Ameloblast Lineage Research Articles

Establishment of inclusive single-cell transcriptome atlases from mouse and human tooth as powerful resource for dental research.

Single-cell (sc) omics has become a powerful tool to unravel a tissue’s cell landscape across health and disease. In recent years, sc transcriptomic interrogation has been applied to a variety of tooth tissues of both human and mouse, which has considerably advanced our fundamental understanding of tooth biology. Now, an overarching and integrated bird’s-view of the human and mouse tooth sc transcriptomic landscape would be a powerful multi-faceted tool for dental research, enabling further decipherment of tooth biology and development through constantly progressing state-of-the-art bioinformatic methods as well as the exploration of novel hypothesis-driven research. To this aim, we re-assessed and integrated recently published scRNA-sequencing datasets of different dental tissue types (healthy and diseased) from human and mouse to establish inclusive tooth sc atlases, and applied the consolidated data map to explore its power. For mouse tooth, we identified novel candidate transcriptional regulators of the ameloblast lineage. Regarding human tooth, we provide support for a developmental connection, not advanced before, between specific epithelial compartments. Taken together, we established inclusive mouse and human tooth sc atlases as powerful tools to potentiate innovative research into tooth biology, development and disease. The maps are provided online in an accessible format for interactive exploration.

Adam10-dependent Notch signaling establishes dental epithelial cell boundaries required for enamel formation

SummaryThe disintegrin and metalloproteinase Adam10 is a membrane-bound sheddase that regulates Notch signaling and ensures epidermal integrity. To address the function of Adam10 in the continuously growing incisors, we used Keratin14Cre/+;Adam10fl/fl transgenic mice, in which Adam10 is conditionally deleted in the dental epithelium. Keratin14Cre/+;Adam10fl/fl mice exhibited severe abnormalities, including defective enamel formation reminiscent of human enamel pathologies. Histological analyses of mutant incisors revealed absence of stratum intermedium, and severe disorganization of enamel-secreting ameloblasts. In situ hybridization and immunostaining analyses in the Keratin14Cre/+;Adam10fl/fl incisors showed strong Notch1 downregulation in dental epithelium and ectopic distribution of enamel-specific molecules, including ameloblastin and amelogenin. Lineage tracing studies using Notch1CreERT2;R26mT/mG mice demonstrated that loss of the stratum intermedium cells was due to their fate switch toward the ameloblast lineage. Overall, our data reveal that in the continuously growing incisors the Adam10/Notch axis controls dental epithelial cell boundaries, cell fate switch and proper enamel formation.

Melatonin-Medicated Neural JNK3 Up-Regulation Promotes Ameloblastic Mineralization.

Introduction: Melatonin, an endogenous neurohormone, modulates the biological circadian rhythms of vertebrates. It functions have been reported in previous stomatological studies as anti-inflammation, antioxidant, osseointegration of dental implants and stimulation to dental pulp stem cells differentiation, but its role in ameloblastic differentiation and mineralization has been rarely studied. Objective: To reveal the effects of melatonin on the mineralization of ameloblast lineage cells (ALCs), and to identify the change in gene expression and the potential mechanism based on ribonucleic acid sequencing (RNA-seq) analysis. Method: ALCs were induced in melatonin-conditioned medium. After 7-days culture, Western blot, real-time PCR, alkaline phosphatase (ALP) activity test, RNA-seq were accordingly used to detect the change in molecular level. After 1-month odontogenic induction in melatonin medium, Alizarin Red-S (ARS) staining showed the changes of mineral nodules. Differentially expressed genes (DEGs), enrichment of functions and signaling pathways analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) database were performed. The JNK3 antagonist (JNK3 inhibitor IX, SR3576) and β-arrestin1 (Arrb1) overexpression were applied to confirm the fluctuation of melatonin-medicated JNK3 and Arrb1 expression. Results: In this study, we found out melatonin contributed to the ameloblastic mineralization, from which we can observed the elevated expression of enamel matrix protein, and increased ALP activity and mineralized nodules formation. RNA-seq analysis showed the up-regulation of neural JNK3 and down-regulation of Arrb1 in ALCs. Meanwhile, phosphorylated JNK3 deficiency (phosphorylated JNK3 inhibitor---SR3576 added to culture medium) led to mineralization delay, and Arrb1 overexpression proved Arrb1 takes bridge between melatonin receptors (MTNR) and JNK3 in MAPK signaling pathway.

