Expression patterns of Sema3F, PlexinA4, -A3, Neuropilin1 and -2 in the postnatal mouse molar suggest roles in tooth innervation and organogenesis.

OBJECTIVE: Semaphorins form a family of axon wiring molecules but still little is known about their role in tooth formation. A class 3 semaphorin, Semaphorin3F (Sema3F), besides acting as a chemorepellant for different types of axons, controls a variety of non-neuronal developmental processes. MATERIALS AND METHODS: Cellular mRNA expression patterns of Sema3F as well as neuropilin 1 (Npn1), neuropilin 2 (Npn2), plexinA3 and plexinA4 receptors were analyzed by sectional in situ hybridization in the mouse molar tooth during postnatal days 0-7. The expression of the receptors was studied in PN5 trigeminal ganglia. RESULTS: Sema3F, Npn1, -2 and plexinA4 exhibited distinct, spatiotemporally changing expression patterns, whereas plexinA3 was not observed in the tooth germs. Besides being expressed in the base of the dental mesenchyme Sema3F, like plexinA4, Npn1 and -2, was present in the ameloblast cell lineage. Npn1 and Npn2 were additionally seen in the pulp horns and endothelial cells and like PlexinA4 in the developing alveolar bone. Npn1, plexinA3 and -A4 were observed in trigeminal ganglion neurons. CONCLUSIONS: Sema3F may act as a tooth target-derived axonal chemorepellant controlling establishment of the tooth nerve supply. Furthermore, Sema3F, like Npn1, -2 and plexinA4 may serve non-neuronal functions by controlling the development of the ameloblast cell lineage. Moreover, Npn1 and Npn2 may regulate dental vasculogenesis and, together with PlexinA4, alveolar bone formation. Further analyses such as investigation of transgenic mouse models will be required to elucidate in vivo signaling functions of Sema3F and the receptors in odontogenesis.

Developmentally regulated expression of Sema3A chemorepellant in the developing mouse incisor.

OBJECTIVE: Semaphorin 3A (Sema3A) is an essential chemorepellant controlling peripheral axon pathfinding and patterning, but also serves non-neuronal cellular functions. Incisors of rodent are distinctive from molars as they erupt continuously, have only one root and enamel is present only on the labial side. The aim of this study is to address putative regulatory roles of Sema3A chemorepellant in the development of incisor innervation and formation. MATERIALS AND METHODS: This study analyzed expression of Sema3A mRNAs during embryonic and early post-natal stages of mouse mandibular incisor using sectional radioactive in situ hybridization. RESULTS: Although Sema3A mRNAs were observed in condensed dental mesenchyme during the early bud stage, they were absent in dental papilla or pulp at later stages. Sema3A mRNAs were observed in the dental epithelium including the cervical loops and a prominent expression was also seen in alveolar bone. Interestingly, transcripts were absent from the mesenchymal dental follicle target area (future periodontal ligament) throughout the studied stages. CONCLUSION: The expression patterns of Sema3A indicate that it may control the timing and patterning of the incisor innervation. In particular, Sema3A appears to regulate innervation of the periodontal ligament, while nerve penetration into the incisor dental pulp appears not to be dependent on Sema3A. Moreover, Sema3A may regulate the functions of cervical loops and the development of alveolar bone. Future study with Sema3A deficient mice will help to elucidate the putative neuronal and non-neuronal functions of Sema3A in incisor tooth development.

Developmentally regulated expression of intracellular Fgf11-13, hormone-like Fgf15 and canonical Fgf16, -17 and -20 mRNAs in the developing mouse molar tooth.

