Abstract
A variety of vertebrate organs, including teeth, begins their development by inductive sequential and reciprocal interactions between epithelium and mesenchyme. In tooth development, the interactions between ectodermal-derived epithelium and the cranial neural crest-derived mesenchyme regulate the shape, position, and size of the tooth crown with a functional cusp. During tooth development, many signaling molecules and transcription factors regulate tooth development and morphogenesis. Recently, we reported Epiprofin, an Sp transcription factor, is expressed during tooth development and exerts critical roles in dental epithelial differentiation and the determination of tooth number. In this review, we describe the expression pattern and functions of Epiprofin in tooth development.
Key words
Tooth Development
The developing tooth is an excellent model to study the molecular mechanisms involved in epithelial-mesenchymal interactions. The first morphological manifestation of tooth development in mice is the formation of the dental lamina, a thickening of the oral epithelium at E11.5. Subsequently, the dental lamina grows into the underlying mesenchyme of the first branchial arch, thereby forming epithelial buds (E13.5). During the cap stage of development, the condensed dental mesenchyme diverges into two different pathways: the dental papilla that gives rise to dentin-secreting odontoblasts and dental pulp fibroblasts; and the dental follicle that contains progenitors for cementoblasts and osteoblasts, and periodontal ligament fibroblasts [1]. After the cap stage, the tooth germ progresses to bell stages, and epithelial cells differentiate into enamel-secreting ameloblasts. Dental epithelium differentiates into ameloblasts through mainly five distinct stages, i.e., (1) proliferative stage, (2) differentiation stage, (3) secretory stage, (4) early maturation stage, and (5) late maturation stage [2]. At the proliferative stage, dental epithelium proliferates rapidly. At the differentiation stage, the cells stop proliferating and differentiate into preameloblasts, which show cellular polarity and begin to secret enamel matrix proteins. At the secretory stage following dentin mineralization, differentiated ameloblasts deposit enamel matrix proteins including amelogenin, ameloblastin, enamelin, and tuftelin, and additionally amelotin are secreted in later stages. During the maturation stage via the transitional stage when ameloblasts eventually undergo apoptosis, the enamel matrix is almost completely replaced by calcium and phosphorous, and ameloblasts eventually give rise to reduced enamel epithelium at the regressive stage [3].
Epiprofin
Epiprofin has been identified through the oral genome project as part of the NIDCR (National Institute of Dental and Craniofacial Research, NIH) initiative for tooth and craniofacial study. To obtain cDNA clones preferentially expressed in tooth, we differentially screened DNA microarrays containing about 12,000 clones from E19.5 molar cDNA library with fluorescently labeled probes from E19.5 molar and E13.5 body mRNA. We identified 197 cDNA clones that are preferentially hybridized to RNA probes from E19.5 molar. The majority of these clones encode enamel matrix proteins, such as ameloblastin, amelogenin, and enamelin, indicating feasibility of the microarray analysis. Finally, 12 out of the 197 clones have been found as unknown proteins or correspond to ESTs previously deposited in GenBank. We have identified a cDNA clone for Epiprofin (Epfn) (NCBI GenBank™ accession number AY338955), which is preferentially expressed in tooth [4]. Epfn is a new member of the Sp/KLF transcription factor family. This family consists of more than 21 proteins in humans and 17 in mice, which have a DNA-binding domain with three tandem C2–H2-type zinc finger motifs at the C-terminus, and transcriptional regulatory domains at the N-terminus [5]. Sp factors comprise nine member gene family encoding transcription factors that play an essential role in regulating a wide variety of developmental and cellular processes, including cell growth, differentiation, apoptosis and tumor formation [6, 7]. Expression of Epfn is detected at the initiation stage of tooth development. Epfn is clearly expressed in dental epithelium of dental lamina and not expressed in dental mesenchyme at the early stage of tooth development. In bud stage, dental epithelial cells are rapidly proliferating to form a tooth bud. During bud stage, Epfn is expressed widely in dental epithelial cells. At the cap stage, dental epithelial cells determine their cell fate into several lineages such as stellate reticulum, and inner and outer enamel epithelium. At this stage, the expression of Epfn is limited in inner enamel epithelium and not expressed in other dental epithelial cell types. At the bell stage, Epfn continuously is expressed in pre-ameloblasts and ameloblasts. Interestingly odontoblasts, which derived from dental mesenchyme, start expressing Epfn.
