Inger Hals Kvinnsland

Nerve fibres and cells immunoreactive to neurochemical markers in developing rat molars and supporting tissues

The distribution of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP), substance P (SP) and neuropeptide Y (NPY) was compared to the general neurochemical markers for nerves and neuroendocrine cells protein gene product 9.5 (PGP 9.5) and neurone-specific enolase (NSE), by use of the avidin-biotin peroxidase complex method in developing dental structures in rats aged 13 to 27 days. A substantially greater part of the nerve fibres was immunoreactive to CGRP and SP than to NPY. In the bell stage, nerve fibres immunoreactive to PGP 9.5, CGRP and SP were found in the dental follicle but not in the dental papilla and stellate reticulum. In the advanced bell stage, after initiation of dentine and enamel formation, PGP 9.5, CGRP- and SP-immunoreactive fibres were found in the dental papilla, while the first NPY-immunoreactive fibres were observed in the papilla when root formation started. Concomitant with the beginning of root development, a subodontoblastic nerve plexus was gradually formed and PGP 9.5-, CGRP- and SP-immunoreactive fibres were found within the dentinal tubules. From the start of root formation, CGRP-, SP- and NPY-immunoreactive nerves were shown in the developing periodontal ligament, although a mature distribution pattern was not observed until root formation was nearly completed. Ameloblasts, odontoblasts and cell-like structures in the outer enamel epithelium and within the dental lamina were PGP 9.5-immunoreactive at the bell stage. As the tooth matured, the immunolabelling gradually decreased, but was still present in some odontoblasts after tooth eruption. NSE-immunoreactive, cell-like structures were found in the periphery of the dental follicle, and persisted close to alveolar bone in the periodontal ligament when the tooth reached occlusion. Hence, it may be concluded that sensory nerves containing SP and CGRP are present in the pulp in advance of sympathetic nerves immunoreactive to NPY.

Regeneration of calcitonin gene-related peptide immunoreactive nerves in replanted rat molars and their supporting tissues

First maxillary right molars in 66 rats were elevated and replanted and the pulps allowed to regenerate for 1-90 days. The contralateral tooth served as control. Regeneration of nerves in the pulp and periodontium was studied by CGRP-immunohistochemistry and the avidin-biotin-peroxidase method. The pulp and periodontium of the controls were richly supplied with CGRP-labelled nerves. One day after replantation the pulp was completely devoid of CGRP-immunoreactive nerves. After 2 days, axon sprouts were present in the apical, regenerated pulp and in the periodontium. From 3-7 days CGRP-immunoreactive axons were regularly seen to have regenerated in front of the cellular inflammation in the pulp. After 10 days, the pulps were reinnervated up to the horns, although more sparsely than in the controls. From day 20-90 there was a marked divergence in pulpal healing: 17 pulps formed irregular postoperative dentine with a gradual increase in nerve density; 16 pulps remained sparsely innervated and were gradually replaced by bone. Root resorption was most extensive in the teeth with bone replacement of pulp. The soft tissue adjacent to extensive resorbing areas had many more CGRP-labelled axons than in the controls. The reinnervation of the regenerating pulp occurred at the same time as pulpal wound healing, but did not achieve the innervation density of the controls.

Nerve fibers immunoreactive to protein gene product 9.5, calcitonin gene-related peptide, substance P, and neuropeptide Y in the dental pulp, periodontal ligament, and gingiva in cats

The distribution patterns of nerve fibers immunoreactive (IR) to calcitonin gene-related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY) in the dental pulp, periodontal ligament (PDL), and gingiva were studied and compared with the complete innervation visualized by antibody to protein gene product (PGP) 9.5 in adult cats. The pulp showed considerably denser nerve supply for PGP 9.5, CGRP, and SP than the periodontal tissues. Most of the pulpal fibers were CGRP-IR, and approximately three to four times more IR fibers were labeled with CGRP than SP. Most fibers in the odontoblast area penetrating into the dentin tubules were CGRP-IR. NPY-IR nerves were mainly observed in connection with the larger blood vessels in pulp and PDL. In the PDL most nerves were localized in the apical third in connection with blood vessels, but CGRP-IR fibers extending close to root cementum were often observed. Immunoreactivity to PGP 9.5 and CGRP was frequently found in cell-like structures in connection with Malassez epithelium in the PDL and in some round epithelial-like cells located in the base of gingival rete pegs.

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

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 Tgfβ1 regulate Sema3a expression in the dental mesenchyme. In addition, Wnt4 stimulates mesenchymal expression of Msx1 transcription factor, which is essential for tooth formation, and Tgfβ1 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.

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 Dynamics 233:161–166, 2005.

Expression of class 3 semaphorins and neuropilin receptors in the developing mouse tooth.

The semaphorins are a large family of secreted or cell-bound signals needed for the development of the nervous system. We compared mRNA expression of class 3 semaphorins (Sema3A, 3B, 3C and 3F) and their two receptors (Neuropilin-1 and -2) in the embryonic mouse first molar tooth germ (E10-18) by radioactive in situ hybridization. All genes showed distinct developmentally regulated expression patterns during tooth organogenesis. Interestingly, Sema3A and 3C were first detected in the early dental epithelium, and later both genes were present in the epithelial primary enamel knot, a putative signaling center of the embryonic tooth regulating tooth morphogenesis. Prior to birth, Sema3A was also observed in tooth-specific cells, preodontoblasts, which later differentiate into odontoblasts secreting dentin, and in the mesenchymal dental follicle cells surrounding the tooth germ. Sema3B appeared transiently in the dental mesenchyme in the bud and cap stage tooth while Sema3F was expressed in both epithelial and mesenchymal components of the tooth. Of note, Npn-1 expression pattern was largely complementary to that of Sema3A, and transcripts were restricted to the dental mesenchymal cells. Npn-1 expression was first seen in the developing dental follicle, and later transcripts also appeared in the dental papilla mesenchyme. In contrast, Npn-2 signal was seen in both epithelial and mesenchymal tissues such as in the primary enamel knot and preodontoblasts.

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. Developmental Dynamics 234:482–488, 2005.

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.

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 (≥35 µm in diameter). Analysis of FG‐labelled neurons revealed that, of the trigeminal neurons supporting the adult dental pulp, ≈ 20% were small‐sized, lacked isolectin B4 binding and did not transport GDNF. Of the large‐sized dental trigeminal neurons ≈ 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.

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.