Differential tissue growth and cell adhesion alone drive early tooth morphogenesis: An ex vivo and in silico study.

From gastrulation to late organogenesis animal development involves many genetic and bio-mechanical interactions between epithelial and mesenchymal tissues. Ectodermal organs, such as hairs, feathers and teeth are well studied examples of organs whose development is based on epithelial-mesenchymal interactions. These develop from a similar primordium through an epithelial folding and its interaction with the mesenchyme. Despite extensive knowledge on the molecular pathways involved, little is known about the role of bio-mechanical processes in the morphogenesis of these organs. We propose a simple computational model for the biomechanics of one such organ, the tooth, and contrast its predictions against cell-tracking experiments, mechanical relaxation experiments and the observed tooth shape changes over developmental time. We found that two biomechanical processes, differential tissue growth and differential cell adhesion, were enough, in the model, for the development of the 3D morphology of the early tooth germ. This was largely determined by the length and direction of growth of the cervical loops, lateral folds of the enamel epithelium. The formation of these cervical loops was found to require accelerated epithelial growth relative to other tissues and their direction of growth depended on specific differential adhesion between the three tooth tissues. These two processes and geometrical constraints in early tooth bud also explained the shape asymmetry between the lateral cervical loops and those forming in the anterior and posterior of the tooth. By performing mechanical perturbations ex vivo and in silico we inferred the distribution and direction of tensile stresses in the mesenchyme that restricted cervical loop lateral growth and forced them to grow downwards. Overall our study suggests detailed quantitative explanations for how bio-mechanical processes lead to specific morphological 3D changes over developmental time.

Signaling Pathways Critical for Tooth Root Formation.

Tooth is made of an enamel-covered crown and a cementum-covered root. Studies on crown dentin formation have been a major focus in tooth development for several decades. Interestingly, the population prevalence for genetic short root anomaly (SRA) with no apparent defects in crown is close to 1.3%. Furthermore, people with SRA itself are predisposed to root resorption during orthodontic treatment. The discovery of the unique role of Nfic (nuclear factor I C; a transcriptional factor) in controlling root but not crown dentin formation points to a new concept: tooth crown and root have different control mechanisms. Further genetic mechanism studies have identified more key molecules (including Osterix, ß-catenin, and sonic hedgehog) that play a critical role in root formation. Extensive studies have also revealed the critical role of Hertwig's epithelial root sheath in tooth root formation. In addition, Wnt10a has recently been found to be linked to multirooted tooth furcation formation. These exciting findings not only fill the critical gaps in our understanding about tooth root formation but will aid future research regarding the identifying factors controlling tooth root size and the generation of a whole "bio-tooth" for therapeutic purposes. This review starts with human SRA and mainly focuses on recent progress on the roles of NFIC-dependent and NFIC-independent signaling pathways in tooth root formation. Finally, this review includes a list of the various Cre transgenic mouse lines used to achieve tooth root formation-related gene deletion or overexpression, as well as strengths and limitations of each line.

Morphological study of tooth development in podoplanin-deficient mice.

Podoplanin is a mucin-type highly O-glycosylated glycoprotein identified in several somatyic cells: podocytes, alveolar epithelial cells, lymphatic endothelial cells, lymph node stromal fibroblastic reticular cells, osteocytes, odontoblasts, mesothelial cells, glia cells, and others. It has been reported that podoplanin-RhoA interaction induces cytoskeleton relaxation and cell process stretching in fibroblastic cells and osteocytes, and that podoplanin plays a critical role in type I alveolar cell differentiation. It appears that podoplanin plays a number of different roles in contributing to cell functioning and growth by signaling. However, little is known about the functions of podoplanin in the somatic cells of the adult organism because an absence of podoplanin is lethal at birth by the respiratory failure. In this report, we investigated the tooth germ development in podoplanin-knockout mice, and the dentin formation in podoplanin-conditional knockout mice having neural crest-derived cells with deficiency in podoplanin by the Wnt1 promoter and enhancer-driven Cre recombinase: Wnt1-Cre;PdpnΔ/Δmice. In the Wnt1-Cre;PdpnΔ/Δmice, the tooth and alveolar bone showed no morphological abnormalities and grow normally, indicating that podoplanin is not critical in the development of the tooth and bone.