Functional studies revealed that Epfn transcription factor promotes cell proliferation, suggesting a role in regulating cell growth during the development of tissues of ectodermal origin [4]. Over-expression of Epfn exerts distinct roles in cell growth by transient or stable expression in dental epithelial cells. By transient expression of Epfn in primary dental epithelial cells were strongly stimulated their cell mitogenic activity. It can be considered that transient expression of Epfn in dental epithelial cells might mimic the progenitor cell types of dental epithelium, which give rise to differentiate into ameloblasts. On the other hand, stable expression of Epfn inhibits cell proliferation. We also demonstrated that over-expression of Epfn promotes dental epithelial cell differentiation into ameloblast, which is in a terminal differentiated phase of dental epithelial cells and stop cell proliferation [4]. Continuous expression of Epfn could induce cell cycle exit due to the rapidly promotion of dental epithelial cell differentiation into ameloblast. More recently, an excess number of teeth, enamel deficiency, defects in tooth cusps and root formation, and abnormal dentin structure have been shown in Epfn knockout (Epfn KO) mice [8].
Regulators in Tooth Development
The interactions between the epithelial and mesenchymal tissues in tooth morphogenesis are mediated by growth factors and signalling molecules [9]. For instance, the role of Fgf8 has been extensively analyzed during the initiation of tooth morphogenesis. Transcripts of Fgf8 are expressed in early dental epithelium and the translated protein induces the expression of a number of genes in the early dental mesenchyme, which are involved in the acquisition of odontogenic competence such as Msx1, Pax9, Activin-βA and Dlx1/Dlx2. The significance of those molecules in early tooth development is demonstrated by analysing their gene targeting mouse models. For example, Msx-1, Lef-1, Pax-9 and Activin-βA knockout mice, tooth development arrests at the bud stage [10–13].
Other signalling molecules in tooth development include Sonic hedgehog (Shh), which is one of the earliest marker for the dental lamina in E11 mouse embryos. During the bud stage, the expression of Shh is reduced, and is subsequently upregulated in the enamel knots at the cap stage. From the first enamel knot (E14.5) the expression spreads to the inner enamel epithelium. It has been suggested that Shh could play a role as an inhibitor regulating the distance between forming cusp [14]. In addition, Shh appears to be an important stimulator of cell proliferation, as shown earlier in other ectodermal organs including skin and hair [15]. Conditional mutant mice have showed that Shh controls the development of tooth shape by differentially regulating growth [16].
Regulators in Tooth Number
Hypodontia (Agenesis of one or more teeth) is currently the most common of human developmental anomalies [17–19]. Failure of one or more of the third molars to form occurs in 20% of the population. The reported incidence of teeth other than third molars being missing varies from 1.6% to 9.6% [18]. Two mutations causing isolated tooth agenesis have been identified. A point mutation in the MSXJ gene (4pl6) was identified in affected members of a family with missing second premolars and third molars [20], and a mutation of PAX9 gene (14q21-ql3) was associated with oligodontia affecting most molars [21]. In addition, a recessive form of tooth agenesis has been mapped to chromosome 16 [22].
Hyperdontia, the formation of one or more supernumerary teeth occurs much less frequently than agenesis. For humans, the most frequent site for these occurrences are the maxillary central or lateral incisor regions (mesiodens). Such teeth may have a highly aberrant form, tucked-in to the lingual of the normal tooth row. They may take on the form of neighboring teeth. Supernumerary teeth often do not erupt, therefore, any survey of living or fossil individuals must necessarily include panoramic radiography to reveal unerupted teeth.
Supernumerary teeth are defined as those that are present in excess of the normal component of human dentition. The pathogenesis of extra teeth formation is poorly understood in human population. In recent years, scientists have found some advances in our understanding of the genetic basis of supernumerary teeth. Runx2 are known to be involved in the regulation of tooth number and they are thought to be transcriptional targets of FGF signaling [23, 24]. And many biological evidences provide insight into why supernumerary tooth formation may occur. Indeed, many of the molecular signaling pathways known to be involved in normal development of the tooth germ can also give rise to additional teeth if inappropriately regulated. These include components of the FGF, Wnt, TNF and BMP families, which provide a useful resource of candidate genes that may potentially play a role in human supernumerary tooth formation.
Cleidocranial dysplasia (CCD, OMIM #119600) is a skeletal disorder with autosomal dominant inheritance. The clinical hallmarks of CCD are short stature, delayed closure of cranial fontanels and sutures, Wormian bones, frontal bossing, supernumerary and late erupting teeth, rudimentary or absent clavicles, wide pubic symphysis, and other skeletal anomalies [25]. RUNX2, located on chromosome 6p21, has been identified as a gene responsible for CCD [26, 27]. Regarding dental abnormality, CCD is associated with supernumerary teeth, and the delayed eruption and impaction of permanent teeth [28–30]. The position and number of supernumerary teeth vary among cases, but they are seen below the permanent teeth (incisors, canines, and bicuspids) that have replaced with the deciduous teeth.