Sox2 controls asymmetric patterning of ameloblast lineage commitment by regulation of FGF signaling in the mouse incisor.

Mouse incisors are covered by enamel only on the labial side and the lingual side is covered by dentin without enamel. This asymmetric distribution of enamel makes it possible to be abrased on the lingual side, generating the sharp cutting edge of incisor on the labial side. The abrasion of mouse incisors is compensated by the continuous growth throughout life. Epithelium stem cells responsible for its continuous growth are reported to localize within the labial cervical loop. The transcription factor Sox2 plays important roles in the maintenance of stem cell pluripotency and organ formation. We previously found that Sox2 mainly expressed in the dental epithelium. Besides, Sox2 has been reported to be a dental epithelium stem cell marker in the incisor. However, the exact mechanism of Sox2 controlling amelogenesis is still not quite clearly elucidated. Here we report that conditional deletion of Sox2 in the dental epithelium using Shhcre caused impaired ameloblast differentiation in the labial side and induced ectopic ameloblast-like cell differentiation on the lingual side. Abnormal FGF gene expression was detected by RNAscope in situ hybridization in the mutant incisor. Collectively, we speculate that asymmetric ameloblast lineage commitment of mouse incisor might be regulated by Sox2 through FGF signaling.

Stage-Specific Role of Amelx Activation in Stepwise Ameloblast Induction from Mouse Induced Pluripotent Stem Cells.

Amelogenin comprises ~90% of enamel proteins; however, the involvement of Amelx transcriptional activation in regulating ameloblast differentiation from induced pluripotent stem cells (iPSCs) remains unknown. In this study, we generated doxycycline-inducible Amelx-expressing mouse iPSCs (Amelx-iPSCs). We then established a three-stage ameloblast induction strategy from Amelx-iPSCs, including induction of surface ectoderm (stage 1), dental epithelial cells (DECs; stage 2), and ameloblast lineage (stage 3) in sequence, by manipulating several signaling molecules. We found that adjunctive use of lithium chloride (LiCl) in addition to bone morphogenetic protein 4 and retinoic acid promoted concentration-dependent differentiation of DECs. The resulting cells had a cobblestone appearance and keratin14 positivity. Attenuation of LiCl at stage 3 together with transforming growth factor β1 and epidermal growth factor resulted in an ameloblast lineage with elongated cell morphology, positivity for ameloblast markers, and calcium deposition. Although stage-specific activation of Amelx did not produce noticeable phenotypic changes in ameloblast differentiation, Amelx activation at stage 3 significantly enhanced cell adhesion as well as decreased proliferation and migration. These results suggest that the combination of inducible Amelx transcription and stage-specific ameloblast induction for iPSCs represents a powerful tool to highlight underlying mechanisms in ameloblast differentiation and function in association with Amelx expression.

Lif Deficiency Leads to Iron Transportation Dysfunction in Ameloblasts

Leukemia inhibitory factor (LIF), a member of the interleukin 6 family of cytokines, is involved in skeletal metabolism, blastocyst implantation, and stem cell pluripotency maintenance. However, the role of LIF in tooth development needs to be elucidated. The aim of the present study was to investigate the effect of Lif deficiency on tooth development and to elucidate the functions of Lif during tooth development and the underlying mechanisms. First, it was found that the incisors of Lif-knockout mice had a much whiter color than those of wild-type mice. Although there were no structural abnormalities or defective mineralization according to scanning electronic microscopy and computed tomography analysis, 3-dimensional images showed that the length of incisors was shorter in Lif−/− mice. Microhardness and acid resistance assays showed that the hardness and acid resistance of the enamel surface of Lif−/− mice were decreased compared to those of wild-type mice. In Lif−/− mice, whose general iron status was comparable to that of the control mice, the iron content of the incisors was significantly reduced, as confirmed by energy-dispersive X-ray spectroscopy (EDS) and Prussian blue staining. Histological staining showed that the cell length of maturation-stage ameloblasts was shorter in Lif−/− mice. Likewise, decreased expression of Tfrc and Slc40a1, both of which are crucial proteins for iron transportation, was observed in Lif−/− mice and Lif-knockdown ameloblast lineage cell lines, according to quantitative reverse transcription polymerase chain reaction, immunohistochemistry, and Western blot. Moreover, the upregulation of Tfrc and Slc40a1 induced by Lif stimulation was blocked by Stattic, a signal transducer and activator of transcription 3 (Stat3) signaling inhibitor. These results suggest that Lif deficiency inhibits iron transportation in the maturation-stage ameloblasts, and Lif modulates expression of Tfrc and Slc40a1 through the Stat3 signaling pathway during enamel development.

