Regulatory role of N-myc downregulated genes in amelogenesis in rats.

Cytodifferentiation of odontogenic cells, a late stage event in odontogenesis is based on gene regulation. However, studies on the identification of the involved genes are scarce. The present study aimed to search for molecules for the cytodifferentiation of ameloblastic cells in rats. Differential display-PCR revealed a differentially expressed gene between cap/early bell stage and hard tissue formation stage in molars. This gene was identified as N-myc Downregulated Gene 1 (Ndrg1), which is the first report in tooth development. Real time PCR and western blotting confirmed that the mRNA level of Ndrg1 was higher during enamel formation than the cap stage. Ndrg1 expression was upregulated in the early bell, crown, and root stages in a time-dependent manner. These patterns of expression were similar in Ndrg2, but Ndrg3 and Ndrg4 levels did not change during the developmental stages. Immunofluorescence revealed that strong immunoreactivity against Ndrg1 were detected in differentiated ameloblasts only, not inner enamel epithelium, odontoblasts and ameloblastic cells in defected enamel regions. Alkaline phosphatase and alizarin red s stains along with real time PCR, revealed that Ndrg1 and Ndrg2 were involved in cytodifferentiation and enamel matrix mineralization by selectively regulating amelogenin and ameloblastin genes in SF2 ameloblastic cells. These results suggest that Ndrg may play a crucial functional role in the cytodifferentiation of ameloblasts for amelogenesis.

Novel Ameloblastin Variants, Contrasting Amelogenesis Imperfecta Phenotypes.

Amelogenesis imperfecta (AI) comprises a group of rare, inherited disorders with abnormal enamel formation. Ameloblastin (AMBN), the second most abundant enamel matrix protein (EMP), plays a critical role in amelogenesis. Pathogenic biallelic loss-of-function AMBN variants are known to cause recessive hypoplastic AI. A report of a family with dominant hypoplastic AI attributed to AMBN missense change p.Pro357Ser, together with data from animal models, suggests that the consequences of AMBN variants in human AI remain incompletely characterized. Here we describe 5 new pathogenic AMBN variants in 11 individuals with AI. These fall within 3 groups by phenotype. Group 1, consisting of 6 families biallelic for combinations of 4 different variants, have yellow hypoplastic AI with poor-quality enamel, consistent with previous reports. Group 2, with 2 families, appears monoallelic for a variant shared with group 1 and has hypomaturation AI of near-normal enamel volume with pitting. Group 3 includes 3 families, all monoallelic for a fifth variant, which are affected by white hypoplastic AI with a thin intact enamel layer. Three variants, c.209C>G; p.(Ser70*) (groups 1 and 2), c.295T>C; p.(Tyr99His) (group 1), and c.76G>A; p.(Ala26Thr) (group 3) were identified in multiple families. Long-read AMBN locus sequencing revealed these variants are on the same conserved haplotype, implying they originate from a common ancestor. Data presented therefore provide further support for possible dominant as well as recessive inheritance for AMBN-related AI and for multiple contrasting phenotypes. In conclusion, our findings suggest pathogenic AMBN variants have a more complex impact on human AI than previously reported.

ASH2L, Core Subunit of H3K4 Methylation Complex, Regulates Amelogenesis.

Histone methylation assumes a crucial role in the intricate process of enamel development. Our study has illuminated the substantial prevalence of H3K4me3 distribution, spanning from the cap stage to the late bell stage of dental germs. In order to delve into the role of H3K4me3 modification in amelogenesis and unravel the underlying mechanisms, we performed a conditional knockout of Ash2l, a core subunit essential for the establishment of H3K4me3 within the dental epithelium of mice. The absence of Ash2l resulted in reduced H3K4me3 modification, subsequently leading to abnormal morphology of dental germ at the late bell stage. Notably, knockout of Ash2l resulted in a loss of polarity in ameloblasts and odontoblasts. The proliferation and apoptosis of the inner enamel epithelium cells underwent dysregulation. Moreover, there was a notable reduction in the expression of matrix-related genes, Amelx and Dspp, accompanied with impaired enamel and dentin formation. Cut&Tag-seq (cleavage under targets and tagmentation sequencing) analysis substantiated a reduction of H3K4me3 modification on Shh, Trp63, Sp6, and others in the dental epithelium of Ash2l knockout mice. Validation through real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence consistently affirmed the observed downregulation of Shh and Sp6 in the dental epithelium following Ash2l knockout. Intriguingly, the expression of Trp63 isomers, DNp63 and TAp63, was perturbed in Ash2l defect dental epithelium. Furthermore, the downstream target of TAp63, P21, exhibited aberrant expression within the cervical loop of mandibular first molars and incisors. Collectively, our findings suggest that ASH2L orchestrates the regulation of crucial amelogenesis-associated genes, such as Shh, Trp63, and others, by modulating H3K4me3 modification. Loss of ASH2L and H3K4me3 can lead to aberrant differentiation, proliferation, and apoptosis of the dental epithelium by affecting the expression of Shh, Trp63, and others genes, thereby contributing to the defects of amelogenesis.