In mouse model, supernumerary teeth have been reported in some mutant mouse lines including Spry2 and Spry4 knockout mice [31]. Spry2 is expressed in dental epithelium, while Spry4 is expressed in dental mesenchyme and both Spry2 and Spry4 act as an antagonist for FGF signaling [31]. Either Spry2 or Spry4 knockout mice develop the supernumerary tooth in diestema by hypersensitivity of FGF signaling in dental epithelium [31].
Ectodysplasin (Eda), a signaling molecule belonging to the tumor necrosis factor family, is required for normal development of several ectodermally derived organs in humans and mice. Studies with mice either lacking the functional proteins of Edar pathway or overexpressing the ligand or receptor suggest that Eda-A1-Edar signaling has multiple roles in ectodermal organ development regulating their initiation, morphogenesis, and differentiation [32]. Over expression of Eda-A1 resulted in supernumerary teeth and mammary placodes, which develop into mature organs [33]. Moreover, Eda-A1 transgenic embryos are characterized by increased placodal size, and treatment of embryonic skin with recombinant Eda-A1 in vitro promotes placodal cell fate in a dose-dependent manner [34]. Forced expression of Eda-A1 or Edar results in a lack of enamel in incisors [33] while high levels of expression of the transgene in wild-type mice result in molar teeth with extra cusps, and in some cases supernumerary teeth, the opposite of the mutant phenotype. The level of activation of Edar thus determines cusp number and tooth number during tooth development [35]. High levels of expression of Eda can cause supernumerary teeth and Shh signaling plays a key role in the process of tooth formation. Ectodysplasin can induce the expression of Shh, which suggests that Shh is a likely transcriptional target of Edar [36].
The canonical Wnt signaling also regulates the number of teeth. In β-cat Δex3K14/+ mice, supernumerary teeth were formed not only by branching of dental epithelium but also by multiple placode formations [37]. Because β-cat Δex3K14/+ mice were created by cross-mating K14-Cre Tg/+ mice and β-cat Δex3flox/+ mice, the entire epithelial tissue including oral ectoderm and dental epithelium was expressing stabilized β-catenin, which mimics the continuous activation of Wnt/β-cat signalling. Therefore the multiple placode formation in epithelium of β-cat Δex3K14/+ mice might be occurred in part through a common mechanism in other Wnt signalling arranged mouse models of ectodermal appendages.
Epfn KO mice develop supernumerary teeth formation in both incisors and molars [8]. Especially, Epfn KO mice keep developing supernumerary incisors with aging and we found nearly 50 incisors in 6-month-old Epfn KO mice [8]. The dental epithelial cells deficient of Epfn fail to polarize and to gain rapid proliferation activity. No enamel formation is observed in Epfn KO mice due to the disturbing the dental epithelial cell differentiation into enamel-secreting ameloblasts. Undifferentiated dental epithelial cells in mutant mice sustain the immature states of cell physiology such as those in bud stage of tooth development. Dental epithelium of mutant mice keeps invaginating into mesenchymal tissue randomly and the interactions between dental epithelium and mesenchyme were sequentially occurred [8]. The mesenchymal cells adjacent of undifferentiated dental epithelium were induced to differentiate into dental mesenchymal cells that further differentiating into odontoblasts. The randomly induced odontoblasts in mutant mice start producing dentin matrix and formed tooth structures such as dentin and dental pulp. This finding suggests that the dental mesenchyme can be induced by interaction with undifferentiated dental epithelial cells and there is no limitation of the dental mesenchymal tissue production.
Conclusions
A variety of molecules contribute to form a functional shape and size of tooth and regulate the proper number of tooth. We have uncovered novel diverse roles of Epfn in tooth development. Epfn is essential for tooth morphogenesis by regulating tooth numbers and dental epithelial cell fate. Our studies provide new dimensions of understanding of the molecular mechanism governing the complex processes in tooth morphogenesis. Furthermore, our findings lead to develop a novel tissue engineering technique of the creation of bioteeth.
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Nakamura, T., Yamada, Y., Fukumoto, S. (2012). Review: The Regulation of Tooth Development and Morphogenesis. In: Sasaki, K., Suzuki, O., Takahashi, N. (eds) Interface Oral Health Science 2011. Springer, Tokyo. https://doi.org/10.1007/978-4-431-54070-0_3
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