Essential roles of G9a in cell proliferation and differentiation during tooth development.

Teeth develop through interactions between epithelial and mesenchymal tissues mediated by a signaling network comprised of growth factors and transcription factors. However, little is known about how epigenetic modifiers affect signaling pathways and thereby regulate tooth formation. We previously reported that the histone 3 lysine 9 (H3K9) methyltransferase (MTase) G9a is specifically enriched in the tooth mesenchyme during mouse development. In this study, we investigated the functions of G9a in tooth development using G9a conditional knockout (KO) mice. We used Sox9-Cre mice to delete G9a in the tooth mesenchyme because Sox9 is highly expressed in the mesenchyme derived from the cranial neural crest. Immunohistochemical analyses revealed that G9a expression was significantly decreased in the mesenchyme of Sox9-Cre;G9afl/fl (G9a cKO) mice compared with that in Sox9-Cre;G9a fl/+(control) mice. Protein levels of the G9a substrate H3K9me2 were also decreased in the tooth mesenchyme. G9a cKO mice showed smaller tooth germ after embryonic day (E) 16.5 and E17.5, but not at E15.5. The developing cusp tips, which were visible in control mice, were absent in G9a cKO mice at E17.5. At 3 weeks after birth, small first molars with smaller cusps and unseparated roots were formed. Organ culture of tooth germs derived from E15.5 cKO mouse embryos showed impaired tooth development, suggesting that tooth development per se is affected independently of skull development. BrdU labeling experiments revealed that the proliferation rates were decreased in the mesenchyme in G9a cKO mice at E17.5. In addition, the proliferation rates in the tooth inner enamel epithelium were also decreased. In situ hybridization revealed altered localization of genes associated with tooth development. In cKO mice, intensively localized expression of mRNAs encoding bone morphogenic protein (Bmp2 and Bmp4) was observed in the tooth mesenchyme at E17.5, similar to the expression patterns observed in control mice at E15.5. Localization of Shh and related signaling components, including Gli1, Ptch1, and Ptch2, in the tooth mesenchyme of cKO mice was generally similar to that at earlier stages in control mice. In addition, expression of Fgf3 and Fgf10 in the mesenchyme was decreased in G9a cKO mice at P0. Expression levels of Fgf9 and p21, both of which were expressed in the secondary enamel-knot, were also decreased. Thus, the expression of genes associated with tooth development was delayed in cKO mice. Our results suggest that H3K9MTase G9a regulates cell proliferation and timing of differentiation and that G9a expression in the tooth mesenchyme is required for proper tooth development.

Expression of transcripts for fibroblast growth factor 18 and its possible receptors during postnatal dentin formation in rat molars.

Fibroblast growth factors (FGFs) regulate the proliferation and differentiation of various cells via their respective receptors (FGFRs). During the early stages of tooth development in fetal mice, FGFs and FGFRs have been shown to be expressed in dental epithelia and mesenchymal cells at the initial stages of odontogenesis and to regulate cell proliferation and differentiation. However, little is known about the expression patterns of FGFs in the advanced stages of tooth development. In the present study, we focused on FGF18 expression in the rat mandibular first molar (M1) during the postnatal crown and root formation stages. FGF18 signals by RT-PCR using cDNAs from M1 were very weak at postnatal day 5 and were significantly up-regulated at days 7, 9 and 15. Transcripts were undetectable by in situ hybridization (ISH) but could be detected by in situ RT-PCR in the differentiated odontoblasts and cells of the sub-odontoblastic layer in both crown and root portions of M1 at day 15. The transcripts of FGFR2c and FGFR3, possible candidate receptors of FGF18, were detected by RT-PCR and ISH in differentiated odontoblasts throughout postnatal development. These results suggest the continual involvement of FGF18 signaling in the regulation of odontoblasts during root formation where it may contribute to dentin matrix formation and/or mineralization.

Liver-type of tissue non-specific alkaline phosphatase is induced during mouse bone and tooth cell differentiation.

BACKGROUND AND OBJECTIVE: Tissue non-specific alkaline phosphatase (TNSALP) contains two types-bone- and liver-type-which are produced from the same gene due to differences in splicing. These two differ in their promoter, but the amino acid sequences of the mature proteins are identical. In this study, we examined the relationship between the two types of TNSALP expression and osteoblast differentiation. DESIGN: Gene expression of the two types of TNSALP was observed by reverse transcription-polymerase chain reaction. MC3T3-NM4 was sub-cloned from an established mouse osteoblastic cell line in which osteoblast characters do not appear without dexamethasone. The C2C12 mouse myoblastic cell line, which can be induced to osteoblasts with bone morphogenic protein 2, and organ-cultured tooth germs were also used in this work. RESULTS: The gene expression of liver-type TNSALP was observed in only MC3T3-NM4 activated by dexamethasone. For C2C12, the gene expression of bone-type TNSALP was observed even in non-induced conditions where myotubes were formed, whereas the liver-type TNSALP mRNA was only expressed when C2C12 differentiated into osteoblasts by bone morphogenic protein 2. Furthermore, in the organ-cultured tooth germs, the liver-type TNSALP mRNA was expressed according to differentiation of tooth germs. CONCLUSION: These results suggest that the liver-type TNSALP mRNA is induced according to differentiation of bone and tooth.