Adult dental epithelial stem cell-derived organoids deposit hydroxylapatite biomineral.

Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. The unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet, partly due to limitations with in vitro experimental models. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When transplanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model to study mineralization process by dental epithelium, which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.

Comment reconnaître et distinguer les anomalies de structure dentaire ?

Introduction: The precise diagnosis of dental structural anomalies is an essential step preceding our restorative and orthodontic therapies. Indeed, first of all, it is necessary to identify the type of structural anomaly and to determine if it is an isolated or a syndromic form: the dental anomaly could be included in a more complex clinical picture combining other clinical signs. Moreover, the establishment of the diagnosis will allow the practitioner to adapt his clinical protocol according to the observed dental structure anomaly. The choice of the bonding material, the type of preparation (no prep, prep less, complete eviction), and the application of a deproteinization protocol with sodium hypochlorite depend to the structural defect. Material and Method: The diagnosis of dental structural anomalies is based on several key points described in this article in order to facilitate the practitioner's diagnostic approach. Conclusion: The diagnosis of amelogenesis or dentinogenesis imperfecta should justify the search for other signs to determine whether the anomaly of tooth structure is isolated or syndromic.

Introduction: Le diagnostic précis des anomalies de structure dentaire est une étape primordiale et préalable à nos thérapeutiques restauratrices et orthodontiques. En effet, dans un premier temps, il conviendra d'identifier le type d'anomalie de structure et de déterminer si celle-ci est isolée ou syndromique, c'est-à-dire si elle s'intègre ou non dans un tableau clinique plus complexe regroupant d'autres signes cliniques. De plus, d'un point de vue thérapeutique, l'établissement du diagnostic permettra au praticien d'adapter son protocole clinique en fonction de l'anomalie observée : choix des matériaux de collage, fraisage sélectif, étape de déprotéinisation à l'hypochlorite de sodium. Matériel et méthode: Le diagnostic des anomalies de structure repose sur plusieurs points clés qui seront abordés au cours de cet article afin de faciliter la démarche diagnostique du praticien. Conclusion: Le diagnostic d'amélogenèses ou de dentinogenèses imparfaites doit justifier la recherche d'autres signes afin de déterminer le caractère isolé ou syndromique de l'anomalie de structure dentaire.

Crucial Role of microRNAs as New Targets for Amelogenesis Disorders Detection.

