importance for their protection and function. To be able to perform their functions, the dental nerve fibers have to navigate, establish their contacts, and survive at the proper sites in the adult tooth. The major target area of trigeminal nerve endings is the dental pulp, where nerve fibers form a dense network in the subodontoblastic region. Nerve fibers are located close to the odontoblasts, with some penetrating into the predentin and dentin (for a review see Hildebrand et al., 1995). The dental sensory nerve fibers mediate mostly painful sensations (for a review see Byers and Narhi, 1999), but some, originating from the large-sized neurons, appear to mediate mechanosensitive stimuli (Kvinnsland et al., 2004). In addition, pulpal sensory fibers possess dynamic plasticity in structure and cytochemistry and are involved in the regulation of blood flow, inflammatory reactions, and tissue repair (Hildebrand et al., 1995; Fristad, 1997; Byers and Narhi, 1999; Heyeraas and Berggreen, 1999). Another major target area for the trigeminal sensory axons is the periodontal ligament, which connects the tooth to the alveolar bone and jaw. The nerve fibers here mediate touch and pressure sensations as well as nociception (Hildebrand et al., 1995). Some nerve fibers from the mesencephalic trigeminal nucleus (TMN) are involved in the sensorimotor control of mastication (for a review see Hildebrand et al., 1995). Sympathetic nerve fibers from the superior cervical ganglion are predominantly found in association with the arterioles and serve mainly vasoregulatory functions, and may modulate inflammation in dental tissues (Hildebrand et al., 1995; Haug and Heyeraas, 2003).
DEVELOPMENTAL ANATOMY OF TOOTH FORMATION AND DENTAL TRIGEMINAL AXON GROWTH AND PATTERNING
The mouse trigeminal system, consisting of the trigeminal ganglion, axons, and their peripheral target tissues of the first branchial arch, has long been an advantageous model for analysis of regulatory mechanisms of development of peripheral innervation (reviewed in Davies, 1988). In particular, the tooth germs, which develop in the oral side of the maxillary and mandibular process, are well-defined target organs for sensory trigeminal innervation. Like the major trigeminal axon trajectories, the dental nerve fibers reach the tooth germs during embryonic development, during which the trigeminal neurons are exposed to the phase of programmed cell death (Davies, 1988). This phase starts soon after the pioneer nerve fibers have reached their peripheral targets, which in mouse takes place at E13. This lasts until E18 during which about 50% of the trigeminal ganglion neurons die. During this time period, the survival of the trigeminal neuronal cells is dependent on target field–derived survival factors of different families (Davies, 1988). Because the final stages of mouse crown morphogenesis as well as crown mineralization, root formation, and eruption take place after birth, axon navigation and their survival in the embryonic tooth target are essential for the establishment of the adult tooth nerve supply (Luukko et al., 1997a).
Several studies of the localization of nerve fibers in human and murine teeth have shown that dental axon growth and patterning take place in a spatiotemporally controlled manner and are tightly linked with advancing tooth morphogenesis (Pearson, 1977; Mohamed and Atkinson, 1983; Tsuzuki and Kitamura, 1991; Fristad et al., 1994; Luukko, 1997; Kettunen et al., 2005). In order to be able to investigate the molecular mechanisms of dental axon guidance, detailed information regarding the localization of nerve fibers at carefully defined tooth developmental stages in particular during pioneer axon growth is of crucial importance. Indeed, earlier localization of nerve fibers in rat and mouse mandibular first molar tooth germ using serially sectioned tooth germs has revealed that, like peripheral axon growth in general, pioneer dental axons follow defined pathways guided by the specific intermediate targets (choice points) in a highly controlled manner (Fig. 1). Significantly, as there is a substantial body of experimental and genetic data regarding the molecular control of tooth formation in the mouse first lower molar (for a review see Miletich and Sharpe, 2003; Thesleff, 2003), the tooth is proving to be an excellent model for analysis of molecular regulatory mechanisms integrating organ formation and axonal guidance and patterning (Kettunen et al., 2005).