Dentin Sialoprotein is a Novel Substrate of Matrix Metalloproteinase 9 in vitro and in vivo.

Dentin sialoprotein (DSP) is essential for dentinogenesis and processed into fragments in the odontoblast-like cells and the tooth compartments. Matrix metalloproteinase 9 (MMP9) is expressed in teeth from early embryonic to adult stage. Although MMP9 has been reported to be involved in some physiological and pathological conditions through processing substrates, its role in tooth development and whether DSP is a substrate of MMP9 remain unknown. In this study, the function of MMP9 in the tooth development was examined by observation of Mmp9 knockout (Mmp9-/-) mouse phenotype, and whether DSP is a substrate of MMP9 was explored by in vitro and in vivo experiments. The results showed that Mmp9-/- teeth displayed a phenotype similar to dentinogenesis imperfecta, including decreased dentin mineral density, abnormal dentin architecture, widened predentin and irregular predentin-dentin boundary. The distribution of MMP9 and DSP overlapped in the odontoblasts, the predentin, and the mineralized dentin, and MMP9 was able to specifically bind to DSP. MMP9 highly efficiently cleaved DSP into distinct fragments in vitro, and the deletion of Mmp9 caused improper processing of DSP in natural teeth. Therefore, our findings demonstrate that MMP9 is important for tooth development and DSP is a novel target of MMP9 during dentinogenesis.

MicroRNA 665 Regulates Dentinogenesis through MicroRNA-Mediated Silencing and Epigenetic Mechanisms.

Studies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis.

Enamelin is critical for ameloblast integrity and enamel ultrastructure formation.

Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta in which the affected enamel is thin or absent. Study of enamelin knockout NLS-lacZ knockin mice revealed that mineralization along the distal membrane of ameloblast is deficient, resulting in no true enamel formation. To determine the function of enamelin during enamel formation, we characterized the developing teeth of the Enam-/- mice, generated amelogenin-driven enamelin transgenic mouse models, and then introduced enamelin transgenes into the Enam-/- mice to rescue enamel defects. Mice at specific stages of development were subjected to morphologic and structural analysis using ß-galactosidase staining, immunohistochemistry, and transmission and scanning electron microscopy. Enamelin expression was ameloblast-specific. In the absence of enamelin, ameloblasts pathology became evident at the onset of the secretory stage. Although the aggregated ameloblasts generated matrix-containing amelogenin, they were not able to create a well-defined enamel space or produce normal enamel crystals. When enamelin is present at half of the normal quantity, enamel was thinner with enamel rods not as tightly arranged as in wild type suggesting that a specific quantity of enamelin is critical for normal enamel formation. Enamelin dosage effect was further demonstrated in transgenic mouse lines over expressing enamelin. Introducing enamelin transgene at various expression levels into the Enam-/- background did not fully recover enamel formation while a medium expresser in the Enam+/- background did. Too much or too little enamelin abolishes the production of enamel crystals and prism structure. Enamelin is essential for ameloblast integrity and enamel formation.

Size and shape variability in human molars during odontogenesis.

Under the patterning cascade model (PCM) of cusp development inspired by developmental genetic studies, it is predicted that the location and the size of later-forming cusps are more variable than those of earlier-forming ones. Here we assessed whether differences in the variability among cusps in total and each particular crown component (enamel-dentin junction [EDJ], outer enamel surface [OES], and cement-enamel junction [CEJ]) could be explained by the PCM, using human maxillary permanent first molars (UM1) and second deciduous molars (um2). Specimens were µCT-scanned, and 3D models of EDJ and OES were reconstructed. Based on these models, landmark-based 3D geometric morphometric analyses were conducted. Size variability in both tooth types was generally consistent with the above prediction, and the differences in size variation among cusps were smaller for the crown components completed in later stages of odontogenesis. With a few exceptions, however, the prediction was unsupported regarding shape variability, and UM1 and um2 showed different patterns. Our findings suggested that the pattern of size variability would be caused by temporal factors such as the order of cusp initiation and the duration from the beginning of mineralization to the completion of crown formation, whereas shape variability may be affected by both topographic and temporal factors.