INTRODUCTION: Amelogenesis imperfecta (AI) refers to a heterogeneous group of conditions with multiple factors which contribute to the hypomineralisation of enamel. Preventive measures are necessary to predict this pathology. Prospects for preventive medicine are closely related to the search for new informative methods for diagnosing a human disease. MicroRNAs are prominent for the non-invasive diagnostic platform. THE AIM OF THE STUDY: The aim of the review is to review the heterogeneous factors involved in amelogenesis and to select the microRNA panel associated with the AI type. METHODS: We used DIANA Tools (algorithms, databases and software) for interpreting and archiving data in a systematic framework ranging from the analysis of expression regulation from deep sequencing data to the annotation of miRNA regulatory elements and targets (https://dianalab. e-ce.uth.gr/). In our study, based on a gene panel associated with the AI types, twenty-four miRNAs were identified for the hypoplastic type (supplement), thirty-five for hypocalcified and forty-- nine for hypomaturation AI. The selection strategy included the microRNA search with multiple targets using the AI type's gene panel. RESULTS: Key proteins, calcium-dependent and genetic factors were analysed to reveal their role in amelogenesis. The role of extracellular non-coding RNA sequences with multiple regulatory functions seems to be the most attractive. We chose the list of microRNAs associated with the AI genes. We found four microRNAs (hsa-miR-27a-3p, hsa-miR-375, hsa-miR-16-5p and hsamiR- 146a-5p) for the gene panel, associated with the hypoplastic type of AI; five microRNAs (hsa- miR-29c-3p, hsa-miR-124-3p, hsa-miR-1343-3p, hsa-miR-335-5p, and hsa-miR-16-5p - for hypocalcified type of AI, and seven ones (hsa-miR-124-3p, hsa-miR-147a, hsa-miR-16-5p, hsamiR- 429, hsa-let-7b-5p, hsa-miR-146a-5p, hsa-miR-335-5p) - for hypomaturation. It was revealed that hsa-miR-16-5p is included in three panels specific for both hypoplastic, hypocalcified, and hypomaturation types. Hsa-miR-146a-5p is associated with hypoplastic and hypomaturation type of AI, which is associated with the peculiarities of the inflammatory response immune response. In turn, hsa-miR-335-5p associated with hypocalcified and hypomaturation type of AI. CONCLUSION: Liquid biopsy approaches are a promising way to reduce the economic cost of treatment for these patients in modern healthcare. Unique data exist about the role of microRNA in regulating amelogenesis. The list of microRNAs that are associated with AI genes and classified by AI types has been uncovered. The target gene analysis showed the variety of functions of selected microRNAs, which explains the multiple heterogeneous mechanisms in amelogenesis. Predisposition to mineralisation problems is a programmed event. Many factors determine the manifestation of this problem. Additionally, it is necessary to remember the variable nature of the changes, which reduces the prediction accuracy. Therefore, models based on liquid biopsy and microRNAs make it possible to take into account these factors and their influence on the mineralisation. The found data needs further investigation.

Single-cell census of human tooth development enables generation of human enamel.

Tooth enamel secreted by ameloblasts (AMs) is the hardest material in the human body, acting as a shield to protect the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here, we use single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) to establish a spatiotemporal single-cell census for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We identify key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in human ameloblast in vitro differentiation from induced pluripotent stem cells (iPSCs). We furthermore develop a disease model of amelogenesis imperfecta in a three-dimensional (3D) organoid system and show AM maturation to mineralized structure in vivo. These studies pave the way for future regenerative dentistry.

Tmem2 Deficiency Leads to Enamel Hypoplasia and Soft Enamel in Mouse.

Teeth consist of 3 mineralized tissues: enamel, dentin, and cementum. Tooth malformation, the most common craniofacial anomaly, arises from complex genetic and environmental factors affecting enamel structure, size, shape, and tooth eruption. Hyaluronic acid (HA), a primary extracellular matrix component, contributes to structural and physiological functions in periodontal tissue. Transmembrane protein 2 (TMEM2), a novel cell surface hyaluronidase, has been shown to play a critical role during embryogenesis. In this study, we demonstrate Tmem2 messenger RNA expression in inner enamel epithelium and presecretory, secretory, and mature ameloblasts. Tmem2 knock-in reporter mice reveal TMEM2 protein localization at the apical and basal ends of secretory ameloblasts. Micro-computed tomography analysis of epithelial-specific Tmem2 conditional knockout (Tmem2-CKO) mice shows a significant reduction in enamel layer thickness and severe enamel deficiency. Enamel matrix protein expression was remarkably downregulated in Tmem2-CKO mice. Scanning electron microscopy of enamel from Tmem2-CKO mice revealed an irregular enamel prism structure, while the microhardness and density of enamel were significantly reduced, indicating impaired ameloblast differentiation and enamel matrix mineralization. Histological evaluation indicated weak adhesion between cells and the basement membrane in Tmem2-CKO mice. The reduced and irregular expressions of vinculin and integrin ß1 suggest that Tmem2 deficiency attenuated focal adhesion formation. In addition, abnormal HA accumulation in the ameloblast layer and weak claudin 1 immunoreactivity in Tmem2-CKO mice indicate impaired tight junction gate function. Irregular actin filament assembly was also observed at the apical and basal ends of secretory ameloblasts. Last, we demonstrated that Tmem2-deficient mHAT9d mouse ameloblasts exhibit defective adhesion to HA-containing substrates in vitro. Collectively, our data highlight the importance of TMEM2 in adhesion to HA-rich extracellular matrix, cell-to-cell adhesion, ameloblast differentiation, and enamel matrix mineralization.