Organ development is a complex process characterized by coordinated growth and differentiation of cells of epithelial and mesenchymal cell lineages. Traditionally, the development of the tooth has been divided into three partially overlapping phases, defined as initiation, morphogenesis, and cell differentiation (Kollar and Lumsden, 1979). The first histological sign of the mouse first molar formation is a local epithelial thickening at the oral side of the mandibular process at E11.5 (Fig. 1A). The pioneer dental nerve fibers emerge from the major mandibular trigeminal nerve trunk (inferior alveolar nerve) at approximately E12 when the tooth germ is at the early bud stage. The turning of the dental axons from the interior alveolar nerve in this intermediate target area is defined as the first choice point, which determines the formation of the dental nerve trajectory of the “molar nerve” (Loes et al., 2002). About half a day later, the molar nerve reaches the tooth target area at approximately the mid-anterior, most developed part of the tooth germ. The nerve trajectory is located under the condensing dental mesenchyme and is advancing buccally (Fig. 1B). Approximately between E12.5 and E13.5, some axons turn lingually beneath the base of the condensed dental mesenchyme (this is termed the second choice point), and at the bud stage (E13.5) the buccal and lingual branches of the molar nerve are growing tangentially next to the dental mesenchyme (Loes et al., 2002) (Fig. 1C). We term the mesenchymal area adjacent to the condensed dental mesenchyme the early dental target field. During subsequent epithelial folding morphogenesis at the cap and bell stages, more nerve fibers are seen in the dental follicle target area around the enamel organ (Fig. 1D and E). Most of the nerve fibers are present in the base of the dental papilla, where they make arborizations and form a nerve plexus (Mohamed and Atkinson, 1983; Loes et al., 2002), but never come in contact with the dental epithelium. Nerve fibers emerging from the basal plexus enter the dental papilla after the onset of dentin and enamel formation, which in mouse takes place about three days postnatally (Mohamed and Atkinson, 1983; Tsuzuki and Kitamura, 1991; Fristad et al., 1994) (Fig. 1F). Thus, the establishment of the molar nerve and timing of molar tooth innervation take place later than the establishment of all other major branches of the mandibular nerve (Lumsden, 1982). Therefore, it is plausible that the trigeminal neurons, which innervate the tooth, are among the last to differentiate and extend their axons. As a consequence, the last dental nerve fibers are suggested to reach the tooth germ at about E15 (Davies, 1988). Thus, because no new trigeminal sensory nerve fibers arrive at the tooth after the early bell stage, the final stages of the establishment of the dental trigeminal axonal patterning such as postnatal nerve fiber penetration to the pulp, formation of the nerve plexus in the pulp-dentin border area, and innervation of the periodontal ligament during root formation (Mohamed and Atkinson, 1983; Tsuzuki and Kitamura, 1991; Fristad et al., 1994), must take place by arborization and reorganization of the existing nerve fibers, which are present in the dental follicle target field and have survived the phase of programmed cell death.
MOLECULAR REGULATION OF DENTAL AXON GUIDANCE AND PATTERNING
During the development of the nervous system, growing nerve fibers are guided to their targets by a combination of positive and negative cues based on diffusible factors and contacts with cell surface and extracellular matrix molecules (Tessier-Lavigne and Goodman, 1996; Varela-Echavarria and Guthrie, 1997; Dickson, 2002). The regulation of the development of tooth sensory nerve supply at a molecular level is also beginning to be elucidated. There is accumulating evidence that the molecules involved in the development of the peripheral nervous system are also involved in dental axon guidance (for a review see Luukko, 1998; Fried et al., 2000). A characteristic feature of the putative dental axon guidance molecules is that they show developmentally regulated expression in the mesenchymal dental axon pathway and/or target field areas or in adjacent exclusion areas at different stages of tooth organ morphogenesis and cell differentiation (Fig. 2).
