Ectodermal organ development is initiated by inductive tissue interactions, and developing teeth, epidermis, hair, and limbs are classic examples of such inductive processes. Tooth development can be divided into the initiation, bud, cap, and bell stages. In mice, tooth development begins with thickening of the dental epithelium. The dental lamina undergoes further proliferation and subsequently develops into the tooth bud and germ. The tooth bud is formed by invagination of the placode and condensation of mesenchyme cells adjacent to the bud. At the cap stage, dental epithelial cells differentiate into several cell types, such as the inner dental epithelium and the enamel knot cells. Cell death by apoptosis within the enamel knot is critical for cusp formation in molars. At the bell stage, the dental mesenchyme differentiates into dentin matrix-secreting odontoblasts, and the inner dental epithelial cells differentiate into enamel matrix-secreting ameloblasts. The goal of this project is to discover novel and previously uncharacterized genes in order to understand how tooth and craniofacial tissues develop, and to define molecular defects underlying the anomalies of these tissues. Epiprofin (Epfn/Sp6), a member of a Sp transcription factor family, is expressed in certain developing ectodermal tissues, such as teeth and limbs. Epfn expression is observed at the early initiation stage in incisors and molars. At the cap stage and at later stages, its expression is restricted to the inner dental epithelium and enamel knot epithelium and to ameloblasts and odontoblasts. We previously identified the essential role of Epfn for tooth morphogenesis by creating Epfn knockout (KO) mice. These Epfn KO mice show developmental retardation of the teeth, but formed excess numbers of incisors and molars with age. These teeth completely lack enamel, due to a defect in ameloblast differentiation, and have defects in dentin structure, due to reduced DSPP expression. In mutant mice, the inner dental epithelium retains the progenitor phenotype but loses its characteristic ability for rapid proliferation. Multiple non-proliferating enamel knot-like structures are also formed ectopically and show reduced Shh expression compared with the high levels of expression in the normal enamel knot. New bud branching likely initiates from the immature inner dental epithelium, which eventually leads to the formation of the excess teeth. Since loss of Epfn causes hyperdontia and severe enamel hypoplasia, we examined whether these abnormal phenotypes might be restored to normal by forced expression of Epfn in dental epithelial cells using a transgenic approach. We created transgenic mice that expressed the Epfn transgene under the control of epithelial cell-specific keratin 5 (K5) promoter, which is active in most dental epithelial cells. We obtained three major mouse lines with low, medium, or high levels of the Epfn transgene. Although the 3rd molars were missing in all three lines, the transgenic mice with low and medium Epfn expression did not show other significant tooth abnormalities. The mice with high expression levels developed an enamel layer on both the labial and lingual sides of the incisors. Wild-type incisors have enamel only on the labial side, suggesting that ectopic Epfn transgene expression in the dental epithelium on the lingual side changes the cell fate to ameloblast differentiation. However, the enamel layers of K5-Epfn teeth with high levels of K5-Epfn expression were thin, suggesting that overexpression of Epfn accelerated differentiation of ameloblasts. We found that the K5-Epfn teeth showed increased proliferation of the dental epithelial cells as well as dental mesenchymal cells. Introduction of the K5-Epfn transgene in Epfn KO mice by mating the K5-Epfn transgenic mice with Epfn KO mice did not rescue the enamel dysplasia seen in the Epfn KO mice. However, the numbers of excess teeth were significantly reduced in these mice. These results suggest that appropriate levels and stages of Epfn expression are well orchestrated during development and are essential for normal tooth numbers and dental cell differentiation.

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National Institute of Dental & Craniofacial Research
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Nakamura, Takashi; Jimenez-Rojo, Lucia; Koyama, Eiki et al. (2017) Epiprofin Regulates Enamel Formation and Tooth Morphogenesis by Controlling Epithelial-Mesenchymal Interactions During Tooth Development. J Bone Miner Res 32:601-610
Yoshizaki, Keigo; Hu, Lizhi; Nguyen, Thai et al. (2017) Mediator 1 contributes to enamel mineralization as a coactivator for Notch1 signaling and stimulates transcription of the alkaline phosphatase gene. J Biol Chem 292:13531-13540
Aurrekoetxea, Maitane; Irastorza, Igor; GarcĂ­a-Gallastegui, Patricia et al. (2016) Wnt/?-Catenin Regulates the Activity of Epiprofin/Sp6, SHH, FGF, and BMP to Coordinate the Stages of Odontogenesis. Front Cell Dev Biol 4:25
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Yoshizaki, Keigo; Hu, Lizhi; Nguyen, Thai et al. (2014) Ablation of coactivator Med1 switches the cell fate of dental epithelia to that generating hair. PLoS One 9:e99991
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Kaneko, Haruka; Ishijima, Muneaki; Futami, Ippei et al. (2013) Synovial perlecan is required for osteophyte formation in knee osteoarthritis. Matrix Biol 32:178-87
Arakaki, Makiko; Ishikawa, Masaki; Nakamura, Takashi et al. (2012) Role of epithelial-stem cell interactions during dental cell differentiation. J Biol Chem 287:10590-601

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