Autophagy Plays a Crucial Role in Ameloblast Differentiation.

Tooth enamel is generated by ameloblasts. Any failure in amelogenesis results in defects in the enamel, a condition known as amelogenesis imperfecta. Here, we report that mice with deficient autophagy in epithelial-derived tissues (K14-Cre;Atg7F/F and K14-Cre;Atg3F/F conditional knockout mice) exhibit amelogenesis imperfecta. Micro-computed tomography imaging confirmed that enamel density and thickness were significantly reduced in the teeth of these mice. At the molecular level, ameloblast differentiation was compromised through ectopic accumulation and activation of NRF2, a specific substrate of autophagy. Through bioinformatic analyses, we identified Bcl11b, Dlx3, Klk4, Ltbp3, Nectin1, and Pax9 as candidate genes related to amelogenesis imperfecta and the NRF2-mediated pathway. To investigate the effects of the ectopic NRF2 pathway activation caused by the autophagy deficiency, we analyzed target gene expression and NRF2 binding to the promoter region of candidate target genes and found suppressed gene expression of Bcl11b, Dlx3, Klk4, and Nectin1 but not of Ltbp3 and Pax9. Taken together, our findings indicate that autophagy plays a crucial role in ameloblast differentiation and that its failure results in amelogenesis imperfecta through ectopic NRF2 activation.

Association between genetic factors and molar-incisor hypomineralisation or hypomineralised second primary molar: A systematic review.

OBJECTIVE: To determine the association between genetic factors and molar-incisor hypomineralisation (MIH) and/or hypomineralised second primary molars by means of a systematic review. DESIGN: A search was performed in Medline-PubMed, Scopus, Embase and Web of Science databases; manual search and search in gray literature were also performed. Selection of articles was performed independently by two researchers. A third examiner was involved in cases of disagreement. Data extraction was performed using an Excel® spreadsheet and independent analysis was performed for each outcome. RESULTS: Sixteen studies were included. There was an association between MIH and genetic variants related to amelogenesis, immune response, xenobiotic detoxification and other genes. Moreover, interactions between amelogenesis and immune response genes, and SNPs in the aquaporin gene and vitamin D receptors were associated with MIH. Greater agreement of MIH was found in pairs of monozygotic twins than dizygotic twins. The heritability of MIH was 20 %. Hypomineralised second primary molars was associated with SNPs in the hypoxia-related HIF-1 gene and methylation in genes related to amelogenesis. CONCLUSION: With very low or low certainty of evidence, an association was observed between MIH and SNPs in genes associated with amelogenesis, immune response, xenobiotic detox and ion transport. Interactions between genes related to amelogenesis and immune response as well as aquaporin genes were associated to MIH. With very low certainty of evidence, hypomineralised second primary molars was associated to a hypoxia-related gene and to methylation in genes related to amelogenesis. Moreover, higher agreement of MIH in pairs of monozygotic twins than dizygotic twins was observed.

Epigenetics in developmental defects of enamel: A scoping review.

OBJECTIVES: The significant role of epigenetics has been revealed in normal enamel formation process and occurrence of developmental defects. This presented literature is aiming at summarizing the regulatory function of epigenetics in physiological amelogenesis process and reviewing the epigenetic mechanisms in occurrence of developmental defects of enamel (DDE), so as to provide biological foundation evidence to support early predication and clinical management of DDE. METHOD: An extensive literature review was conducted using electronic databases MEDLINE (through PubMed), Web of Science and EMBASE up to November 30, 2022. Studies about epigenetic effects on enamel tissue or cells associated with amelogenesis, including in vivo studies using human or animal models, and in vitro studies, are selected. RESULTS: A total of 22 studies were included. Epigenetic factors or effects specifically activate or silence certain genes, which may regulate related biological activities including cell proliferation, cell differentiation, enamel secretion, and mineralization during the process of amelogenesis. Once the status of epigenetic modification is altered, the quantity and quality of enamel may both be disturbed, which can finally result in DDE. CONCLUSION: Epigenetics plays a noteworthy role of regulating the amelogenesis process and DDE potentially by altering the expression levels of genes related to enamel formation, providing a new perspective of early predication and clinical management of DDE.