OBJECTIVE: To investigate and compare the cellular expression of non-secreted Fgf11-14 and secreted Fgf15-18 and -20 mRNAs during tooth formation. MATERIALS AND METHODS: mRNA expression was analyzed from the morphological initiation of the mouse mandibular first molar development to the onset of crown calcification using sectional in situ hybridization. RESULTS: This study found distinct, differentially regulated expression patterns for the Fgf11-13, -15-17 and -20, in particular in the epithelial-mesenchymal interface, whereas Fgf14 and 18 mRNAs were not detected. Fgf11, -15, -16, -17 and -20 were seen in the epithelium, whereas Fgf12 and -13 signals were restricted to the mesenchymal tissue component of the tooth. Fgf11 was observed in the putative epithelial signaling areas, the tertiary enamel knots and enamel free areas of the calcifying crown. Fgf15, Fgf17 and -20 were transiently colocalized in the thickened dental epithelium at E11.5. Later Fgf15 and -20 were exclusively expressed in the epithelial enamel knot signaling centers. In contrast, Fgf13 was present in the dental mesenchyme including odontoblasts cell lineage, whereas Fgf12 appeared transiently in the preodontoblasts. CONCLUSIONS: The expression of the Fgf11-13, -15, -17 and -20 in the epithelial signaling centers and/or epithelial-mesenchymal interfaces at key stages of the tooth formation suggest important functions in odontogenesis. Future analyses of the transgenic mice will help elucidate in vivo functions of the studied Fgfs during odontogenesis and whether any of the functions of the tooth expressed epithelial and mesenchymal Fgfs of different sub-families are redundant.

Development of the pioneer sympathetic innervation into the dental pulp of the mouse mandibular first molar.

OBJECTIVE: Our goal was to study the development of pioneer sympathetic innervation of dental pulp of mouse mandibular first molar. DESIGN: We used double fluorescent immunohistochemistry with tyrosine hydroxylase (TH) and anti-medium-chain neurofilament (2H3) antibodies to detect sympathetic and sensory nerve fibres. Serial sections of whole teeth from postnatal days (PN) 0-14, trigeminal and sympathetic superior cervical ganglia of PN 15 mice were examined with confocal microscope. RESULTS: There were two main findings. The unexpected finding was that 2H3 antibody was specific only for sensory nerve fibres and neurons and failed to stain either sympathetic nerve fibres or neurons. The main finding was that although both sympathetic and sensory nerve fibres were already seen near the tooth germ at the newborn stage, the pioneer sympathetic nerve fibres were first observed in the dental pulp only after the onset of root formation on day 9, in contrast to sensory nerve fibres which entered the tooth already on day 4. CONCLUSION: Pioneer sympathetic innervation of dental pulp starts on postnatal day 9 and follows sensory innervation. This indicates differential developmental regulation of the initial sensory and sympathetic innervation of teeth and provides essential background data for further studies on the molecular regulation of pulp innervation.

Fgfr2b mediated epithelial-mesenchymal interactions coordinate tooth morphogenesis and dental trigeminal axon patterning.

Dental trigeminal nerve fiber growth and patterning are strictly integrated with tooth morphogenesis, but it is still unknown, how these two developmental processes are coordinated. Here we show that targeted inactivation of the dental epithelium expressed Fgfr2b results in cessation of the mouse mandibular first molar development at the degenerated cap stage and the failure of the trigeminal molar nerve to establish the lingual branch at E13.5 stage while the buccal branch develops properly. This axon patterning defect correlates to the histological absence of the mesenchymal dental follicle and adjacent Semaphorin3A-free dental follicle target field as well as appearance of ectopic Sema3A expression domain in the lingual side of the epithelial bud. Although the mesenchymal ligands for Fgfr2b, Fgf3 and -10 were present in the Fgfr2b(-/)(-) dental mesenchyme, mutant dental epithelium showed dramatically reduced proliferation and the lack of Fgf3. Tgfbeta1, which controls Sema3A was absent from the Fgfr2b(-/-) tooth germ, and Sema3A was specifically downregulated in the dental mesenchyme at the bud and cap stage. In addition, the epithelial primary enamel knot signaling center although being molecularly present neither was histologically detectable nor expressed Bmp4 and Fgf3 as well as Fgf4, which is essential for tooth morphogenesis and stimulates mesenchymal Fgf3 and Tgfbeta1. Fgf4 beads rescued Tgfbeta1 in the Fgfr2b(-/-) dental mesenchyme explants and Tgfbeta1 induced de novo Sema3A expression in the dental mesenchyme. Collectively these results demonstrate that epithelial Fgfr2b controls tooth morphogenesis and dental axon patterning, and suggests that Fgfr2b, by mediating local epithelial-mesenchymal interactions, integrates these two distinct developmental processes during odontogenesis.

Histological development and dynamic expression of Bmp2-6 mRNAs in the embryonic and postnatal mouse cranial base.

