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

  • Author Footnotes
    # This author contributed equally to this work.
    Furong Xie
    Footnotes
    # This author contributed equally to this work.
    Affiliations
    Department of Pediatric Dentistry, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology. Shanghai, 200011, China
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  • Author Footnotes
    # This author contributed equally to this work.
    Xueqin Zhu
    Footnotes
    # This author contributed equally to this work.
    Affiliations
    Department of Pediatric Dentistry, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology. Shanghai, 200011, China
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  • Xiao Liu
    Affiliations
    Department of Pediatric Dentistry, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology. Shanghai, 200011, China
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  • Hui Chen
    Correspondence
    Corresponding author: Hui Chen. Email:
    Affiliations
    Department of Pediatric Dentistry, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology. Shanghai, 200011, China
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  • Jun Wang
    Correspondence
    Corresponding author: Jun Wang, Email:
    Affiliations
    Department of Pediatric Dentistry, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology. Shanghai, 200011, China
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  • Author Footnotes
    # This author contributed equally to this work.
Open AccessPublished:November 17, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102715

      Abstract

      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 knockout (cKO) 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.

      Keywords

      Introduction

      As the most mineralized tissue in mammal, dental enamel provides maximum durability that allows teeth to function as weapons and/or tools as well as for food processing. The development and mineralization of enamel is a complex and elaborate process which is tightly regulated by ameloblasts. Tooth development sequentially undergoes bud stage, cap stage, early late bell stage. At bell stage, ameloblast differentiate from inner enamel epithelium(IEE)(
      • Zeichner-David M.
      • Diekwisch T.
      • Fincham A.
      • Lau E.
      • MacDougall M.
      • Moradian-Oldak J.
      • et al.
      Control of ameloblast differentiation.
      ) and possess a life cycle of several stages including presecretory ameloblasts, secretory ameloblasts and mature ameloblasts. Secretory ameloblasts are columnar, tall, polarized cells, which can secretory extracellular matrix proteins such as amelogenin (AMELX)
      • Shibata S.
      • Suzuki S.
      • Tengan T.
      • Yamashita Y.
      A histochemical study of apoptosis in the reduced ameloblasts of erupting mouse molars.
      ,
      • Bartlett J.D.
      Dental enamel development: proteinases and their enamel matrix substrates.
      . The fine mechanisms under the alteration from dental epithelial stem cells to differentiated ameloblasts that leads to enamel formation remains unknown.
      N6-methyladenosine (m6A) which was first discovered in 1970s(
      • Desrosiers R.C.
      • Friderici K.H.
      • Rottman F.M.
      Characterization of Novikoff hepatoma mRNA methylation and heterogeneity in the methylated 5' terminus. Biochemistry. Research Support, U.S. Gov't.
      ), is the most abundant internal mRNA modification. It is a key determinant of post-transcriptional mRNA regulation and function in the regulation in cell fate determination. In mammals, m6A methylation is mediated by the methyltransferase complex, which is composed of METTL3 (methyltransferase-like 3), METTL14, and subunit wilms tumor 1-associated protein (WTAP), KIAA1429, RNA-binding motif protein 15 (RBM15), and its paralog (RBM15B) (
      • Patil D.P.
      • Chen C.K.
      • Pickering B.F.
      • Chow A.
      • Jackson C.
      • Guttman M.
      • et al.
      m(6)A RNA methylation promotes XIST-mediated transcriptional repression.
      ). Nevertheless, methylation is erased by the demethylases fat mass and obesity-associated (FTO) and alkB homolog (5ALKBH5) (
      • Zhao B.S.
      • Roundtree I.A.
      • He C.
      Post-transcriptional gene regulation by mRNA modifications.
      ). At molecular level, m6A modification influences the mRNA metabolisms, including regulating RNA stability (
      • Wang X.
      • Lu Z.
      • Gomez A.
      • Hon G.C.
      • Yue Y.
      • Han D.
      • et al.
      N6-methyladenosine-dependent regulation of messenger RNA stability. Nature. Research Support, N.I.H..
      ) and RNA splicing (
      • Xiao W.
      • Adhikari S.
      • Dahal U.
      • Chen Y.S.
      • Hao Y.J.
      • Sun B.F.
      • et al.
      Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing.
      ), as well as mRNA translation efficiency
      • Wang X.
      • Zhao B.S.
      • Roundtree I.A.
      • Lu Z.
      • Han D.
      • Ma H.
      • et al.
      N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency. Cell. Research Support, Non-U.S. Gov't Research Support, U.S. Gov't.
      ,
      • Zhou J.
      • Wan J.
      • Gao X.
      • Zhang X.
      • Jaffrey S.R.
      • Qian S.B.
      Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature. Research Support, N.I.H..
      , thus affect the cellular function, identity and stemness of their residing cells. Many important biological processes are known to be regulated by m6A, including cell fate determination (
      • Geula S.
      • Moshitch-Moshkovitz S.
      • Dominissini D.
      • Mansour A.A.
      • Kol N.
      • Salmon-Divon M.
      • et al.
      Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation.
      ) and embryonic development
      • Zhao B.S.
      • Wang X.
      • Beadell A.V.
      • Lu Z.
      • Shi H.
      • Kuuspalu A.
      • et al.
      m(6)A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition. Nature. Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov′t Research Support, U.S. Gov′t.
      ,