Combination of Runx2 and Cbfβ upregulates Amelotin gene expression in ameloblasts by directly interacting with cis‑enhancers during amelogenesis.

Amelotin (Amtn) is a recently identified enamel protein secreted by ameloblasts at late stage of enamel development. Runt‑related transcription factor 2 (Runx2) in combination with the coactivator core‑binding factor β (Cbfβ) regulates the early stages of tooth development. The aim of the present study was to investigate the role of Runx2 in the regulation of Amtn gene expression in ameloblasts. Immunohistochemistry was performed and the results revealed that Runx2 protein was predominantly expressed in the nuclei of ameloblasts during the transition stage and the maturation stage of enamel development, whereas Cbfβ was expressed in ameloblasts from the secretory stage to the maturation stage. Reverse transcription‑quantitative polymerase chain reaction results demonstrated that Runx2 knockdown decreased Amtn expression in ameloblast‑lineage cells and co‑expression of Runx2 and Cbfβ in ameloblast lineage cells induced an upregulation in Amtn gene expression. Two putative Runx2‑binding sites within the Amtn promoter were identified using bioinformatics analysis. Results of an electrophoretic mobility shift assay and chromatin immunoprecipitation indicated that Runx2/Cbfβ bound to specific DNA sequences. Site‑directed mutagenesis of the Runx2 binding sites within the Amtn promoter resulted in decreased basal promoter activity and did not affect the overexpressed Runx2/Cbfβ. The results of the present study suggest that Runx2 upregulates Amtn gene expression via binding directly to Runx2 sites within the Amtn promoter during amelogenesis.

Plasticity within the niche ensures the maintenance of a Sox2+ stem cell population in the mouse incisor.

In mice, the incisors grow throughout the animal's life, and this continuous renewal is driven by dental epithelial and mesenchymal stem cells. Sox2 is a principal marker of the epithelial stem cells that reside in the mouse incisor stem cell niche, called the labial cervical loop, but relatively little is known about the role of the Sox2+ stem cell population. In this study, we show that conditional deletion of Sox2 in the embryonic incisor epithelium leads to growth defects and impairment of ameloblast lineage commitment. Deletion of Sox2 specifically in Sox2+ cells during incisor renewal revealed cellular plasticity that leads to the relatively rapid restoration of a Sox2-expressing cell population. Furthermore, we show that Lgr5-expressing cells are a subpopulation of dental Sox2+ cells that also arise from Sox2+ cells during tooth formation. Finally, we show that the embryonic and adult Sox2+ populations are regulated by distinct signalling pathways, which is reflected in their distinct transcriptomic signatures. Together, our findings demonstrate that a Sox2+ stem cell population can be regenerated from Sox2- cells, reinforcing its importance for incisor homeostasis.

The Fam50a positively regulates ameloblast differentiation via interacting with Runx2.

Differentiated ameloblasts secret enamel matrix proteins such as amelogenin, ameloblastin, and enamelin. Expression levels of these proteins are regulated by various factors. To find a new regulatory factor for ameloblast differentiation, we performed 2D-PAGE analysis using mouse ameloblast lineage cell line (mALCs) cultured with mineralizing medium. Of identified proteins, family with sequence similarity 50 member A (Fam50a) was significantly increased during differentiation of mALCs. Fam50a protein was also highly expressed in secretory ameloblasts of mouse tooth germs. In mALCs cultures, forced expression of Fam50a up-regulated the expression of enamel matrix protein genes such as amelogenin, ameloblastin, and enamelin. In addition, up-regulation of Fam50a also increased ALP activity and mineralized nodule formation in a dose-dependent manner. In contrast, knockdown of Fam50a decreased expression levels of enamel matrix protein genes, ALP activity, and mineralized nodule formation. By fluorescence microscopy, endogenous Fam50a protein was found to be localized to the nucleus of ameloblasts. In addition, Fam50a synergistically increased Ambn transactivation by Runx2. Moreover, Fam50a increased binding affinity of Runx2 to Ambn promoter by physically interacting with Runx2. Taken together, these results suggest Fam50a might be a new positive regulator of ameloblast differentiation.