Conditional knockout of transient receptor potential melastatin 7 in the enamel epithelium: Effects on enamel formation.

Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase-dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14-Cre;Trpm7fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red-positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis.

Ift88 regulates enamel formation via involving Shh signaling.

OBJECTIVES: The ciliopathies are a wide spectrum of human diseases, which are caused by perturbations in the function of primary cilia. Tooth enamel anomalies are often seen in ciliopathy patients; however, the role of primary cilia in enamel formation remains unclear. MATERIALS AND METHODS: We examined mice with epithelial conditional deletion of the ciliary protein, Ift88, (Ift88fl / fl ;K14Cre). RESULTS: Ift88fl / fl ;K14Cre mice showed premature abrasion in molars. A pattern of enamel rods which is determined at secretory stage, was disorganized in Ift88 mutant molars. Many amelogenesis-related molecules expressing at the secretory stage, including amelogenin and ameloblastin, enamelin, showed significant downregulation in Ift88 mutant molar tooth germs. Shh signaling is essential for amelogenesis, which was found to be downregulated in Ift88 mutant molar at the secretory stage. Application of Shh signaling agonist at the secretory stage partially rescued enamel anomalies in Ift88 mutant mice. CONCLUSION: Findings in the present study indicate that the function of the primary cilia via Ift88 is critical for the secretory stage of amelogenesis through involving Shh signaling.

Regulation of Hydroxyapatite Nucleation In Vitro through Ameloblastin-Amelogenin Interactions.

Amelogenin (Amel) and ameloblastin (Ambn) are two primary extracellular enamel matrix proteins that play crucial roles for proper thickness, prismatic structure, and robust mechanical properties. Previous studies have shown that Amel and Ambn bind to each other, but the effect of their coassembly on the nucleation of hydroxyapatite (HAP) is unclear. Here, we systematically investigated the coassembly of recombinant mouse Amel and Ambn in various ratios using in situ atomic force microscopy, dynamic light scattering, and transmission electron microscopy. The size of protein particles decreased as the Ambn:Amel ratio increased. To define the coassembly domain on Ambn, we used Ambn-derived peptides and Ambn variants to examine their effects on the amelogenin particle size distribution. We found that the peptide sequence encoded by exon 5 of Ambn affected Amel self-assembly but the variant lacking this sequence did not have any effect on Amel self-assembly. Furthermore, through monitoring the pH change in bulk mineralization solution, we tracked the nucleation behavior of HAP in the presence of Ambn and Amel and found that their coassemblies at different ratios showed varying abilities to stabilize amorphous calcium phosphate. These results demonstrated that Ambn and Amel coassemble with each other via a motif within the sequence encoded by exon 5 of Ambn and cooperate in regulating the nucleation of HAP crystals, enhancing our understanding of the important role of enamel matrix proteins in amelogenesis.

Effects of Fam83h truncation mutation on enamel developmental defects in male C57/BL6J mice.

Truncation mutations in family with sequence similarity, member H (FAM83H) gene are considered the main cause of autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI); however, its pathogenic mechanism in amelogenesis remains poorly characterized. This study aimed to investigate the effects of truncated FAM83H on developmental defects in enamel. CRISPR/Cas9 technology was used to develop a novel Fam83h c.1186C > T (p.Q396*) knock-in mouse strain, homologous to the human FAM83H c.1192C > T mutation in ADHCAI. The Fam83hQ396⁎/Q396⁎ mice showed poor growth, a sparse and scruffy coat, scaly skin and early mortality compared to control mice. Moreover, the forelimbs of homozygous mice were swollen, exhibiting a significant inflammatory response. Incisors of Fam83hQ396⁎/Q396⁎ mice appeared chalky white, shorter, and less sharp than those of control mice, and energy dispersive X-ray spectroscopy (EDS) analysis and Prussian blue staining helped identify decreased iron and increased calcium (Ca) and phosphorus (P) levels, with an unchanged Ca/P ratio. The expression of iron transportation proteins, transferrin receptor (TFRC) and solute carrier family 40 member 1 (SLC40A1), was decreased in Fam83h-mutated ameloblasts. Micro-computed tomography revealed enamel defects in Fam83hQ396⁎/Q396⁎ mice. Fam83hQ396⁎/Q396⁎ enamel showed decreased Vickers hardness and distorted enamel rod structure and ameloblast arrangement. mRNA sequencing showed that the cell adhesion pathway was most notably clustered in LS8-Fam83h-mutated cells. Immunofluorescence analysis further revealed decreased protein expression of desmoglein 3, a component of desmosomes, in Fam83h-mutated ameloblasts. The FAM83H-casein kinase 1α (CK1α)-keratin 14 (K14)-amelogenin (AMELX) interaction was detected in ameloblasts. And K14 and AMELX were disintegrated from the tetramer in Fam83h-mutated ameloblasts in vitro and in vivo. In secretory stage ameloblasts of Fam83hQ396⁎/Q396⁎ mice, AMELX secretion exhibited obvious retention in the cytoplasm. In conclusion, truncated FAM83H exerted dominant-negative effects on gross development, amelogenesis, and enamel biomineralization by disturbing iron transportation, influencing the transportation and secretion of AMELX, and interfering with cell-cell adhesion in ameloblasts.