Proteolytic processing of dentin sialophosphoprotein (DSPP) is essential to dentinogenesis.

DSPP, which plays a crucial role in dentin formation, is processed into the NH(2)-terminal and COOH-terminal fragments. We believe that the proteolytic processing of DSPP is an essential activation step for its biological function in biomineralization. We tested this hypothesis by analyzing transgenic mice expressing the mutant D452A-DSPP in the Dspp-knock-out (Dspp-KO) background (referred to as "Dspp-KO/D452A-Tg" mice). We employed multipronged approaches to characterize the dentin of the Dspp-KO/D452A-Tg mice, in comparison with Dspp-KO mice and mice expressing the normal DSPP transgene in the Dspp-KO background (named Dspp-KO/normal-Tg mice). Our analyses showed that 90% of the D452A-DSPP in the dentin of Dspp-KO/D452A-Tg mice was not cleaved, indicating that D452A substitution effectively blocked the proteolytic processing of DSPP in vivo. While the expression of the normal DSPP fully rescued the dentin defects of the Dspp-KO mice, expressing the D452A-DSPP failed to do so. These results indicate that the proteolytic processing of DSPP is an activation step essential to its biological function in dentinogenesis.

Constitutive stabilization of ß-catenin in the dental mesenchyme leads to excessive dentin and cementum formation.

Wnt/ß-catenin signaling plays an important role in morphogenesis and cellular differentiation during development. Essential roles of Wnt/ß-catenin signaling in tooth morphogenesis have been well known, but the involvement of Wnt/ß-catenin signaling in the dental hard tissue formation remains undefined. To understand roles of Wnt/ß-catenin signaling in dentin and cementum formation, we generated and analyzed the conditional ß-catenin stabilized mice in the dental mesenchyme. The OC-Cre;Catnb(lox(ex3)/+) mice exhibited malformed teeth characterized by aberrantly formed dentin and excessively deposited cementum. Large amount of dentin was rapidly formed with widened predentin and numerous globular calcifications in the crown. Whereas roots of molars were short and covered with the excessively formed cellular cementum. With age, the coronal pulp chamber and periodontal space were narrowed by the excessively formed dentin and cementum, respectively. To compare the changes of gene expression in the mutant mice, Col1a1 expression was increased but that of Dspp was decreased in the odontoblasts. However, both of Col1a1 and Bsp expression was increased in the cementoblasts. The gene expression changes were consistent with the localization of matrix proteins. Biglycan and PC-1 was increased but Phex was decreased in the odontoblasts and dentin matrix, respectively. TNAP was increased but Dmp1 and FGF23 was decreased in the cementoblasts and cementum matrix, respectively. Our results indicate that persistent stabilization of ß-catenin in the dental mesenchyme leads to premature differentiation of odontoblasts and differentiation of cementoblasts, and induces excessive dentin and cementum formation in vivo. These results suggest that temporospatial regulation of Wnt/ß-catenin signaling plays critical roles in the differentiation of odontoblasts and cementoblasts, and that inhibition of Wnt/ß-catenin signaling may be important for the formation of dentin and cementum during tooth development. Local modulation of Wnt/ß-catenin signaling has therapeutic potential to improve the regeneration of dentin and periodontium.

SMAD4-mediated WNT signaling controls the fate of cranial neural crest cells during tooth morphogenesis.