Secreted Gdnf (glial cell line-derived neurotrophic factor) and, in particular, Ngf (nerve growth factor) neurotrophic factors appear to be key regulators of dental axon guidance, patterning, and establishment of the tooth sensory nerve supply (Luukko et al., 1997a, b; Nosrat et al., 1998; Matsuo et al., 2001; Kvinnsland et al., 2004). Ngf is specifically expressed in the mesenchymal dental axon pathway when pioneer dental axons are navigating towards the early bud stage tooth germ (Luukko et al., 1997a). Subsequently, Ngf becomes expressed together with Gdnf in the dental follicle target field (Luukko et al., 1997a, b; Nosrat et al., 1998). Postnatally, both genes are upregulated in the odontoblastic target area of the dental papilla consisting of dentin, predentin, odontoblasts, and the underlying mesenchymal subodontoblastic area before axon ingrowth to the dental papilla (Luukko et al., 1997a, b), though they appear not to initiate axon growth to the dental papilla (Lillesaar et al., 1999). Among the other neuroregulatory molecules implicated in dental axon guidance are Ncam (neural cell adhesion molecule), which appears to mediate contact attraction on growing axons, and secreted Netrin-3 expressed in dental target fields (Obara and Takeda, 1993; Loes et al., 2003). Cell membrane–bound ephrin ligands and their Eph receptor tyrosine kinases regulate axon guidance by acting as repellants, but they can also function as axonal attractors (Kullander and Klein, 2002). Localization of ephrin-A ligands and their EphA receptors, in particular EphA3 and -A7 as well as EphrinA5 in the developing tooth, suggests that they may contribute to dental axon guidance as well as defasciculation and arborization of dental axons (Luukko et al., 2005).
Collectively, these results suggest that different families of neuroregulatory molecules and axon guidance mechanisms act simultaneously and in a coordinated manner to direct dental trigeminal axon pathfinding and establishment of the tooth innervation pattern. The finding that key molecules such as Ngf and Gdnf are first expressed in the dental follicle target field and later postnatally upregulated in the odontoblastic target area in the dental papilla (Luukko et al., 1997a, b) indicates that the same neuroregulatory molecules are repetitively used at different stages of tooth innervation development. In addition, these results suggest that local regulation of their expression at defined sites is crucial for the establishment of tooth nerve supply. However, until the recent analysis of the functions and regulation of Semaphorin3A (Sema3A), evidence regarding the basal regulatory mechanisms of establishment of tooth innervation was lacking.
SEMAPHORIN3A CONTROLS THE TIMING OF TOOTH INNERVATION AND NERVE FIBER PATTERNING
Sema3A is a secreted chemorepellant, which regulates the patterning and fasciculation of the peripheral nerve fibers including the trigeminal nerves (Taniguchi et al., 1997; Ulupinar et al., 1999). In the developing molar tooth, Sema3A shows a distinct, developmentally regulated cellular expression pattern in areas that harbor the mesenchymal dental axon pathway and dental follicle target field (Loes et al., 2001; Kettunen et al., 2005) (Fig. 2). Sema3A is first detected in the presumptive dental mesenchyme under the thickened dental epithelium, and, subsequently, it is expressed in the mesenchymal areas devoid of the dental nerve fibers (Kettunen et al., 2005). Analysis of transgenic Sema3A-deficient mice revealed that numerous trigeminal nerve fibers were prematurely present in the presumptive dental mesenchyme under the oral epithelium. During the bud, cap, and bell stages, many nerve fibers were ectopically premature located in the condensed dental mesenchyme and the dental papilla mesenchyme. These results indicate that Sema3A acts as a local repellant that, by forming dynamic restriction areas, regulates the timing of tooth innervation as well as dental axon navigation and patterning during advancing tooth morphogenesis. The absence of Sema3A from the sites of the developing root canals suggests that Sema3A may participate in defining the sites through which the nerve fibers enter the dental papilla postnatally.