Polymorphisms in genes expressed during amelogenesis and their association with dental caries: a case-control study.

OBJECTIVES: Dental caries is a widespread multifactorial disease, caused by the demineralization of hard dental tissues. Susceptibility to dental caries is partially genetically conditioned; this study was aimed at finding an association of selected single nucleotide polymorphisms (SNPs) in genes encoding proteins involved in amelogenesis with this disease in children. MATERIALS AND METHODS: In this case-control study, 15 SNPs in ALOX15, AMBN, AMELX, KLK4, TFIP11, and TUFT1 genes were analyzed in 150 children with primary dentition and 611 children with permanent teeth with/without dental caries from the European Longitudinal Study of Pregnancy and Childhood (ELSPAC) cohort. RESULTS: Dental caries in primary dentition was associated with SNPs in AMELX (rs17878486) and KLK4 (rs198968, rs2242670), and dental caries in permanent dentition with SNPs in AMELX (rs17878486) and KLK4 (rs2235091, rs2242670, rs2978642), (p ≤ 0.05). No significant differences between cases and controls were observed in the allele or genotype frequencies of any of the selected SNPs in ALOX15, AMBN, TFIP11, and TUFT1 genes (p > 0.05). Some KLK4 haplotypes were associated with dental caries in permanent dentition (p ≤ 0.05). CONCLUSIONS: Based on this study, we found that although the SNPs in AMELX and KLK4 are localized in intronic regions and their functional significance has not yet been determined, they are associated with susceptibility to dental caries in children. CLINICAL RELEVANCE: AMELX and KLK4 variants could be considered in the risk assessment of dental caries, especially in permanent dentition, in the European Caucasian population.

PMCA Ca2+ clearance in dental enamel cells depends on the magnitude of cytosolic Ca2.

Enamel formation (amelogenesis) is a two-step process whereby crystals partially grow during the secretory stage followed by a significant growth expansion during the maturation stage concurrent with an increase in vectorial Ca2+ transport. This requires tight regulation of cytosolic Ca2+ (c Ca2+ ) concentration in the enamel forming ameloblasts by controlling Ca2+ influx (entry) and Ca2+ extrusion (clearance). Gene and protein expression studies suggest that the plasma membrane Ca2+ -ATPases (PMCA1-4) are likely involved in c Ca2+ extrusion in ameloblasts, yet no functional analysis of these pumps has been reported nor whether their activity changes across amelogenesis. PMCAs have high Ca2+ affinity and low Ca2+ clearance which may be a limiting factor in their contribution to enamel formation as maturation stage ameloblasts handle high Ca2+ loads. We analyzed PMCA function in rat secretory and maturation ameloblasts by blocking or potentiating these pumps. Low/moderate elevations in c Ca2+ measured using the Ca2+ probe Fura-2-AM show that secretory ameloblasts clear Ca2+ faster than maturation stage cells through PMCAs. This process was completely inhibited by an external alkaline (pH 9.0) solution or was significantly delayed by the PMCA blockers vanadate and caloxin 1b1. Eliciting higher c Ca2+ transients via the activation of the ORAI1 Ca2+ channel showed that the PMCAs of maturation ameloblasts were more efficient. Inhibiting PMCAs decreased the rate of Ca2+ influx via ORAI1 but potentiation with forskolin had no effect. Our findings suggest that PMCAs are functional Ca2+ pumps during amelogenesis regulating c Ca2+ upon low and/or moderate Ca2+ stimulus in secretory stage, thus participating in amelogenesis.