TGFß/BMP signaling regulates the fate of multipotential cranial neural crest (CNC) cells during tooth and jawbone formation as these cells differentiate into odontoblasts and osteoblasts, respectively. The functional significance of SMAD4, the common mediator of TGFß/BMP signaling, in regulating the fate of CNC cells remains unclear. In this study, we investigated the mechanism of SMAD4 in regulating the fate of CNC-derived dental mesenchymal cells through tissue-specific inactivation of Smad4. Ablation of Smad4 results in defects in odontoblast differentiation and dentin formation. Moreover, ectopic bone-like structures replaced normal dentin in the teeth of Osr2-IresCre;Smad4(fl/fl) mice. Despite the lack of dentin, enamel formation appeared unaffected in Osr2-IresCre;Smad4(fl/fl) mice, challenging the paradigm that the initiation of enamel development depends on normal dentin formation. At the molecular level, loss of Smad4 results in downregulation of the WNT pathway inhibitors Dkk1 and Sfrp1 and in the upregulation of canonical WNT signaling, including increased ß-catenin activity. More importantly, inhibition of the upregulated canonical WNT pathway in Osr2-IresCre;Smad4(fl/fl) dental mesenchyme in vitro partially rescued the CNC cell fate change. Taken together, our study demonstrates that SMAD4 plays a crucial role in regulating the interplay between TGFß/BMP and WNT signaling to ensure the proper CNC cell fate decision during organogenesis.

The morphology and mineralization of dental hard tissue in the offspring of passive smoking rats.

OBJECTIVE: To study the effects of maternal passive smoking on the morphology and mineralization of dental hard tissue in offspring rats. DESIGN: We have established a maternal passive smoking model. Offspring rats were sacrificed on the 20th day of gestation (E20) or the 3rd (D3) or 10th day (D10) after birth. We observed hard tissue morphology using Haematoxylin-Eosin (H&E) staining sections, used micro computer tomography (Micro-CT) to measure hard tissue thickness and volume on the mandibular first molars of the offspring rats, and used Micro-CT and energy dispersive X-ray spectroscopy with scanning electron microscopy (SEM/EDS) to determine the hard tissue mineral density and the ratio of calcium atom number/calcium atom+phosphorus atom number (Ca(2+)/P(3-)+Ca(2+)). RESULTS: Overall, the development of dental hard tissue was delayed in the offspring of passive smoking rats. The thickness and volume of hard tissue were lower in the offspring of the maternal passive smoking group than in the offspring of the control group. Mineral density of the hard tissue and the ratio of (Ca(2+)/P(3-)+Ca(2+)) were also reduced in the offspring of the maternal passive smoking group. CONCLUSION: Maternal passive smoking inhibits the morphological development and mineralization level of hard tissue on the mandibular first molars of offspring rats.

Spatiotemporal expression of sclerostin in odontoblasts during embryonic mouse tooth morphogenesis.

INTRODUCTION: Sclerostin is the product of the SOST gene. Loss-of-function mutations in the SOST gene result in a high bone mass phenotype, thus confirming that sclerostin is a negative regulator of bone mass. SOST knockdown in humans also causes oral and dental malformations. However, the relationship between sclerostin and tooth development is unclear. METHODS: Using immunohistochemical techniques, we investigated sclerostin expression during fetal mouse tooth development and adult mouse tooth morphogenesis. RESULTS: Sclerostin was expressed in the secretory odontoblasts located along the ameloblasts of fetal mouse tooth germ and adult incisor. Sclerostin expression was also observed in the fetal and adult osteocytes in the jaw bone. CONCLUSION: These results suggest that sclerostin, one of the important regulatory factors of differentiated odontoblast function, may usable in vital pulp therapy.

Early morphogenesis of heterodont dentition in minipigs.

The minipig provides an excellent experimental model for tooth morphogenesis because its diphyodont and heterodont dentition resemble that of humans. However, little information is available on the processes of tooth development in the pig. The purpose of this study was to classify the early stages of odontogenesis in minipigs from the initiation of deciduous dentition to the late bell stage when the successional dental lamina begins to develop. To analyze the initiation of teeth anlagens and the structural changes of dental lamina, a three-dimensional (3D) analysis was performed. At the earliest stage, 3D reconstruction revealed a continuous dental lamina along the length of the jaw. Later, the dental lamina exhibited remarkable differences in depth, and the interdental lamina was shorter. The dental lamina grew into the mesenchyme in the lingual direction, and its inclined growth was underlined by asymmetrical cell proliferation. After the primary tooth germ reached the late bell stage, the dental lamina began to disintegrate and fragmentize. Some cells disappeared during the process of lamina degradation, while others remained in small islands known as epithelial pearls. The minipig can therefore, inter alia, be used as a model organism to study the fate of epithelial pearls from their initiation to their contribution to pathological structures, primarily because of the clinical significance of these epithelial rests.

