Tooth development is a highly reguated process orchestrated by a number of genes that encode growth factors, transcription factors and extracellular matrix proteins. Molecular mechanisms underlying the noral and abnormal tooth development is not well characterized. We have initiated studies that focus on the candidate genes for the common tooth disorders such as dentinogenesis imperfecta (DGI), dentin dysplasia, ?amelogenesis imperfecta, and osteognensis imperfcta as well as growth factor genes involved in tooth mineralization. Dentin sialophosphoprotein: Dentinogenesis imperfecta (DGI) is one of the two major genetic disorders which affect dentin with an estimated incidence of 1 in 6000-8000. DGI is clinically characterized by an opalescent dentin resulting in discoloration of the teeth. The dentin is poorly formed due to the irregular arrangement of dentinal tubules with abnormally low mineralization. In this disorder, teeth usually wear down rapidly, leaving short and brown stumps. Mutation and/or aberrant expression of functional dentin phosphoprotein (DPP), a noncollagenous protein, has been implicated in DGI-II disorder. Odontoblasts, ectomesenchymal cells originating from the neural crest, secrete several collagenous and non-collagenous proteins (NCPs) to form predentin. There are several NCPs present in the bone and dentin extracellular matrix (DECM) and many of them have been thought to play potential roles in biomineralization. The exact biological functions of these proteins are not clearly understood. One category of mineralized tissue-specific proteins of dentin NCPs, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), are expressed exclusively by odontoblasts. It has been postulated, based on the physicochemical and biochemical properties, that DPP is a nucleator or a mediator of hydroxyapatite crystal formation leading to the mineralized dentin. We had previously reported cloning and characterization of the murine dentin sialophospho protein gene (dspp) to establish the structure, regulation and functions associated with the gene products. We have now adapted a functional genomics approach to delineate precisely the biological functions of these protein in vivo. Our previous results have clearly demonstrated the basal promoter, enhancer and suppressor elements within 5' upstream sequences using a series of deletions in the established mouse odontoblast cell line MO6-G3. We have developed a transgenic animal model with a reporter gene (?-galactosidase) under the control of 5.7 kb 5? flanking sequences to establish the presence of all the necessary elements within the promoter that would to mimic the temporal and spatial expression pattern of the endogenous gene. Two independent transgenic lines harboring the transgenes were analyzed for the DSPP-LacZ expression profiles. Developmental expression patterns of the transgene was found to be very similar to the endogenous DSPP gene. These studies were further extended to generate the targeted ES cell clones to obtain DSPP knockout mice which will serve as DGI-II model. We have constructed two targeting vectors to disrupt and also monitor the dspp promoter in vivo. The targeted clones were injected and many overt chimeras have been obtained. Amelogenin: Amelogenesis Imperfecta (MIM 301200) is a phenotypically diverse hereditary disorder affecting enamel structure. Deletions or point mutations in the X-chromosomal amelogenin gene are believed to cause the defect, however the precise function of the amelogenin proteins in enamel formation is not well defined. Amelogenin proteins constitute 90% of the enamel organic matrix, and are subject to extensive, controlled proteolysis during enamel maturation, as enamel develops into the most highly mineralized tissue in the body. We have disrupted the amelogenin locus in the mouse and the amelogenin null mice display distinctly abnormal teeth with chalky-white discoloration of incisors. Microradiographic analysis revealed significant attrition of the tips of incisors and molars. SEM analysis indicated disorganized hypoplastic enamel. The amelogenin null phenotype resembles amelogenesis imperfecta and will be valuable to gain insights into the pathogenesis underlying this disorder. Transforming growth factor-?1: The strength and hardness of teeth are due to the orderly mineralization of a specialized dentin extracellular matrix (DECM). Autosomal tooth disorders such as dentinogenesis imperfecta (DGI, MIM 125490 and 12550), and dentin dysplasia (DD, MIM 125400 and 125420) are characterized by discoloration and fractures of teeth associated with poor mineralization of DECM. Mutations in the Col1A1 and Col1A2 genes encoding collagen I resulting in increased deposition and altered assembly of collagen fibers, major components of DECM, have been described for DGI-I associated with osteogenesis imperfecta (OI, MIM 166240). However, the molecular defects in DGI and DD are not known, and the involvement of pleiotropic growth factors in the molecular processes underlying these disorders is not clearly understood. A number of growth factors and other regulatory molecules are expressed by the DECM-producing odontoblasts during the process of dentinogenesis and mineralization. Transforming growth factor-?1 (TGF-?1) is one of these growth factors that is expressed in teeth during development and mineralization of teeth. TGF-?1 is known to regulate a wide range of cellular processes including cell proliferation, differentiation, embryonic development and apoptosis. In addition, TGF-?1 has profound effects on synthesis, assembly and deposition of DECM components. During tooth development, TGF-?1 is expressed initially in the oral epithelium (mouse embryonic day 13, E13) and later in the oral mesenchyme (E18) and confined to ectomesenchymal layer (odontoblasts). However, TGF-?1 expression continues at a low level in mesenchymal derived dental pulp. Even though TGF-?1 has been implicated in dental tissue repair processes by the induction of reactionary and reparative dentinogenesis, its precise functions in teeth are not yet clear. Surprisingly, the targeted disruption of TGF-?1 did not result in a gross tooth abnormality. However, subtle changes such as attrition, and reduced mineralization were observed in the TGF-?1 knockout mice. It is not clear whether these subtle changes are the result of a partial rescue of knockout embryos by maternal transfer of TGF-?1 or possibly due to redundant roles of the TGF-? isoforms and/or lethal multifocal-inflammation observed in the TGF-?1 knockout mice. To gain more insight into the specific roles of TGF-?1 during tooth development, we chose to elevate its levels in odontoblasts from E17 (late bell stage) by expressing active TGF-?1 under the control of the upstream regulatory sequences of the dentin sialophosphoprotein (Dspp) gene in transgenic mice. These animals exhibit a novel tooth phenotype that resembles hereditary dental disorders such as DD and DGI. To our knowledge, this is the first report in which targeted expression of a pleiotropic growth factor in teeth leads to a phenotype that resembles the tooth disorders. These mice represent a valuable animal model to study the role of TGF-?1 in tooth development and to understand the molecular processes that lead to dysplastic dentin that cause not only disfigurement, but also significant functional deficits commonly observed in inherited tooth disorders. We further carried out proteomic analysis of dTGF-b1 mouse teeth using two-dimensional gel electrophoresis and mass spectrum analysis. We have identified the novel expression of members of the crystallin family in the developing teeth. aB and b- crystallins were found to be elevated in dTGF-b1 mouse teeth whereas g- crystallins (gB, gC and gF), markers of cell differentiation, were significantly reduced. Crystallins are
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