Enamelin is the largest protein in the enamel matrix of developing teeth. In the pig, enamelin is secreted as 186-kDa phosphorylated glycoprotein, which is rapidly processed by enamel proteinases into smaller cleavage products. During the secretory stage of enamel formation, enamelin is found among the crystallites in the rod and interrod enamel and comprises roughly 5% of total matrix protein. Although the function of enamelin is unknown, it is thought to participate in enamel crystal nucleation and extension, and the regulation of crystal habit. Here we report the results of enamelin in situ hybridization in a day 1 mouse developing incisor that shows that enamelin is expressed by ameloblasts, but not by odontoblasts or other cells in the dental pulp. The restricted pattern of enamelin expression makes the human enamelin gene a prime candidate in the etiology of amelogenesis imperfecta (AI), a genetic disease in which defects of enamel formation occur in the absence of non-dental symptoms. We have cloned and characterized a full-length human enamelin cDNA and determined by radiation hybrid mapping and fluorescent in situ hybridization (FISH) that the gene is located on chromosome 4q near the ameloblastin gene in a region previously linked to local hypoplastic AI in six families. These findings will facilitate the search for specific mutations in the enamelin gene in kindreds suffering from amelogenesis imperfecta.

The transcription factor Osf2/Cbfa1 is a key regulator of osteogenic differentiation while BSP, a major non-collagenous protein, is a marker of osteoblastic differentiation. To determine the relationship between Osf2/Cbfa1 and the formation of mineralized tissues in tooth development we have studied the temporal expression of Osf2/Cbfa1 and BSP mRNA using in situ hybridization. These studies show that Osf2/Cbfa1 is expressed early in mesenchymal and epithelial tissues destined to form the mineralized tissues of the tooth and periodontal tissues, whereas BSP provides a specific marker for the differentiated cells in each of these tissues. Expression of Osf2/Cbfa1, but not BSP, was observed in the periodontal ligament indicating that expression of Osf2/Cbfa1 is not restricted to mineralizing tissues.

Enamel matrix serine proteinase 1 (EMSP1) is a proteolytic enzyme that has been isolated from the developing enamel of pig teeth. Its apparent function is to degrade the organic matrix in preparation for enamel maturation. The expression of EMSP1 has never been investigated in another organism besides the pig, and EMSP1 expression in the enamel organ has never been specifically demonstrated in ameloblasts. Here we report the expression of recombinant pig EMSP 1 (rpEMSP 1), the generation of rabbit polyclonal antibodies against rpEMSP1, the characterization of the antibodies and EMSP1 expression by Western blot and immunohistochemical analyses, the cloning and characterization of a full-length cDNA encoding mouse EMSP1, and the localization of EMSP1 expression in ameloblasts in mouse day 14 first and second molars by in situ hybridization. The full-length mouse EMSP1 cDNA clone has 1,237 nucleotides, excluding the poly(A+) tail, and encodes a preproprotein of 255 amino acids. Mouse EMSP1 shares 75% amino acid identity with pig EMSP1 and has three potential N-linked glycosylation sites, two of which are conserved in the pig homologue. Western blot analysis shows that the polyclonal antibodies are specific for EMSP1 and do not cross-react with trypsin. Immunohistochemistry of pig incisors shows discrete staining in the surface enamel at the earliest part of the maturation stage. In mouse molars, in situ hybridization gives a distinct and specific signal in maturation-stage ameloblasts, and in the junctional epithelium following tooth eruption. We conclude that EMSP1 is expressed by pig and mouse ameloblasts during the early maturation stage of amelogenesis.

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HF: Enamel epithelium expresses hone sialoprolein ( 8 P). Eur J Oral Sci /99 : 106 (suppll ): 33 1 336. \.. Eur J Oral ci. 199Bone sialoprotein ( B P) is a major non-collagenou extracellular matrix protein in bone and other mineralized connective ti ues. B P i synthesized and secreted by bone-. dentin-and cementum-forming cells. In this study we hypothesized that B P may be also involved in enamel formation through it postulated role in matrix mineralization. In situ hybridization with cR A probes for rat and hamster BSP. respectively. showed strong mRNA signals in ameloblas ts actively synthesizing enamel matrix in developing incisors. However. no hybridization signals were observed at an earlier developmental tage when bell-shaped molar tooth germs were being formed . Immun ohistochemical analysis of tooth tis ue from transgenic mice harboring a 2.7 kb rat BSP promoter ligated to a lucifera e reporter gene revealed trong staining for luciferase in the enamel epithelium of the developing tooth germ . Interestingly. BSP expre ion was al ·o observed in epit helial cell of an ameloblastoma. The neoplastic epithelial nest and cords demonstrated strong mRNA signal to the human BSP probe while the connective tissue stroma showed only a background level of silver gra ins. Immune taining also howed deposition of B P by the odontogenic cells of the tumor. These results demon trate that B Pis expre sed by the enamel-forming epi thelium of developing teeth. sugge ting a pos ·ible role for B P in enamel formation and it subsequent mineraliza tion . xpre sion of the B P gene in ameloblastoma is consi tent with the expression of B P by the enamel epithelium and al o with the expression of BSP by neopla ·tic ti ·sues. suggesting a possible role in tumorigenesis.