      Lence T, Akhtar J, Bayer M, Schmid K, Spindler L, Ho CH, et al. m(6)A modulates neuronal functions and sex determination in Drosophila. Nature. Research Support, Non-U.S. Gov′t Dec 8 2016;540(7632):242-247. Epub 2016/12/06.

      . Mammalian WTAP was first identified as a proteins associated with Wilms’tumor-1 (WT1) (
      • Little N.A.
      • Hastie N.D.
      • Davies R.C.
      Identification of WTAP, a novel Wilms' tumour 1-associating protein.
      ). Widely knockout of Wtap in mice will lead to embryonic death between embryonic day 6.5 –10.5 and show serious defects in cell proliferation, which in turn leads to defects in endoderm and mesoderm formation
      • Horiuchi K.
      • Umetani M.
      • Minami T.
      • Okayama H.
      • Takada S.
      • Yamamoto M.
      • et al.
      Wilms' tumor 1-associating protein regulates G2/M transition through stabilization of cyclin A2 mRNA.
      ,
      • Fukusumi Y.
      • Naruse C.
      • Asano M.
      Wtap is required for differentiation of endoderm and mesoderm in the mouse embryo.
      . WTAP is also a regulatory subunit of the RNA N6-methyladenosine methyltransferase, plays an important enzymatic role in METTL3-METTL14-WTAP methyltransferases complex(
      • Ping X.L.
      • Sun B.F.
      • Wang L.
      • Xiao W.
      • Yang X.
      • Wang W.J.
      • et al.
      Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase.
      ). Recently, many studies proved that WTAP regulated dynamic m6A level, which can not only determines the fate of stem cells and regulate mammalian development, but also be involved in carcinogenesis
      • Chen Y.
      • Peng C.
      • Chen J.
      • Chen D.
      • Yang B.
      • He B.
      • et al.
      WTAP facilitates progression of hepatocellular carcinoma via m6A-HuR-dependent epigenetic silencing of ETS1.
      ,
      • Bertero A.
      • Brown S.
      • Madrigal P.
      • Osnato A.
      • Ortmann D.
      • Yiangou L.
      • et al.
      The SMAD2/3 interactome reveals that TGFbeta controls m(6)A mRNA methylation in pluripotency.
      .
      In this study, Wtap conditional knockout mutant mice were generated. Loss of m6A by inactivation of Wtap led to severe amelogenesis imperfecta, which reveals the critical functions of m6A modification and its binding protein WTAP in dental enamel development.

      Results

      WTAP is widely expressed at early stage of tooth development

      In order to study the role of WTAP in tooth development, we firstly analyzed the expression pattern of WTAP in mice and human sample. At E11.5 (dental lamina), E13.5 (bud stage), E14.5 (cap stage), E16.5 (bell stage), E18.5 (late bell stage) and P0 (late bell stage) mice, we detected ubiquitous occupation of WTAP in inner enamel epithelium by immunostaining (sFig. 1A-F), indicating that WTAP might be involved in enamel development. In human reduced dental epithelium of unerupted 3rd molar tooth, we also detected strong expression of WTAP protein (sFig. 2), proving that expression pattern of WTAP was similar between mice and human.