Mesoscale structural gradients in human tooth enamel.

The outstanding mechanical and chemical properties of dental enamel emerge from its complex hierarchical architecture. An accurate, detailed multiscale model of the structure and composition of enamel is important for understanding lesion formation in tooth decay (dental caries), enamel development (amelogenesis) and associated pathologies (e.g., amelogenesis imperfecta or molar hypomineralization), and minimally invasive dentistry. Although features at length scales smaller than 100 nm (individual crystallites) and greater than 50 µm (multiple rods) are well understood, competing field of view and sampling considerations have hindered exploration of mesoscale features, i.e., at the level of single enamel rods and the interrod enamel (1 to 10 µm). Here, we combine synchrotron X-ray diffraction at submicrometer resolution, analysis of crystallite orientation distribution, and unsupervised machine learning to show that crystallographic parameters differ between rod head and rod tail/interrod enamel. This variation strongly suggests that crystallites in different microarchitectural domains also differ in their composition. Thus, we use a dilute linear model to predict the concentrations of minority ions in hydroxylapatite (Mg2+ and CO32-/Na+) that plausibly explain the observed lattice parameter variations. While differences within samples are highly significant and of similar magnitude, absolute values and the sign of the effect for some crystallographic parameters show interindividual variation that warrants further investigation. By revealing additional complexity at the rod/interrod level of human enamel and leaving open the possibility of modulation across larger length scales, these results inform future investigations into mechanisms governing amelogenesis and introduce another feature to consider when modeling the mechanical and chemical performance of enamel.

N6-methyladenosine (m6A) RNA methylation mediated by methyltransferase complex subunit WTAP regulates amelogenesis.

N6-methyladenosine (m6A) RNA methylation, one of the most widespread posttranscriptional modifications in eukaryotes, plays crucial roles in various developmental processes. The m6A modification process is catalyzed by a methyltransferase complex that includes Wilms tumor 1-associated protein (WTAP) as a key component. Whether the development of dental enamel is regulated by m6A RNA methylation in mammals remains unclear. Here, we reveal that WTAP is widely expressed from the early stage of tooth development. Specific inactivation of Wtap in mouse enamel epithelium by the Cre/loxp system leads to serious developmental defects in amelogenesis. In Wtap conditional KO mice, we determined that the differentiation of enamel epithelial cells into mature ameloblasts at the early stages of enamel development is affected. Mechanistically, loss of Wtap inhibits the expression of Sonic hedgehog (SHH), which plays an important role in the generation of ameloblasts from stem cells. Together, our findings provide new insights into the functional role of WTAP-mediated m6A methylation in amelogenesis in mammals.

Enamel defects in Acp4R110C/R110C mice and human ACP4 mutations.

Human ACP4 (OMIM*606362) encodes a transmembrane protein that belongs to histidine acid phosphatase (ACP) family. Recessive mutations in ACP4 cause non-syndromic hypoplastic amelogenesis imperfecta (AI1J, OMIM#617297). While ACP activity has long been detected in developing teeth, its functions during tooth development and the pathogenesis of ACP4-associated AI remain largely unknown. Here, we characterized 2 AI1J families and identified a novel ACP4 disease-causing mutation: c.774_775del, p.Gly260Aspfs*29. To investigate the role of ACP4 during amelogenesis, we generated and characterized Acp4R110C mice that carry the p.(Arg110Cys) loss-of-function mutation. Mouse Acp4 expression was the strongest at secretory stage ameloblasts, and the protein localized primarily at Tomes' processes. While Acp4 heterozygous (Acp4+/R110C) mice showed no phenotypes, incisors and molars of homozygous (Acp4R110C/R110C) mice exhibited a thin layer of aplastic enamel with numerous ectopic mineralized nodules. Acp4R110C/R110C ameloblasts appeared normal initially but underwent pathology at mid-way of secretory stage. Ultrastructurally, sporadic enamel ribbons grew on mineralized dentin but failed to elongate, and aberrant needle-like crystals formed instead. Globs of organic matrix accumulated by the distal membranes of defective Tomes' processes. These results demonstrated a critical role for ACP4 in appositional growth of dental enamel probably by processing and regulating enamel matrix proteins around mineralization front apparatus.