P38/JNK signaling pathway mediates the fluoride-induced down-regulation of Fam83h

Background/AimThe similar clinical and pathological feature in fluorosis and amelogenesis imperfect with FAM83H mutations imply that excess fluoride could have effects on the expression of FAM83H and could elaborate this process by some signal pathways regulation. The present study aims to investigate the effects of fluoride on Fam83h expression and try to explore the molecular signaling regulation between them as well as the association of high concentration fluoride with mineralization in ameloblast lineage cells. MethodsProtein expression and signaling pathways of mouse ameloblast-like LS8 cells, exposed to fluoride or MAPK inhibitors, were compared to control cells without exposure. Fam83h, proteins of MAPK signal pathways (ERK, P38 and JNK) were examined by Quantitative real-time PCR and/or Western-blot. ALP activity and ALP staining were used to detect the mineralization in the cells with exposure during 7-day mineralization inducing differentiation. ResultsThe results showed that Fam83h protein level in LS8 cells decreased in the presence of fluoride and MAPK inhibitors. Down-regulation of Fam83h by fluoride was related to suppression of JNK and P38 phosphorylation, and the descending degree of P38 was more obvious. Fluoride and MAPK inhibitors treatment significantly decreased the mineralization level in LS8 cells. ConclusionThe findings suggest that JNK and P38 could be key regulatory element for Fam83h expression, and that LS8 cells can respond to fluoride by down-regulating Fam83h expression through the regulation of JNK and p38 signaling pathways.

Isolation and characterization of embryonic ameloblast lineage cells derived from tooth buds of fetal miniature swine.

Dental enamel formation, known as "amelogenesis," is initiated by cytodifferentiation of the ectodermally derived dental epithelium. Enamel cannot regenerate itself because once it is completely formed, ameloblasts are lost as the tooth erupts. Rodent teeth have been useful for studying the mechanisms of amelogenesis because ameloblast cell lines can be derived from the ever-growing incisors. However, higher mammals such as humans have no growing teeth, and cell lines derived from larger animals that are more similar to humans are required for higher fidelity studies. Here, we isolated embryonic enamel epithelium-derived epithelial cells from fetal swine. The explant culture of the developing deciduous molars that had been removed from the dental papilla-derived mesenchymal tissue and cells inside the tooth buds provided the epithelial cell population for the primary culture. To isolate the cell population, we performed a unique cell isolation technique called cell fishing. The isolated cells showed clear embryonic-stage ameloblast characteristics with appropriate gene/protein expressions of enamel matrix and proteinases, abundant glycogen pools, and secretory granular materials. They could be continuously subcultured several times and are presently being maintained. This cell population will facilitate the establishment of a stable cell line and allow us to characterize the definitive phenotype and functional behavior of porcine ameloblasts, which, in turn, promises to yield useful and practical findings that are more relevant than those provided by rodent studies. Finally, analysis of in vitro enamel formation will be important for engineering "bio-enamel" as a new dental therapy to restore enamel defects.

Transforming growth factor-β1 regulates expression of the matrix metalloproteinase 20 (Mmp20) gene through a mechanism involving the transcription factor, myocyte enhancer factor-2C, in ameloblast lineage cells

Matrix metalloproteinase-20 (Mmp20) plays an essential role in amelogenesis during tooth development and is regulated by transforming growth factor-β1 (TGF-β1) in mouse ameloblast lineage cells (ALCs). The objective of this study was to explore the role of myocyte enhancer factor-2C (MEF2C), a key transcription factor in craniofacial development, in TGF-β1-induced Mmp20 gene expression. We investigated Mmp20 expression in ALCs over-expressing MEF2C and in ALCs with MEF2C knocked down. We also analyzed activity of the Mmp20 promoter using a transient reporter gene-expression assay in cultured ALCs. Putative transcription factor-binding sites for MEF2C and TGF-β1 on the Mmp20 promoter were analyzed with bioinformatics tools and examined using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The expression of Mmp20 was induced, in a dose-dependent manner, by MEF2C over-expression, and TGF-β1-induced Mmp20 expression was blocked by MEF2C knockdown in ALCs. There was a TGF-β1/MEF2C-responsive region, including a putative MEF2-binding site, between base pairs -356 and -73 of the Mmp20 promoter. Mutation of the putative MEF2-binding site significantly reduced Mmp20 promoter activity upon activation with MEF2C or TGF-β1. In conclusion, TGF-β1-induced Mmp20 expression in ALCs was regulated through the MEF2-binding site on the Mmp20 promoter and thus mediated by the MEF2C signaling pathway.

