Odontogenic cells express many genes spatiotemporally through complex and intricate processes during tooth formation. Therefore, investigating them during the tooth development has been an important subject for the better understanding of tooth morphogenesis. The present study was performed to identify the genetic profiles which are involved in the morphological changes during the different stages of rat tooth development using the Agilent Rat Oligonucleotide Microarrays. Morphologically, the maxillary 3rd molar germ at 10 days post-partum (dpp) was at the cap/bell stage. In contrast, the maxillary 2nd molar germ showed the root development stage. After microarray analysis, there were a considerable number of up- or down-regulated genes in the 3rd and the 2nd molar germ cells during tooth morphogenesis. Several differentially expressed genes for nerve supply were further studied. Among them, neuroligin 1 (Nlgn 1) was gradually downregulated during tooth development both at the transcription and the translation level. Also, Nlgn 1 was mostly localized in the dental sac, which is an important component yielding the nerve supply. This genetic profiling study proposed that many genes may be implicated in the biological processes for the dental hard tissue formation and, furthermore, may allow the identification of the key genes involved in the nerve supply to the dental sac.

The salivary gland undergoes complex process of growth and differentiation of the branching morphogenesis of ductal system during the prenatal and early postnatal periods which are regulated by various elements in the extracellular matrix. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell adhesion molecule. In the present study, localization and expression of EMMPRIN in development and effects of chorda-lingual denervation and cyclosporine A (CsA) treatment on the EMMPRIN expression were investigated. Immunohistochemistry, RT-PCR and Western blot were used to determine expression level. Immunohistochemistry revealed that EMMPRIN was localized specifically in the cytoplasm of ductal cells, not acini of the submandibular gland all the postnatal periods. At prenatal day 18, when the formation of ducts was not definite, no immunoreactivity was observed. Both Western blot and RT-PCR analyses revealed that EMMPRIN expression was maintained up to postnatal day 7, decreased after postnatal day 10. The EMMPRIN expression was upregulated by the surgical denervation of the chorda-lingual nerve in the gland as well as by the CsA treatment. The present study suggests that EMMPRIN is a crucial molecule for maintaining physiological functions of the salivary gland.

The deleted in colorectal cancer (DCC) protein mediates attractant responses to netrin during axonogenesis. In the rat trigeminal ganglia (TG), axons must extend toward and grow into the trigeminal nerve to innervate target tissues such as dental pulp. Our present study aimed to investigate the exp- ression of DCC in the TG. Four developmental timepoints were assessed in the experiments: postnatal days 0, 7 and 10 and adulthood. RT-PCR and western blotting revealed that the expression of DCC mRNA and protein does not signi- ficantly change throughout development. Immunohistoche- mistry demonstrated that DCC expression in the TG was detectable in the perikarya region of the ganglion cells du- ring development. Nerve injury at 3 and 5 days after the man- dibular nerve had been cut did not induce altered expression of DCC mRNA in the TG. Moreover, DCC-positive cell bodies also showed similar immunoreactive patterns after a nerve cut injury. The results of this study suggest that DCC constituti- vely participates in an axonogenesis attractant in ways other than expression regulation.

The effects of the an immunosuppressive drug cyclos- porine A (CsA), on the salivary gland are largely unknown, even though clinical trials for the stimulation of salivation using CsA have been attempted. Cyclophilin A (CypA) is known to be a binding protein for CsA. CypA has cell proliferation and tissue matrix change activities. In our present study, the presence of CypA in the gland and effects of CsA on CypA expression were investigated by immu- nohistochemistry, immunoblotting and RT-PCR analyses. CypA was immunohistochemically detected in various kinds of ducts in the submandibular glands of Sprague Dawley rats. The CypA mRNA level was highest at postnatal day 1 and gradually decreased in a time-dependent manner up to adulthood. The expression of CypA increased after a 10 day subcutaneous administration of CsA in postnatal day 1 rats. Surgical sections of the chorda-lingual nerve with impaired salivation showed no changes in CypA expression. A cell proliferation assay using PCNA anti-serum showed inc- reased cell division following CsA treatment. These results suggest that CsA and CypA may act on ductal cells to regulate saliva composition rather than salivation levels.

Tooth development involves bud, cap, bell and hard tissue formation stages, each of which is tightly controlled by regulatory molecules. The aim of this study was to identify genes that are differentially expressed during dental hard tissue differentiation. Sprague-Dawley rats at postnatal days 3, 6 and 9 were used in the analysis. Differential display RT-PCR (DD-PCR) was used to screen differentially expressed genes between the 2nd (root formation stage, during mineralization) and 3rd (cap stage, before minerali-zation) molar germs at postnatal day 9. The DNA detected in the 2nd molar germs showed homology to osteonectin only (GenBank accession no. NM_012656.1). The level of osteonectin mRNA expression was much higher in the 2nd molar germs than in the 3rd molar germs and was found to increase in a time-dependent manner from the early bell stage to the root formation stage in the 2nd molar germs. The pattern of osteonectin protein expression was consistent with these RT-PCR results. Osteonectin protein was found by immunofluorescent analysis to localize in odontoblasts and preodontoblasts rather than the dentin matrix itself. Further studies are needed to validate the involvement of osteonectin in mineralization and root formation.

Matrix metalloproteinases (MMPs) have been implicated in tissue development and re-modeling. Dynamic morphological changes of tooth germs reflect involvement of these enzymes during odontogenesis. The present study was performed to investigate expression and localization of MMP-2 and MMP-9, which have been known to have type IV collagenase activities, in rat tooth germs at different developmental stages. MMP-2 expression was increased gradually in the tooth germs from cap to crown staged germs at both transcription and translation levels. The localization of this molecule was detected in secretory ameloblasts and preameloblasts. The strong immunoreactivities were occasionally seen along the basement membrane between ameloblasts (or preameloblasts) and odontoblasts (preodontoblasts). However, weak reactivity was detected in odontoblasts and reduced enamel epithelium. The level of MMP-9 expression in the tooth germs was higher in cap stage than in crown staged germs at both transcription and translation levels. They were strongly expressed in both ameloblasts and odontoblasts. Even though reduced enamel epithelium after enamel formation and inner enamel epithelium at the cap stage exhibited weak reactivity, strong reactivity was detected in dental follicles and perifollicular tissues surrounding cap staged germs. These results suggested that MMP-2 may involve degradation of the basement membrane during hard tissue formation, whereas MMP-9 might be involved in remodeling of follicular tissues.