CHARACTERIZATION OF THE INNER ENAMEL EPITHELIUM IN THE ENAMEL-FREE AREA BASED ON THE ABILITY TO SECRETE ENAMEL PROTEIN DEMONSTRATED BY IN-SITU HYBRIDIZATION AND IMMUNOHISTOCHEMISTRY

Both the expression of amelogenin mRNA and secretion of amelogenin wer e investigated in rat molars by in situ hybridization and immunohistoc hemistry. Probes were designed by multiple-labeling of oligonucleotide probes for in situ hybridization. Amelogenin mRNA first appeared in d ifferentiating ameloblasts of the distal region and some inner enamel epithelial cells of enamel-free area (EFA cells) of the second cusp at postnatal day 0. At the same time, amelogenin protein was detected in the extracellular matrix between dentin and differentiating ameloblas ts' and in some EFA cells of the second cusp. At postnatal day 1-3, am elogenin was expressed in the secretory ameloblasts, and in the matrix beneath these cells. Both amelogenin mRNA and amelogenin were detecte d in the EFA cells and their extracellular matrix. After postnatal day 5, amelogenin mRNA and amelogenin were detected in the secretory ameo loblasts and extracellular matrix in the enamel-forming region, respec tively. At this time, amelogenin mRNA was not detected in the EFA cell s, but a small amount of amelogenin was found in the matrix beneath th e EFA cells. These findings suggest that EFA cells differentiate into amelogenin-secreting cells, i.e. ameloblasts, but that the secretion l asts for only a short period at the early stage of tooth development.