It was also observed that nerve fibers showed increasingly correct localization in Sema3A−/− tooth, namely in the dental follicle target field as odontogenesis proceeded. The findings that Ngf, Gdnf, Ncam, Lanr, and Net3 showed proper expression in the dental follicle target field area in Sema3A−/− tooth (Kettunen et al., 2005) indicate that their expression is not controlled by Sema3A signaling and suggests that local, target field–derived molecules act to rescue the axonal patterning phenotype in Sema3A−/− teeth. These findings provide genetic support for the model that dental axon guidance and establishment of tooth nerve supply involves concerted, redundant signaling activity of neuroregulatory molecules of different families.
EPITHELIAL-MESENCHYMAL INTERACTIONS REGULATE FORMATION OF TOOTH NERVE SUPPLY
The findings that precisely regulated expression domain of Sema3a is crucial for the timing of tooth innervation as well as dental axon guidance and patterning suggest that the developing tooth controls the development of its own nerve supply. This hypothesis receives support from the earlier experimental findings, which have suggested that local mechanisms but not growing nerve fibers control formation of tooth innervation (Luukko et al., 1996, 1997a; Lillesaar and Fried, 2004; Erdelyi et al., 1987; Holland and Robinson, 1987).
Cell interactions are considered the single most important mechanism regulating development in vertebrates (Gurdon, 1992; Saxén, 2001). The series of inductive events during organ formation is termed “secondary induction” to distinguish it from primary induction, which leads to the formation of the neural ectoderm in vertebrate embryos (Spemann and Mangold, 1924). The tooth develops as a result of tissue interactions between the epithelial and cranial neural crest–derived ectomesenchymal cells (Mina and Kollar, 1987; Lumsden, 1988). The interactions control all stages of tooth formation including dental patterning, tooth initiation, morphogenesis, and dental cell differentiation (for a review see Thesleff and Nieminen, 2001). The finding that developmentally changing expression domains of Sema3A during tooth morphogenesis are essential for its functions made it an excellent marker gene for analysis of the basal regulatory mechanism of tooth innervation development. Tissue recombination experiments showed that the early odontogenic epithelium induces Sema3A expression in the early presumptive dental mesenchyme that is before the establishment of the molar nerve (Kettunen et al., 2005). Later during dental axon growth, the dental epithelium continues to control Sema3A expression in the dental mesenchyme. Given the essential function of Sema3A in tooth innervation development, these results provided the first evidence that local epithelial-mesenchymal interactions control the formation of tooth nerve supply.
The tooth is a highly specialized organ and trigeminal sensory nerve fibers innervate the structures not found in other parts of the animal, such as dentin and periodontal ligament. As a consequence of this, the composition and function of the trigeminal innervation supplying dental target areas differs from that innervating other organs and tissues (for a review see Fried et al., 2000; Kvinnsland et al., 2004). Collectively, the tooth sensory system as a whole is unique and not found in other parts of the body. It is obvious that these distinct features must result in tooth-specific molecular regulation of the development of the trigeminal nerve supply and that this involves instructions from the tooth target organ itself. Classical tissue recombination studies and evidence accumulated from molecular and cellular studies have established that E10–E11 mouse oral and early dental epithelium possesses the instructive information for tooth organ formation (Mina and Kollar, 1987; Lumsden, 1988; Tucker et al., 1998). The finding that the early odontogenic epithelium controls tooth innervation development indicates that, in addition to the odontogenic potential, the dental epithelium possesses the “neurogenic” information to instruct dental trigeminal axon guidance and patterning, and most probably the tooth-specific sensory trigeminal nerve supply as well.
