Ectodermal organ development is initiated by inductive tissue interactions. Developing teeth, epidermis, hair, and limbs are classic examples of these types of inductive processes. In mice, tooth development begins at embryonic day (E) 11.5 with the thickening of the dental epithelium. The dental lamina undergoes further proliferation, subsequently developing into the tooth bud and germ. The tooth bud is formed by the invagination of the placode and the condensation of mesenchyme cells adjacent to the bud. At the cap stage (E14.5), dental epithelial cells differentiate into several cell types, such as inner dental epithelium (IDE), outer dental epithelium, stellate reticulum, and stratum intermedium. At the bell stage (E17.5), the IDE cells differentiate into enamel matrix-secreting ameloblasts and the dental mesenchyme differentiates into dentin matrix-secreting odontoblasts. The goal of this project is to understand how tooth and craniofacial tissues develop and to define molecular defects underlying anomalies of these tissues. Epfn and Tbx1 regulates dental epithelial stem cell commitment to ameloblast fate and differentiation: We previously identified epiprofin (Epfn/Sp6), a member of the Sp transcription family, in developing teeth. We showed that Epfn exerts multiple functions in keratinocyte development through changing its expression levels. In tooth development, Epfn is continuously expressed from an early stage of the ameloblast lineage to the maturation stage, and its expression level progressively increases. Our previous study revealed that Epfn is required for both proliferation and differentiation of the IDE to ameloblasts. Therefore, similar Epfn functions may regulate keratinocyte and ameloblast development. However, we also predict cell type-specific differences in Epfn functions between these tissues due to cell-type specific factors and genes with which Epfn interacts and targets during development of the two different ectodermal tissues. T-Box 1 (Tbx1) is a member of the T-Box transcription family and is associated with DiGeorge syndrome, a developmental disorder that affects the teeth, heart, thymus, parathyroid, and face. Tbx1 is expressed in the cervical loop of incisors and the IDE cells, and its expression in inhibited in differentiated ameloblasts. K14Cre-mediated Tbx1 cKO mice show enamel defects due to reduced IDE proliferation and differentiation, similar to Epfn KO teeth. Therefore, we hypothesized that genetic and functional interactions of Epfn and Tbx1 regulate stem cell fate and ameloblast development through cooperative and/or independent pathways. We obtained K14Cre-Tbx1 cKO mice from Dr. Antonio Baldini at the University Federico II, Italy and Dr. Brad Amendt at the University of Iowa. We find that Tbx1 is expressed in Sox2-expressing stem cells and most strongly in the IDE, and its expression deceases dramatically in secretory stage ameloblasts. In Epfn-/- teeth, Tbx1 expression was reduced, whereas Epfn remained expressed in K14Cre-Tbx1 KO teeth, suggesting that Epfn may regulate Tbx1 expression, at least at the IDE stage. ChIP assays revealed that Epfn bound to a Tbx1 promoter region (R2 site). In embryonic day 17 (E17) Epfn-/- molars, Sox2-expressing stem cells expanded into the presumptive IDE layer, suggesting that Epfn inhibits Sox2 expression. In P1 incisors of K14Cre-Tbx1 KO mice, the size of the cervical loop was smaller compared with WT incisors and the number of Sox2-expressing stem cells was reduced, suggesting that Tbx1 contributes to maintenance of the stem cells. In transfection experiments using the dental epithelial stem cell line termed CLDE cells, which we established from the cervical loop region of mouse incisors, we found that a low level of Epfn expression induced Tbx1 mRNA expression, whereas high-level Epfn expression abrogated Tbx1 induction, and instead induced expression of ameloblast differentiation markers, such as ameloblastin and amelogenin. A low level, but not a high level, of expression of Tbx1 in CLDE cells induced Sox2 and stem cell related-genes such as Lef1 and beta-catenin. However, low-level Epfn expression abrogated Tbx1-mediated induction of these genes. We found that Epfn physically bound to Tbx1, likely inhibiting Tbx1-mediated Sox2 inducing transcriptional activity. We also found that relatively low-level Epfn expression plus higher-level Tbx1 expression promoted CLDE cell proliferation. Our results suggest that Tbx1 maintains Sox2-expressing stem cells. When Epfn is expressed at low levels, it promotes stem cell commitment to the ameloblast lineage by reducing Sox2 expression, at least in part by binding and inhibiting Tbx1 transcriptional activity. However, Epfn also increases Tbx1 expression, and both Epfn and Tbx1 promote proliferation of IDE cells. Thus, Epfn and Tbx1 function cooperatively during early ameloblast development. Thus, different levels of Epfn expression dictate different Epfn functions at different stages during ameloblast development. This mechanism is conceptually similar to Epfns functions in keratinocyte development. However, Epfn target genes are likely different at the differentiation stage between the two cell types.

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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
Miyazaki, Kanako; Yoshizaki, Keigo; Arai, Chieko et al. (2016) Plakophilin-1, a Novel Wnt Signaling Regulator, Is Critical for Tooth Development and Ameloblast Differentiation. PLoS One 11:e0152206
Lu, Xuanyu; Li, Wenjin; Fukumoto, Satoshi et al. (2016) The ameloblastin extracellular matrix molecule enhances bone fracture resistance and promotes rapid bone fracture healing. Matrix Biol 52-54:113-126
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
Nakamura, Takashi; Yoshitomi, Yasuo; Sakai, Kiyoshi et al. (2014) Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation. J Cell Sci 127:5261-72
Teepe, John D; Schmitz, James E; Hu, Yuanyuan et al. (2014) Correlation of ameloblastin with enamel mineral content. Connect Tissue Res 55 Suppl 1:38-42
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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