[Theoretical and practical principles of dentinogenesis: hypotheses and confirmed clinically reality].

The problem of maintaining dental vitality and stimulating reparative processes is a priority in modern odontology. Restorative processes depend not only on the type and size of tissue damage, but also on the protection capacity and integrity of the structural/functional pulp-dentin boundary. Primary dentin that is initiated in the intrauterine period has unique structure and composition. Secondary dentin continues to form after the tooth is erupted, then after root formation is finished, and throughout life. Actually the primary and secondary dentins have similar tissue structures developed at different stages of dentinogenesis. Primary dentinogenesis is initiated by odontoblasts located in the periphery of dental pulp. Secondary dentin as a structure already exists once root formation is complete, but at that stage is has low levels of mineralization. Formation of tertiary dentin is always reactionary to different pathologies and is initiated by so called "transitional odontoblasts" (odontoblast-like cells) and partially fibroblasts. Odontotropic and anti-inflammatory medications strongly change structural characteristics of the dentin. Pulpal ability to produce dentin-like matrix (tertiary dentin) is an important component of the pulp-dentin reparative capacity. Only specific characteristics of the dentin can account for indications and contraindications for using restorative liners and explain the impact of adhesive systems on these. In this context, the interest is high to the dentin and its response and change in reaction to different stimuli. Dental caries and other pathological processes (abrasion, erosion, attrition) seriously affect dentin vital activity causing it to change to the "emergency" mode. This process is viewed not as resulting from pulp medication but as reactionary, aimed for self-preservation. In such cases the major focus is not on drug composition but on pulpal response. The pulp may be said to "form tertiary dentin for self-protection". In conclusion, the tertiary dentin that forms as a result of pathological processes (express-dentin, reparatory dentin) could be identified as a perfect barrier for the pulp necessary for keeping it vital. And investigation of mechanisms causing primary stimulation of odontoblasts and triggering the reparative processes remains a pressing problem in modern odontology.

Distribution pattern of cholinesterase enzymes in human tooth germs.

The two distinct molecular forms of cholinesterase (ChE) are acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Our previous studies have reported that ChE is involved in tooth development. However, further experiments are needed to understand the precise action of ChE in tooth development. This study aimed to localise types of ChE in human tooth germs, and identify their distribution pattern. ChE were localised in frozen sections of jaws which were prepared from dead fetuses, neonates and stillborns who were free from visible abnormalities by Karnovsky and Root method. AChE was identified in the inner and outer enamel epithelia including the cervical loop region, stratum intermedium and preameloblasts of tooth germs at bell stage. Secretory ameloblasts were free from staining. The bud and cap stages of permanent tooth germs showed AChE activity on the lingual aspect and top surface of the epithelial ingrowths, respectively. BuChE activity was localised in the degenerating dental lamina. Our study reported the first evidence of localisation of ChE in human tooth development and identified the possible molecular form of ChE in tooth germs as AChE. Also, our results have provided strong evidence to speculate the action of AChE is on the cells of enamel organ during tooth development.

Spatial and temporal expression of KLF4 and KLF5 during murine tooth development.