EPITHELIAL-MESENCHYMAL INTERACTIONS MEDIATED BY SIGNALING MOLECULES COORDINATE TOOTH FORMATION AND ESTABLISHMENT OF TOOTH TRIGEMINAL NERVE SUPPLY
There is increasing experimental and genetic evidence that secreted signals, in particular belonging to Fgf (fibroblast growth factors), Bmp (bone morphogenetic proteins), hh (Hedgehog), and Wnt families, mediate tissue interactions and regulate gene expression in the presumptive dental mesenchyme (Miletich and Sharpe, 2003; Thesleff, 2003). Analysis of the effects of signaling molecules belonging to these families revealed that Wnt4 and Tgfβ1 regulate Sema3A expression. Epithelial Wnt4 induced Sema3A in the presumptive molar mesenchyme at E10.5. Later at E11.5 and E12.5, Wnt4 and Tgfβ1 (Cam et al., 1990; Vaahtokari et al., 1991), which become expressed at the epithelial bud, stimulated Sema3A in the dental mesenchyme. Thus, Wnt4 and Tgfβ1 are putative in vivo signals, which mediate tissue interactions and control Sema3A. Results from the other developing tissues and organs suggest that Wnt4 and Tgfβ1 may also regulate local expression of other key neuroregulatory molecules during establishment of tooth nerve supply (Fig. 3). For instance, in the developing limb epithelial Wnt4 controls mesenchymal Nt3, which appears to be essential for initial stages of tooth innervation (Luukko et al., 1997a; Patapoutian et al., 1999). In the developing skin, Tgfβ1–3 upregulate Ngf and downregulate Nt3 levels in vitro (Buchman et al., 1994), and epithelial signaling controls Nt3 in the maxillary process mesenchyme (O'Connor and Tessier-Lavigne, 1999).
Besides regulating tooth innervation, Wnt and Tgfβ signaling is needed for tooth formation (van Genderen et al., 1994; Andl et al., 2002; Kratochwil et al., 2002; Ito et al., 2001). In addition, Wnt4 promoted expression of Msx1 transcription factor, which is essential for tooth development, in the early mandibular mesenchyme (Satokata and Maas, 1994; Kettunen et al., 2005), while Tgfβ1 stimulated cell proliferation, which is crucial for organ morphogenesis (Kettunen et al., 2005). Thus, Wnt4 and Tgfβ1 are putative signals to coordinate tooth morphogenesis with dental trigeminal axon growth and patterning. Because Wnt4 and Tgfβ1 are part of the larger signaling networks regulating tooth organogenesis (Vainio et al., 1993; Kratochwil et al., 2002; Aberg et al., 2004), collectively, these results indicate that signaling pathways regulating formation of tooth nerve supply are integrated with signaling networks controlling tooth organogenesis. This is proposed to provide a central mechanism whereby dental axon growth and patterning as well as the development of tooth-specific innervation are spatiotemporally coordinated with tooth organ formation. However, it is obvious that many of the signals, signaling networks, and feedback loops involved in this process are yet to be discovered.
Molecular and genetic analyses have provided evidence that signaling pathways regulating tooth organ formation are integrated at many levels and form complex networks. It has been known for a long time that tooth organ development depends on a series of reciprocal inductive interactions between epithelial and mesenchymal tissue components. Recent advances in developmental neurobiology have provided evidence that developing tooth controls the formation of its own nerve supply by epithelial-mesenchymal interactions. These “neurogenic” interactions appear to be mediated by diffusible signals, which are part of signaling networks regulating the formation of the tooth organ proper. As tissue interactions regulate organogenesis, it is possible that they provide a central mechanism to coordinate organ formation and the development of their nerve supply. Interestingly, the formation of blood vessels is also tightly coupled with organ formation and there is evidence, for instance, from the developing lung that tissue interactions and locally expressed signal molecules regulate lung angiogenesis (reviewed in Warburton et al., 2000). Moreover, many axon guidance molecules expressed in the developing organs such as Sema3A have been recently found to regulate angiogenesis (Serini et al., 2003) suggesting common regulatory mechanisms for the establishment of vascular and neural networks (for a review see Eichmann et al., 2005). Collectively, these results suggest that local tissue interactions may provide a general mechanism to orchestrate development of the organs and their supporting tissues.
We thank Ms. Kjellfrid Haukanes and Ms. Helen Olsen for their skillful technical assistance.