Tooth development shows dynamic morphological changes from the stages of cap to hard tissue formation and is strictly regulated during development. In the present study, we compared expression and localization of 3 major enamel matrix proteins in rats: amelogenin, enamel and ameloblastin. DD-PCR and RT-PCR revealed differential expression of the major proteins from the cap stage to root stage. Immunofluorescence staining results indicated that amelogenin was not detected in either inner enamel epithelium or reduced enamel epithelium, but highly immunoreactive in preameloblasts and ameloblasts; in addition, it was sporadically expressed in preodontoblasts abutting preameloblasts. Ameloblastin expression was also observed in not only differentiated ameloblasts but also osteoblasts. Immunoreactivity to ameloblastin in ameloblasts was strong in Tomes' processes. Enamelin was exclusively localized along the entire newly formed and maturing enamel. Enamelin was largely localized in near Tomes' processes and enamel rods in maturing enamel. Alendronate treatment resulted in down-regulation of amelogenin and ameloblastin at both transcription and translation levels; whereas, enamelin expression was unchanged in response to the treatment. These results suggested that amelogenin, ameloblastin and enamelin might be implicated in cell differentiation, adhesion of ameloblasts to enamel and enamel crystallization during enamel matrix formation, respectively.

Tooth bud is initiated by dental epithelium thickening and invaginated epithelium forms cap-like structure with condensed underlying mesenchyme. From this cap stage, specific epithelial cells form a cluster of non-dividing cells, primary enamel knot (PEK), and this PEK expresses abundant signaling molecules and functions as a signaling center for tooth development. Recently, hundreds of genes involved in tooth development have been reported by advanced genome-wide screening technology, but still remained unidentified genes obstruct elucidation of the precise molecular mechanisms underlying tooth development. In this work, we examined the expression patterns and developmental functions of a novel tooth germ-expressing gene, Dachshund1 (Dach1). Dach1, known as the cell fate regulator via controlling the transcription in various cell types, expressed in PEK region from cap stage to bell stage. In order to evaluate the developmental functions of Dach1 in tooth development, we cultivated the lower first molar at E12.5 for 2 and 4 days with or without treatments of antisense-oligodeoxynucleotides (AS-ODNs) against Dach1. After the knocking down of Dach1, morphological changes and cell physiology such as proliferation, cell death and migration patterns were examined. In addition, the expression levels of PEK-expressing genes such as Bmp2, Bmp7, Fgf4, Shh and Wnt5a were examined by using RT-qPCR and in situ hybridization methods. Based on these results, we suggest that Dach1 would involve in mice tooth morphogenesis through regulating the expressions of PEK related genes and cellular events to form the proper tooth structural formation.