OBJECTIVE: KLF4 and KLF5, members of the Krüppel-like factor (KLF) family, play key roles in proliferation, differentiation and apoptosis during development. In order to determine if these transcription factors are associated with tooth development, we examined the expression pattern of KLF4 and KLF5 during murine tooth development. DESIGN: In situ hybridization and immunohistochemistry were performed to detect the expression pattern of KLF4 and KLF5 from E12.5 to PN3 during murine tooth development. RESULTS: In situ hybridization analysis revealed that Klf4 was specifically expressed in polarizing odontoblasts from E16.5 (incisor) or E18.5 (first molar) to PN3. Immunohistochemistry staining showed that KLF4 was specifically expressed in both polarizing odontoblasts and ameloblasts at the same stages. KLF5 was mainly expressed from E18.5 to PN3 in secretory ameloblasts when enamel mineralization occurs and in secretory odontoblasts. However, an expression of KLF5 was also observed at earlier stages (E14.5 and E16.5) mainly in proliferating epithelial cells. CONCLUSIONS: These results suggest that the expression of KLF4 is closely correlated to the growth-arrest and the first step of odontoblast and ameloblast differentiation. Furthermore, KLF5 maybe involved in proliferation at the early stages of tooth development and related to mineralization of both enamel and dentin matrices at later stages.

The effect of retinoic acid on mouse mandibular molar development in vitro, using alkaline phosphatase as a molecular indicator of differentiation.

INTRODUCTION: An excellent model system that links evolutionary biology and developmental biology in seeking to understand evolutionary diversity is the study of tooth development in mammals. These studies reflect the diversity of mammalian radiations which bear on the interpretation of South African fossil hominids. Tooth development occurs via epithelio-mesenchymal interactions and involves the production of many substances, including alkaline phosphatase, which is necessary for dentine and enamel formation. Retinoic acid is a known morphogen and is important in tooth development. In excess, retinoic acid has been found to alter the formation of teeth. OBJECTIVES: In order to determine whether retinoic acid has any effect on tooth morphology, exogenous retinoic acid was administered to developing mouse molar teeth in vitro, and alkaline phosphatase was utilized as an indicator of differentiation. METHODS: Molars were microdissected from 15.5 day mouse embryo mandibles and cultured at the air: medium interface with or without retinoic acid for seven days. Following fixation and embedding, the explants were sectioned for morphological analysis. Alkaline phosphatase activity was detected using a modified Gomori's histochemical method. RESULTS AND CONCLUSION: Retinoic acid appeared to retard the growth and differentiation of the molar explants. This was coincident with reduced alkaline phosphatase.

Mouse models of tooth abnormalities.

Tooth number is abnormal in about 20% of the human population. The most common defect is agenesis of the third molars, followed by loss of the lateral incisors and loss of the second premolars. Tooth loss appears as both a feature of multi-organ syndromes and as a non-syndromic isolated character. Apart from tooth number, abnormalities are also observed in tooth size, shape, and structure. Many of the genes that underlie dental defects have been identified, and several mouse models have been created to allow functional studies to understand, in greater detail, the role of particular genes in tooth development. The ability to manipulate the mouse embryo using explant culture and genome targeting provides a wealth of information that ultimately may pave the way for better diagnostics, treatment or even cures for human dental disorders. This review aims to summarize recent knowledge obtained in mouse models, which can be used to gain a better understanding of the molecular basis of human dental abnormalities.

Transcription factor epiprofin is essential for tooth morphogenesis by regulating epithelial cell fate and tooth number.

In tooth morphogenesis, the dental epithelium and mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth. We previously identified epiprofin (Epfn), a gene preferentially expressed in dental epithelia, differentiated ameloblasts, and certain ectodermal organs. To identify the role of Epfn in tooth development, we created Epfn-deficient mice (Epfn-/-). Epfn-/- mice developed an excess number of teeth, enamel deficiency, defects in cusp and root formation, and abnormal dentin structure. Mutant tooth germs formed multiple dental epithelial buds into the mesenchyme. In Epfn-/- molars, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Formation of the enamel knot, a signaling center for cusps, whose cells differentiate from the dental epithelium, was also inhibited. However, multiple premature nonproliferating enamel knot-like structures were formed ectopically. These dental epithelial abnormalities were accompanied by dysregulation of Lef-1, which is required for the normal transition from the bud to cap stage. Transfection of an Epfn vector promoted dental epithelial cell differentiation into ameloblasts and activated promoter activity of the enamel matrix ameloblastin gene. Our results suggest that in Epfn-deficient teeth, ectopic nonproliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.