Ectodermal organ development is initiated by inductive tissue interactions. Developing teeth, epidermis, hair, and limbs are classic examples of these types of inductive processes. Tooth development can be divided into the initiation, bud, cap, and bell stages. In mice, tooth development begins at embryonic day (E) 11.5 with the 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 the invagination of the placode and the condensation of mesenchyme cells adjacent to the bud. The dental epithelium differentiates into four types of epithelial cells, including the inner enamel epithelium, the stratum intermedium, the stellate reticulum, and the outer enamel epithelium. Among these cell types, the inner enamel epithelium differentiates into enamel matrix-secreting ameloblasts. 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 anomalies of these tissues. Members of the bHLH transcription factor family regulate cell fate specification, differentiation, and morphogenesis in the development of various tissues. In these processes, tissue-specific gene transcription is regulated either positively or negatively by the formation of heterodimers between tissue-specific Class B bHLH (e.g., MyoD, NeuroD) and ubiquitous Class A bHLH proteins (e.g., E12, E47). These heterodimers interact with cis-acting DNA elements (E-box) in the promoter and enhancer regions of tissue-specific genes to induce target gene expression. We identified a novel basic helix-loop-helix (bHLH) factor from a tooth germ cDNA library using the HLH protein interaction domain of the E12 protein as bait in a yeast two-hybrid system. This factor was tentatively named CartD/Dentix, as it is expressed strongly in developing teeth but weakly in some other tissues. The mouse CartD gene is a homologue of the human ASCL5 gene, which was identified by a gene family homology search. However, its function remains unknown. Transfection analysis showed that CartD activates an E-box reporter construct with E12, suggesting that CartD functions as a Class A bHLH factor. Immunostaining and in situ hybridization in mouse embryos revealed that CartD is expressed strongly in the inner dental epithelium, a progenitor of ameloblasts, but not at the late differentiation stage of ameloblasts. This differs from the expression of epiprofin (Epfn), which we previously identified as an Sp zinc finger family transcription factor that is essential for ameloblast differentiation and tooth morphogenesis. CartD may regulate the commitment and differentiation of dental epithelial cells into the ameloblast cell lineage. We previously identified epiprofin (Epfn/Sp6) as a member of a Sp zinc-finger transcription factor family that is expressed in certain developing ectodermal tissues, such as teeth, hair follicles, skin, and limbs. Loss of Epfn caused thickened epidermal layers, hairlessness, enamel hypoplasia, and excess numbers of teeth. In the skin, Epfn is expressed in keratinocytes of the epidermis. The epidermis arises from a single ectodermal layer and renews by highly coordinated regulatory programs controlling proliferation and differentiation. The epidermis consists of four distinct layers mainly composed of keratinocytes at different stages of maturation. The epidermal basal layer contains slowly renewing stem cells and rapidly proliferating transit-amplifying (TA) cells. P63 is expressed mainly in the basal layer and required for maintenance of stem cells and TA cells. Notch1 is expressed in the suprabasal layer and regulates the commitment of keratinocyte differentiation. P63 and Notch1 are mutually regulated through a negative loop. In Epfn-deficient skin, the number of rapidly proliferating TA cells was reduced, p63-expressing basal layer keratinocytes formed multiple layers, and Notch1-expressing suprabasal layer keratinocytes were reduced. These results suggest that Epfn is involved in the regulation of p63/Notch expression and orchestrates skin development and morphogenesis by controlling cell proliferation and differentiation.

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National Institute of Dental & Craniofacial Research
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Aurrekoetxea, Maitane; Irastorza, Igor; García-Gallastegui, Patricia et al. (2016) Wnt/β-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
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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Nakamura, Takashi; Yoshitomi, Yasuo; Sakai, Kiyoshi et al. (2014) Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation. J Cell Sci 127:5261-72
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Ishijima, Muneaki; Suzuki, Nobuharu; Hozumi, Kentaro et al. (2012) Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation. Matrix Biol 31:234-45

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