Development and terminal differentiation of pulp and periodontal nerve elements in subcutaneous transplants of molar tooth germs and incisors of the rat.

Ectopic tooth transplants are known to receive rich innervation of local neurons, but the precise location and structural features of neurites in the pulp and periodontal ligament (PDL) of such transplants are unclear. In this experiment, the molar tooth germs of rat embryos and incisors of young rats were subcutaneously transplanted into the dorsal regions of rats and processed, at various time intervals, for immunohistochemical demonstration of neural elements. Teeth with periodontal tissue elements developed in most of the molar transplants in 6 or 8 wk and received rich innervation, including some autonomic fibres, in the pulp. Nerve elements were also confirmed to be present in the PDL of these transplants, including specialized nerve ending-like structures reminiscent of the periodontal Ruffini endings. Mechanoreceptor-like structures were also induced in the regenerated PDL of similarly transplanted incisors, although the success rate was low. We conclude that rich and highly ordered innervation of the pulp, and occasional development of mechanoreceptors in the regenerated PDL of ectopic dental transplants, imply a high probability of successful induction of teeth with both nociceptive and mechanical sensations in the ectopic tooth and/or tooth germ transplant systems, although differentiation of mechanoreceptor-like nerve endings occurred in only a few rare cases.

Expression of alternatively spliced RNA transcripts of amelogenin gene exons 8 and 9 and its end products in the rat incisor.

In addition to seven known exons of the amelogenin gene, recent studies have identified two exons downstream of amelogenin exon 7 in genomic DNA of mouse and rat. Here the spatial and temporal expression of mRNAs and of the translated proteins derived from alternative splicing of the amelogenin gene ending with exon 8 and exon 9 were examined by in situ hybridization (ISH) and immunohistochemistry (IHC). RNA signals for exons 8 and 9 were expressed in the ameloblast layer extending from early presecretory to postsecretory transitional stages of amelogenesis. IHC of amelogenin proteins that include sequences encoded by these exons demonstrated identical localization of these proteins in the ameloblast layer corresponding to RNA signals identified by ISH. There was intense immunostaining of the enamel matrix secreted by these cells. Western blotting analysis of rat enamel proteins revealed three distinct protein bands with sequences encoded by the new exons. These data confirmed the existence of the transcripts of alternatively spliced mRNAs coding for exons 8 and 9 of the amelogenin gene in rat tooth germs and suggest that the translated proteins contribute to the heterogeneity of amelogenins and have some significant roles in enamel formation and mineralization.

The influence of parathyroid hormone-related protein (PTHrP) on tooth-germ development and osteoclastogenesis in alveolar bone of PTHrP-knock out and wild-type mice in vitro.

In a previous study, it was shown that tooth germs of neonatal homozygous parathyroid hormone-related protein (PTHrP)-knockout mice are penetrated or compressed by the surrounding alveolar bone, suggesting an important role for PTHrP in the formation and activation of osteoclasts around growing tooth germs. In order to elucidate the role of PTHrP during the development of the tooth germ and related structures, mandibular explants containing cap stage tooth germs of embryonic day 14, homozygous mice were here cultured with or without surrounding alveolar bone. There was no difference in the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells around the first molars of homozygous and wild-type mice. After 10 days of culture, osteoclastic cells were rarely present in explants from homozygous mice and penetration of alveolar bone into the dental papilla was observed. The decline in osteoclast number was partly restored by the addition of PTHrP to the culture. Tooth germs of both wild-type and homozygous mice cultured without alveolar bone developed well, with no apparent structural abnormality; dentine formation was evident after 10 days. These data suggest that PTHrP is not required for the development of the tooth germ proper but is indispensable in promoting the osteoclast formation required to accommodate that development.

Formation of acellular cementum-like layers, with and without extrinsic fiber insertion, along inert bone surfaces of aging c-Src gene knockout mice.

To investigate the long-term effects of c-src deficiency on skeletal and dental tissues, we examined the lower jaws and long bones of c-src gene knockout (c-src KO) mice by histological and histochemical methods. Numerous multinucleated osteoclasts were distributed throughout the mandible in 5-wk-old c-src KO mice, but by 14 wk they had almost completely disappeared from the alveolar bone, leaving tartrate-resistant acid phosphatase (TRAP)-positive layers along the bone surface. Deposition of osteopontin-positive mineralized tissue, reminiscent of acellular afibrillar cementum (AAC), was confirmed along the TRAP-positive bone surface at 14 wk. The layer progressively thickened up to 21 months. A comparable mineralized layer was noted along the trabeculae of long bones as thickened cement lines. In the periostin-rich areas of jaw bones, but not in the long bones, portions of AAC-like mineralized layers were often replaced with and/or covered by acellular extrinsic fiber cementum (AEFC)-like tissue. These data suggest that the deposition of AAC-like mineralized tissue is a general phenomenon that may occur along inert or slowly remodeling bone surfaces under conditions characterized by reduced bone-resorbing activity, whereas the induction of AEFC-like tissue seems to be associated with the expression of certain molecules that are particularly abundant in the microenvironment of the periodontal ligament.