The cranial base is formed by endochondral ossification and is characterized by the presence of the synchondrosis growth centers. The aim of this study was to describe the histological development of the mouse midsagittal cranial base area from embryonic day 10 (E10) to the postnatal age of 2 months. The Bmp family of signaling molecules serves important functions in embryo and bone development and may therefore play a significant role in the early formation of the cranial base. To investigate this, we analyzed the mRNA pattern of expression of Bmp2-6 in the mouse cranial base from E10 to 5 days postnatally using radioactive in situ hybridization. We found that the formation of the mouse cranial base corresponds to that of rat and proceeds in a caudorostral sequence. Moreover, all Bmps studied showed distinct and overlapping developmentally regulated expression domains. Bmp2, Bmp5, and Bmp6 were expressed in the early mesenchymal condensations. Later, Bmp2, Bmp3, Bmp4, and Bmp5 were detected in the perichondrium and in the adjacent mesenchyme. Subsequently, Bmp2 and Bmp6 expressions were confined to hypertrophic chondrocytes, while Bmp3, Bmp4, and Bmp5 were expressed in the osteoblasts of the trabecular bone and bone collar. Interestingly, Bmp3 was uniquely expressed postnatally in the resting zone of the synchondrosis growth center, suggesting a role in the regulation of cranial base growth. These results suggest that Bmp signaling may serve specific and synergistic functions at different key stages of cranial base development and growth.

Tissue interactions in the regulation of axon pathfinding during tooth morphogenesis.

Like many other organs, the tooth develops as a result of the epithelial-mesenchymal interactions. In addition, the tooth is a well-defined peripheral target organ for sensory trigeminal nerves, which are required for the function and protection of the teeth. Dental trigeminal axon growth and patterning are tightly linked with advancing tooth morphogenesis and cell differentiation. This review summarizes recent findings on the regulation of dental axon pathfinding, which have provided evidence that the development of tooth trigeminal innervation is controlled by epithelial-mesenchymal interactions. The early dental epithelium possesses the information to instruct tooth nerve supply, and signals mediating these interactions are part of the signaling networks regulating tooth morphogenesis. Tissue interactions, thus, appear to provide a central mechanism of spatiotemporally orchestrating tooth formation and dental axon navigation and patterning.

Developmentally regulated expression of Shh and Ihh in the developing mouse cranial base: comparison with Sox9 expression.

The cranial base, located between the cranial vault and the facial bones, plays an important role in integrated craniofacial development and growth. Transgenic Shh and Sox9-deficient mice show similar defects in cranial base patterning. Therefore, in order to examine potential interactions of Shh, Ihh, another member of the Hh family, and Sox9 during cranial base development and growth, we investigated their cellular mRNA expression domains in the embryonic (E) and early postnatal (PN) cranial base from E10 to PN5 using sectional radioactive 35-S in situ hybridization. Of the Hhs, Shh was observed in the foregut epithelium and the notochord, while Sox9 showed broad expression in the loose mesenchyme of the cranial base area during E10-E11. Subsequently, from E12 onward, all genes were observed in the developing cranial base, and after birth the genes were prominently colocalized in the prehypertrophic chondrocytes of the synchondroses. Collectively, these data suggest that Hh-Sox9 auto- and paracrine signaling interactions may provide a critical mechanism for regulating the patterning of the cranial base as well as for its development and growth.

Dynamic expression of Wnt signaling-related Dickkopf1, -2, and -3 mRNAs in the developing mouse tooth.

Wnt signaling is essential for tooth formation. Members of the Dickkopf (Dkk) family modulate the Wnt signaling pathway by binding to the Wnt receptor complex. Comparison of Dkk1, -2, and -3 mRNA expression during mouse tooth formation revealed that all three genes showed distinct spatiotemporally regulated expression patterns. Dkk1 was prominently expressed in the distal, incisor-bearing mesenchyme area of the mandibular process during the initial stages of tooth formation. During molar morphogenesis Dkk1 was detected in the dental mesenchyme, including the preodontoblasts. Dkk2 was seen in the dental papilla, whereas Dkk3 was specifically expressed in the putative epithelial signaling centers, the primary and secondary enamel knots. Postnatally, Dkk1 was prominently expressed in the preodonto- and odontoblasts, while Dkk3 mRNAs were transiently seen in the preameloblasts before the onset of enamel matrix secretion. These results suggest that modulation of Wnt-signaling by Dkks may serve important functions in patterning of dentition as well as in crown morphogenesis and dental hard-tissue formation.