Inductive Ability of Human Developing and Differentiated Dental Mesenchyme

The development of cell-based therapeutic strategies to bioengineer tooth tissue is a promising approach for the treatment of lost or damaged tooth tissue. The lack of a readily available cell source for human dental epithelial cells (ECs) severely constrains the progress of tooth bioengineering. Previous studies in model organisms have demonstrated that developing dental mesenchyme can instruct nondental epithelium to differentiate into enamel-forming epithelium. In this study, we characterized the ability of fetal and adult human dental mesenchyme to promote differentiation of human embryonic stem cell (hESC)-derived ECs (ES-ECs) into ameloblast-lineage cells. ES-ECs were co-cultured either with human fetal dental mesenchymal cells (FDMCs) or with adult dental mesenchymal cells (ADMCs) in either a three-dimensional culture system, or in the renal capsules of SCID mice. When co-cultured with FDMCs in vitro, ES-ECs polarized and expressed amelogenin. Tooth organ-like structures assembled with epithelium and encased mesenchyme and developing enamel-like structures could be detected in the complexes resulting from in vitro and ex vivo co-culture of ES-ECs and FDMCs. In contrast, co-cultured ES-ECs and ADMCs formed amorphous spherical structures and occasionally formed hair. Transcription factors were significantly upregulated in FDMCs compared to ADMCs including MSX1, GLI1, LHX6, LHX8,LEF1 and TBX1. In summary, FDMCs but not ADMCs had the capacity to induce differentiation of ES-ECs into ameloblast lineage cells. Further characterization of the functional differences between these two types of dental mesenchyme could enable reprogramming of ADMCs to enhance their odontogenic inductive competence.

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.

Transforming growth factor‐β1 expression is up‐regulated in maturation‐stage enamel organ and may induce ameloblast apoptosis

Transforming growth factor-beta1 (TGF-beta1) regulates a variety of cellular responses that are dependent on the developmental stage and on the origins of the cell or the tissue. In mature tissues, and especially in tissues of epithelial origin, TGF-beta1 is generally considered to be a growth inhibitor that may also promote apoptosis. The ameloblast cells of the enamel organ epithelium are adjacent to and responsible for the developing enamel layer on unerupted teeth. Once the enamel layer reaches its full thickness, the tall columnar secretory-stage ameloblasts shorten, and a portion of these maturation-stage ameloblasts become apoptotic. Here we investigate whether TGF-beta1 plays a role in apoptosis of the maturation-stage ameloblasts. We demonstrate in vitro that ameloblast lineage cells are highly susceptible to TGF-beta1-mediated growth arrest and are prone to TGF-beta1-mediated cell death/apoptosis. We also demonstrate in vivo that TGF-beta1 is expressed in the maturation-stage enamel organ at significantly higher levels than in the earlier secretory-stage enamel organ. This increased expression of TGF-beta1 correlates with an increase in expression of the enamel organ immediate-early stress-response gene and with a decrease in the anti-apoptotic Bcl2 : Bax expression ratio. We conclude that TGF-beta1 may play an important role in ameloblast apoptosis during the maturation stage of enamel development.

Calcium‐mediated differentiation of ameloblast lineage cells in vitro

Calcium is a key component of the mineralized enamel matrix, but may also have a role in ameloblast cell differentiation. In this study we used human ameloblast lineage cells to determine the effect of calcium on cell function. Primary human ameloblast lineage cells were isolated from human fetal tooth buds. Cells were treated with calcium ranging from 0.05 to -1.8 mM. Cell morphology was imaged by phase contrast microscopy, and amelogenin was immunolocalized. Proliferation of cells treated with calcium was measured by BrdU immunoassay. The effect of calcium on mRNA expression of amelogenin, Type 1 collagen, DSPP, amelotin, and KLK-4 was compared by PCR analysis. Von Kossa staining was used to detect mineral formation after cells were pretreated with calcium. Calcium induced cell organization and clustering at 0.1 and 0.3 mM concentrations. Increasing concentrations of calcium significantly reduced ameloblast lineage cell proliferation. The addition of 0.1 mM calcium to the cultures upregulated expression of amelogenin, Type I collagen, and amelotin. After pretreatment with 0.3 mM calcium, the cells could form a mineralized matrix. These studies, which utilized human ameloblast lineage cells grown in vitro, showed that the addition of calcium at 0.1 and 0.3 mM, induced cell differentiation and upregulation of amelogenin Type I collagen and amelotin.

