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 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. The Mediator is a multi-protein complex that functions as a transcriptional coactivator. The Mediator interacts with the core transcriptional machinery, including RNA polymerase II. Med-1 is a subunit of the Mediator and was recently shown to be critical for hair formation, adipogenesis, and thyroid gland development. In collaboration with Dr. Yuko Oda at UCSA and VA Medical Center in San Francisco, we found that Med-1 determines the cell fate of the dental epithelium to the Si cell linage and is required for enamel mineralization. Rodent incisors, unlike molars, grow continuously throughout life because of a continuous supply of dental epithelial stem cells that are located in the cervical loop of the incisors. Sox2-expressing dental epithelia stem cells give rise to all of the differentiated dental epithelial cells, including Si cells and ameloblasts. A layer of Si cells is located adjacent to the ameloblast layer. We found that in conditional Med1 knockout (Med1CKO) mice, epithelial cells develop ectopic formation of multiple hairs in their incisors and a defect in enamel mineralization. We found that the Med1 deficiency targeting the epithelium caused dental epithelial stem cells to fail to commit to the Si cell lineage. Instead, it induced the dental epithelium to differentiate into epidermal keratinocyte and hair follicle cells. Our data demonstrated that 1) Med1 ablation in the dental epithelium inhibited Notch signaling that prevented Si cell differentiation and that 2) Sox2-expressing dental epithelial stem cells continued to be propagated in the presumptive Si cell linage layer and eventually differentiated into the epidermal and hair follicle cell lineages. Using dental epithelial progenitor cells in culture, we were able to reproduce this in vivo cell lineage switch and confirmed that Med1 is critical for the Si cell lineage specification through Notch1 signaling. Our dental epithelial cell culture data showed that Notch1 was a target molecule of Med1 and that the Med1/Notch1 axis is determined by the Si lineage. The addition of Ca2+ to conditioned media switched the Med1-deficient dental cells to the epidermal linage. These data suggest that Ca2+ signaling induces the dental epithelial stem cells to commit to epidermal and hair follicle cells in the absence of Med1.
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