Developmental expression of Dkk1-3 and Mmp9 and apoptosis in cranial base of mice.

The Dickkopf (Dkk) family and Mmp9 are important for apoptosis and a number of other developmental processes. However, little is known about their roles in the development of cranial base, which is an important structure for coordinated development and growth of the craniofacial skeletons. In order to establish whether and in what way these genes are involved in cranial base development, we examined their expression patterns and cell apoptosis. Dkk1 was first seen in the perichondral mesenchyme in restricted domains from E14, and later in the migrating mesenchymal cells within the cartilage. Thereafter, it was widespread throughout the bones of the cranial base. The expression was downregulated in postnatal stages. Dkk2 was localized in the perichondral mesenchyme outlining the anterior cranial base in embryogenesis. Dkk3 was mainly detected in the occipital-vertebral joint at E13 and E14. Mmp9 transcripts were clustered in the inner layer of perichondral mesenchyme, juxtaposed with the terminally differentiated hypertrophic chondrocytes from E14. Later Mmp9-expressing cells were found at the sites of chondrocyte apoptosis. This was particularly clear at the distal ends of the synchondroses. These data indicate that Mmp9 regulates skeletogenesis in cranial base in a manner that is largely similar to that of the appendicular skeletons. Expression of Dkks suggests other roles that remain to be defined.

Expression of ephrin-A ligands and EphA receptors in the developing mouse tooth and its supporting tissues.

Ephrins are cell-membrane-bound ligands for Eph receptor tyrosine kinases and regulate a variety of developmental processes. In order to investigate the potential roles of the ephrin-Eph system in tooth formation, we studied the cellular mRNA expression of Ephrin-A1-A5 and EphA2, EphA3, EphA4, EphA7, and EphA8 receptors during embryonic histomorphogenesis of the mouse first molar (embryonic days 11.5-18.5). Ephrin-A1, ephrin-A5, EphA2, EphA3, EphA4, and EphA7 were expressed in the tooth germ at the epithelial thickening stage, and later, ephrin-A1, ephrin-A5, EphA2, EphA4, and EphA7 showed distinct expression patterns in the enamel organ undergoing epithelial folding morphogenesis. Prior to birth, ephrin-A1, ephrin-A5, EphA2, and EphA4 transcripts were present in the cuspal area of the dental papilla including the preodontoblasts. In addition, ephrin-A1 and ephrin-A5 were seen in the forming blood vessels and alveolar bone, respectively. In contrast, ephrin-A2, ephrin-A3, and ephrin-A4 showed ubiquitous expression during odontogenesis, whereas EphA8 transcripts were not observed. During dental trigeminal axon pathfinding (embryonic days 12.5-13.5), ephrin-A2, ephrin-A4, and ephrin-A5 were evenly distributed in the trigeminal ganglion, whereas EphA7 was expressed in a subset of ganglion cells. These results suggest regulatory roles for ephrin-A/EphA signaling in the formation of the tooth organ proper and its supporting tissues.

Coordination of trigeminal axon navigation and patterning with tooth organ formation: epithelial-mesenchymal interactions, and epithelial Wnt4 and Tgfbeta1 regulate semaphorin 3a expression in the dental mesenchyme.

During development, trigeminal nerve fibers navigate and establish their axonal projections to the developing tooth in a highly spatiotemporally controlled manner. By analyzing Sema3a and its receptor Npn1 knockout mouse embryos, we found that Sema3a regulates dental trigeminal axon navigation and patterning, as well as the timing of the first mandibular molar innervation, and that the effects of Sema3a appear to be mediated by Npn1 present in the axons. By performing tissue recombinant experiments and analyzing the effects of signaling molecules, we found that early oral and dental epithelia, which instruct tooth formation, and epithelial Wnt4 induce Sema3a expression in the presumptive dental mesenchyme before the arrival of the first dental nerve fibers. Later, at the bud stage, epithelial Wnt4 and Tgfbeta1 regulate Sema3a expression in the dental mesenchyme. In addition, Wnt4 stimulates mesenchymal expression of Msx1 transcription factor, which is essential for tooth formation, and Tgfbeta1 proliferation of the dental mesenchymal cells. Thus, epithelial-mesenchymal interactions control Sema3a expression and may coordinate axon navigation and patterning with tooth formation. Moreover, our results suggest that the odontogenic epithelium possesses the instructive information to control the formation of tooth nerve supply.

