Ectodermal organ development is initiated by inductive tissue interactions. Developing teeth, epidermis, hair, and limbs are examples of these types of inductive processes. The overall goal of this project is to discover novel molecular mechanisms underlying the development of ectodermal organs by identifying novel functions of previously unstudied genes and the extracellular matrix (ECM). These tissues and organs develop through highly coordinated stem cell commitment, proliferation, and differentiation programs, which are regulated by multiple protein factors and ECM. Tooth development is a particularly attractive system for understanding ectodermal organ development because of its well-defined stages and distinctive differentiated cell types. Teeth consist of two hard tissues, enamel and dentin. Dental epithelial stem cells differentiate into all dental epithelial cell types to form the enamel organ. Ameloblasts produce enamel matrix to form enamel. Stratum intermedium (SI) cells are located adjacent to ameloblasts and produce alkaline phosphatase (Alp1) for enamel matrix mineralization. Mesenchyme stem cells differentiate into odontoblasts that secrete dentin matrix to form dentin. Tooth development can be divided into the initiation, bud, cap, and bell stages. In mice, tooth development begins 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. At the cap stage, dental epithelial cells differentiate into several cell types, such as the inner dental epithelium (IDE) 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 odontoblasts, and the inner dental epithelial cells differentiate into ameloblasts. Med1 and Notch1 are required for stem cell commitment to SI cell fate and differentiation: Rodent incisors grow continuously throughout their lives because of a continuous supply of dental epithelial and mesenchyme stem cells that are located in the cervical loop and the surrounding region, respectively. Sox2 stem cell marker-expressing dental epithelia cells give rise to all of the differentiated dental epithelial cells, including SI cells and ameloblasts. However, the factors that regulate stem cell commitment to the dental epithelial cell fate are largely unknown. We found that Med1 and Notch1 are essential for stem cell commitment to the SI lineage and differentiation in collaboration with Dr. Yuko Oda at UCSF and Veterans Affairs Medical Center. Mediator (Med) is a multiprotein complex that plays an important role in cell fate specification and differentiation. Mediator 1 (Med1) is a subunit of the Med complex. K15Cre-Med1 conditional knockout (Med1 KO) mice specific to the epithelium develop ectopic formation of multiple hairs in their incisors and a defect in enamel mineralization. We found that the Med1 deficiency that targeted the epithelium caused a failure of dental epithelial stem cells 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 continue to be propagated in the presumptive SI cell linage layer and eventually differentiated into the epidermal lineage. We further tested the function of Med1/Notch1 in SI differentiation using the clonal primary dental epithelial CLDE cell line, which we had established from stem cell-rich cervical loop regions of E15 mouse incisors. About 80% of our CLDE cells are double-positive for Sox2 and CDy1, a chemical compound bound specifically to stem cells. In CLDE cell cultures, we found that adding high Ca2+ to the medium decreases Sox2 expression, while it induces the expression of alkaline phosphatase (Alp), a marker of differentiated SI cells. Activation of Notch1 is required for this Alp induction. When endogenous Med1 expression is knocked down in CLDE cells by siRNA, Sox2 expression remains prolonged during culture at even high Ca2+ concentration, and both Notch1 activation and Alp induction are inhibited. Instead, CLDE cells differentiate into keratinocytes. Our findings reveal that Med1 regulates dental epithelial stem cell commitment to the SI cell fate and differentiation through Notch1 signaling. Epfn orchestrates proliferation and differentiation regulatory processes in epidermal keratinocyte development: We previously identified epiprofin (Epfn/Sp6) as a member of the Sp zinc-finger transcription factor family that is expressed in certain developing ectodermal tissues, including teeth, skin, hair follicles, and limbs. During tooth development, Epfn is expressed in the dental epithelium at the initiation stage. Later, its expression is restricted to the ameloblast lineage, including IDE and secretory and mature ameloblasts with increasing levels of expression. Epfn is also expressed in mature odontoblasts. We previously created Epfn knockout (Epfn-/-) mice to study its in vivo functions. Epfn-/- mice show small body size, severe enamel hypoplasia, excess teeth, impaired dentin structure, hyperplastic epidermis and hyperkeratosis of the skin, hairlessness, and digit fusion. We explored the function of Epfn in keratinocyte development to obtain mechanistic insights into Epfn functions that are common versus unique in tooth and skin development. We discovered a novel mechanism that sequentially regulates keratinocyte proliferation and differentiation through multiple distinct functions of Epfn as a cell cycle regulator and a transcription factor. Differing expression levels of Epfn alter its functions in keratinocyte development. The basal single cell layer of the epidermis contains stem cells and transit amplifying (TA) cells that rapidly proliferate with limited cell cycles, eventually stop proliferating, and differentiate further into the upper layers of the epidermis. The TA cells in the epidermis are equivalent to the IDE cells in developing teeth. p63 and Notch1 are two major regulators in this process through a mutually exclusive negative regulatory loop. p63 is expressed in the basal layer and is required for the maintenance of stem cells and TA cells, while Notch1 is primarily expressed in the upper layers and essential for differentiation. We found that Epfn-/- mice have a thickened epidermis, in which p63-expressing basal cells formed multiple layers due to accumulation of premature TA cells with reduced proliferation, and a reduction in differentiating keratinocytes expressing Notch1. Using cell culture, we discovered that low levels of Epfn expression promote proliferation while high levels of Epfn expression promote keratinocyte differentiation. We found that Epfn forms a complex with cyclinD1/Cdk4, which results in super-phosphorylation of Rb. This Rb phosphorylation releases E2F from the inactive E2F/Rb complex, which induces genes for cell cycle progression. In contrast, when Epfn expression levels are increased, Epfn binds to E2F, which inhibits E2F transactivation and promotes cell cycle exit. Epfn also induces Notch1 expression by binding to the Notch1 promoter. Consequently, the switch from proliferation to differentiation is regulated in part by the level of Epfn expression. Thus, Epfn plays multiple roles in orchestrating keratinocyte proliferation and differentiation crucial to epidermal homeostasis and morphogenesis.
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