Enamel Tissue Engineering Using Subcultured Enamel Organ Epithelial Cells in Combination with Dental Pulp Cells

We describe a strategy for the in vitro engineering of enamel tissue using a novel technique for culturing enamel organ epithelial (EOE) cells isolated from the enamel organ using 3T3-J2 cells as a feeder layer. These subcultured EOE cells retain the capacity to produce enamel structures over a period of extended culture. In brief, enamel organs from 6-month-old porcine third molars were dissociated into single cells and subcultured on 3T3-J2 feeder cell layers. These subcultured EOE cells were then seeded onto a collagen sponge in combination with primary dental pulp cells isolated at an early stage of crown formation, and these constructs were transplanted into athymic rats. After 4 weeks, complex enamel-dentin structures were detected in the implants. These results show that our culture technique maintained ameloblast lineage cells that were able to produce enamel in vivo. This novel subculture technique provides an important tool for tooth tissue engineering.

A regulatory relationship between Tbx1 and FGF signaling during tooth morphogenesis and ameloblast lineage determination

The Tbx1 gene is a transcriptional regulator involved in the DiGeorge syndrome, which affects normal facial and tooth development. Several clinical reports point to a common enamel defect in the teeth of patients with DiGeorge syndrome. Here, we have analyzed the expression, regulation, and function of Tbx1 during mouse molar development. Tbx1 expression is restricted to epithelial cells that give rise to the enamel producing ameloblasts and correlates with proliferative events. Tbx1 expression in epithelium requires mesenchyme-derived signals: dental mesenchyme induces expression of Tbx1 in recombined dental and non-dental epithelia. Bead implantation experiments show that FGF molecules are able to maintain epithelial Tbx1 expression during odontogenesis. Expression of Tbx1 in dental epithelium of FGF receptor 2b −/− mutant mice is downregulated, showing a genetic link between FGF signaling and Tbx1 in teeth. Forced expression of Tbx1 in dental explants activates amelogenin expression. These results indicate that Tbx1 expression in developing teeth is under control of FGF signaling and correlates with determination of the ameloblast lineage.

JNK/c-Jun signaling pathway mediates the fluoride-induced down-regulation of MMP-20 in vitro

Delayed removal of amelogenins, which are initially hydrolyzed by matrix metalloproteinase MMP-20, is a characteristic of enamel fluorosis. In this study, we investigated the regulation of MMP-20 and possible effects of fluoride on MMP-20 expression in human ameloblast lineage cells. Protein expression and signaling pathways of human ameloblast lineage cells, exposed to 10 μM fluoride, were compared to control cells without fluoride exposure. The role of activator protein-1 in MMP-20 regulation was analyzed by DNA-protein affinity precipitation and luciferase reporter gene assays. MMP-20 protein levels in human ameloblast lineage cells decreased in the presence of fluoride, while amelogenin and TIMP-2 were not altered. Fluoride also decreased the transcription of a luciferase reporter gene driven by the MMP-20 promoter. Down-regulation of MMP-20 by fluoride was related to suppression of JNK/c-Jun phosphorylation. In contrast, the JNK activator elevated the expression of MMP-20. Three c-Jun binding sites on the MMP-20 promoter were identified for the first time, and were occupied by c-Jun as MMP-20 was induced. Deletion of any one of AP-1 binding sites on the MMP-20 promoter significantly reduced the transcription of downstream luciferase reporter. These in vitro findings suggest that c-Jun is a key regulatory element for MMP-20 expression, and human ameloblast lineage cells can respond to fluoride by down-regulating MMP-20 transcription through the JNK/c-Jun signaling pathway.

Stratum intermedium lineage diverges from ameloblast lineage via Notch signaling

The stratum intermedium develops as flattened cell layers on the proximal side of the ameloblast layer during tooth development. However, little information is available regarding the origin and the role. In this study, we indicate that some stratum intermedium cells originate from the inner enamel epithelium (IEE) in rat incisor organ cultures using DiI as a tracer. Immunohistochemical and in situ hybridization studies showed that the stratum intermedium cells express the Notch1 protein and Hes1 mRNAs, while the IEE and ameloblasts express the Jagged1. Further, we examined the role of Notch signaling using the dental epithelial cell line HAT-7. Recombinant Jagged1 protein enhanced the appearance of stratum intermedium cells in HAT-7 cultures and neutralization with an anti-Jagged1 antibody inhibited these effects. Additionally, overexpression of the Notch1 internal domain increased the number of stratum intermedium cells. We hypothesize that the stratum intermedium lineage differentiates from the ameloblast lineage via Notch signaling.