Glial cell line-derived neurotrophic factor (GDNF) from adult rat tooth serves a distinct population of large-sized trigeminal neurons.

Glial cell line-derived neurotrophic factor (GDNF) mediates trophic effects for specific classes of sensory neurons. The adult tooth pulp is a well-defined target of sensory trigeminal innervation. Here we investigated potential roles of GDNF in the regulation of adult trigeminal neurons and the dental pulp nerve supply of the rat maxillary first molar. Western blot analysis and radioactive 35S-UTP in situ hybridization revealed that GDNF in the dental pulp and its mRNAs were localized with Ngf in the coronal pulp periphery, in particular in the highly innervated subodontoblast layer. Retrograde neuronal transport of iodinated GDNF and Fluorogold (FG) from the dental pulp indicated that GDNF was transported in about one third of all the trigeminal dental neurons. Of the GDNF-labelled neurons, nearly all (97%) were large-sized (> or =35 microm in diameter). Analysis of FG-labelled neurons revealed that, of the trigeminal neurons supporting the adult dental pulp, approximately 20% were small-sized, lacked isolectin B4 binding and did not transport GDNF. Of the large-sized dental trigeminal neurons approximately 40% transported GDNF. About 90% of the GDNF-accumulating neurons were negative for the high-temperature nociceptive marker VRL-1. Our results show that a subclass of large adult trigeminal neurons are potentially dependent on dental pulp-derived GDNF while small dental trigeminal neurons seems not to require GDNF. This suggests that GDNF may function as a neurotrophic factor for subsets of nerves in the tooth, which apparently mediate mechanosensitive stimuli. As in dorsal root ganglia both small- and large-sized neurons are known to be GDNF-dependent; these data provide molecular evidence that the sensory supply in the adult tooth differs, in some aspects, from the cutaneous sensory system.

CGRP1 and NK1 receptors in postnatal, developing rat dental tissues.

There is little evidence that neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) participate in the regulation of tooth development. The aim of this study was to analyse the expression of their respective receptors, neurokinin (NK) 1 and CGRP1 receptor, in postnatal developing rat molars and supporting tissues, thereby localizing the target areas for neuropeptide activity. Mol:WIST rats were killed at 7, 14 and 21 d after birth and upper and lower jaws were processed for immunohistochemistry. At early crown stage (P7), only a few individual cells in the dental follicle were receptor positive. At the onset of root formation (P14), post-secretory ameloblasts, cells in the stratum intermedium, the reduced enamel epithelium and the developing alveolar bone demonstrated both NK1 and CGRP1 receptor immunoreactivity. The CGRP1 receptor sites were occasionally evident on cells in the odontoblast layer. At advanced root development (P21), neuropeptide receptor expression was evident on cells close to the developing dentin, cementum and alveolar bone. These data demonstrate dynamic changes in the localization of NK1 and CGRP1 receptors in developing rat dental tissues and indicate an active role for their ligands in the regulation of crown and root development.

Identification of a novel putative signaling center, the tertiary enamel knot in the postnatal mouse molar tooth.

The final shape of the molar tooth crown is thought to be regulated by the transient epithelial signaling centers in the cusp tips, the secondary enamel knots (SEKs), which are believed to disappear after initiation of the cusp growth. We investigated the developmental fate of the signaling center using the recently characterized Slit1 enamel knot marker as a lineage tracer during morphogenesis of the first molar and crown calcification in the mouse. In situ hybridization analysis showed that after Fgf4 downregulation in the SEK, Slit1 expression persisted in the deep compartment of the knot. After the histological disappearance of the SEK, Slit1 expression was evident in a novel epithelial cell cluster, which we call the tertiary enamel knot (TEK) next to the enamel-free area (EFA)-epithelium at the cusp tips. In embryonic tooth, Slit1 was also observed in the stratum intermedium (SI) and stellate reticulum cells between the parallel SEKs correlating to the area where the inner enamel epithelium cells do not proliferate. After birth, the expression of Slit1 persisted in the SI cells of the transverse connecting lophs of the parallel cusps above the EFA-cells. These results demonstrate the presence of a novel putative signaling center, the TEK, in the calcifying tooth. Moreover, our results suggest that Slit1 signaling may be involved in the regulation of molar tooth shape by regulating epithelial cell proliferation and formation of EFA of the crown.