The induction of enamel and dentin complexes by subcutaneous implantation of reconstructed human and murine tooth germ elements.

Tooth induction by xenogenic graft of reconstructed human tooth germ components has never been attempted. Here we report our first attempt at a transplantation of human tooth germ components, heterologously recombined with mouse dental epithelia, into immunocompromised animals. Human third molar tooth germs enucleated from young patients as prophylactic treatment for orthodontic reasons were collected. The whole or minced human dental papilla was reconstructed with human- or mouse molar enamel epithelium, and transplanted in the dorsal aspect of C.B-17/Icr-scid Jcl mice. The transplant of human dental papilla reconstructed with human enamel epithelium formed thin dentin and immature enamel layers by 3 to 4 weeks, but remained extremely small in quantity due to a shortage of epithelial components in the graft. The addition of E16 mouse molar enamel organs (n=10-12) to each graft augmented the formation of tooth germ-like structures, but the differentiation of mouse molar ameloblasts was suppressed. However, once a solid layer of mineralized dentin was established, mouse ameloblasts accelerated their differentiation, and completed the enamel matrix formation and maturation within the following 4 weeks, whereas human ameloblasts, which had interacted with human dental papilla, remained in the stage of matrix formation during the same period. These data imply that, in reconstructed transplants, the differentiation of mouse dental epithelia is restrained by putative suppressive factors derived from human dental papilla until they are separated by mineralized dentin layers that serve as a diffusion barrier. The mouse enamel organ nevertheless retains its own phenotypic characteristics and intrinsic timing of cell differentiation and function.

Expression of dentin matrix protein 1 (DMP1) in nonmineralized tissues.

Dentin matrix protein 1 (DMP1) is an Arg-Gly-Asp-containing acidic phosphoprotein that was originally identified from a rat incisor cDNA library and was thought to be a dentin-specific protein. DMP1 was later shown to express in a number of hard tissue-forming cells, including osteoblasts, osteocytes, ameloblasts, and cementoblasts, and was considered to play important roles in mineralization. Further, DMP1 gene expression was also detected in fetal bovine brain and in newborn mouse brain. These findings indicate the possibility of DMP1 expression in other soft tissues. In the present study, to clarify the significance of DMP1 expression in nonmineralized tissues, we made a specific antibody to mouse DMP1 peptides and demonstrated that DMP1 protein was localized in mouse brain, pancreas, and kidney by immunohistochemistry. Further DMP1 mRNA was detected in nonmineralized mouse tissues including liver, muscle, brain, pancreas, and kidney by RT-PCR. Based on the evidence that the localization and the expression of DMP1 are not restricted to mineralized tissues, we assume that DMP1 may have functions other than the regulation of mineralization.

Possible role of dentin matrix in region-specific deposition of cellular and acellular extrinsic fibre cementum.

The mechanism whereby a region-specific deposition of the two types of cementum (cellular cementum and acellular extrinsic fibre cementum) is regulated on the growing root surface was tested using bisphosphonate-affected teeth of young rats and guinea pigs. The animals were injected subcutaneously with 8 or 10 mg P x kg body weight(-1) x day(-1) of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) for 1 or 2 weeks. In rat molars, HEBP prevented mineralization of newly formed root dentin matrix and totally inhibited de novo deposition of acellular extrinsic fibre cementum. Instead, thick cellular cementum was induced on the non-mineralized root dentin surface, irrespective of the position of the root. In both animals, cellular cementum was also induced on the non-mineralized surface of root analogue dentin in HEBP-affected incisors, where only acellular extrinsic fibre cementum is deposited under normal conditions. In normal rat molars, dentin sialoprotein (DSP) was concentrated along the dentin-cellular cementum border, but not that of dentin and acellular extrinsic fibre cementum. In HEBP-affected rat incisors, DSP was shown to penetrate through the non-mineralized dentin into the surrounding tissues, but not through the mineralized portions. These data suggest that, at the site of cellular cementum formation, putative inducing factors for cellular cementum might diffuse into the periodontal space through the newly deposited mantle dentin matrix before it is mineralized. At earlier stages of root formation, mantle dentin might mineralize more promptly not to allow such diffusion. The timing of mineralization of mantle dentin matrix might be the key determinant of the types of the cementum deposited on the growing root surface.