      Wtap conditional knockout mice displayed amelogenesis imperfecta phenotypes

      In order to study the biological function of the m6A writer WTAP in enamel development, Wtap floxed mice bearing loxP sites flanking exons 4–5 of the Wtap gene were obtained from professor Minghan Tong’s Laboratory. The precise methods were previously described by Tong’s group(
      • Jia G.X.
      • Lin Z.
      • Yan R.G.
      • Wang G.W.
      • Zhang X.N.
      • Li C.
      • et al.
      WTAP Function in Sertoli Cells Is Essential for Sustaining the Spermatogonial Stem Cell Niche.
      ). The resulting Wtapflox/flox mice were crossed with K14-cre transgenic mice to generate Wtap conditional knockout mice (genotype Wtapflox/flox; K14-Cre, named as cKO) (sFig. 3). Littermates of the cKO mice identified with Wtapflox/flox genotypes were used as control subjects. PCR and immunohistochemical staining (Fig. 1A, C, sFig 1G) results showed that the cKO mice failed to produce WTAP protein in ameloblasts. Nevertheless, WTAP protein remained unperturbed in other dental germ tissues, such as dental mesenchyme.
      Figure thumbnail gr1
      Figure 1Knock out of Wtap in dental epitherlial cells resulted in enamel malformation. A: Expression of WTAP protein in dental germ of newborn(0.5dpn) mice were detected via immunofluorescence staining (A1: control mice; A2: knockout mice; EO: enamel organ; M: dental mesenchymal tissue; white arrow: inner enamel epithelial cells). B: Photo of control and knockout mice on birth day. C: The weight of knockout mice was significantly lower compared with control mice. (*: p<0.05). D: Observation of mandibular mandibular first molar via H-E staining. (D1, 2: mandibular first molar of control mice; D3, 4: mandibular first molar of knockout mice). E: Enamel formation of mandibular incisors was observed via masson staining (E1: picture of tip of mandibular incisor from control mice; E2: high magnification of black frame part from E1; E3: picture of tip of mandibular incisor from knockout mice; E4: high magnification of black frame part from E2. Black arrow: deposition of enamel matrix)
      Compared to the controls, cKO mice had a notably decreased body weight at P0 (Fig. 1B, C). No obvious phenotypic changes were found in the head, leg and size by gross observation. However, at P0, we found severely enamel developmental failure in all of cKO mice. The tooth size and enamel thickness were observed largely deceased in both incisors and molars (Fig 1D, Fig 2C ). The cusps of molars changed to small and blunt (Fig. 1D3,4). The degree of enamel mineralization in mutant and control teeth was detected by MASSON trichrome staining on P0 incisor. Dentin formation was present on control and cKO mice (Fig.1E). However, enamel was undetectable in the cKO incisor (Fig.1E3,4), indicating that disruption of the Wtap gene results in enamel malformation during the early developmental stages of amelogenesis.
      Figure thumbnail gr2
      Figure 2Wtap deletion in enamel epithelium affects the polarization of ameloblasts. A: Observation of sagittal histology of mandibular incisor from control mice; A1, A2, A3, A4: the high magnification observation results of the red boxs in A respectively(×400); B: Observation of coronal histology of mandibular incisor from control mice(×400). Transection is made between A3 and A4; C: Observation of sagittal histology of mandibular incisor from mutant mice; C1, C2, C3, C4: the high magnification observation results of the red boxs in C respectively (×400); D: Observation of coronal histology of mandibular incisor from mutant mice (×400). Transection is made between C3 and C4.
      Figure thumbnail gr3
      Figure 3WTAP promoted differentiation and proliferation of dental inner enamel epithelial cells (IEEs). A: Expression of AMELX of control mandibular incisor; B: Expression of DSPP of control mandibular incisor; C, D: Expression of BrdU of control mandibular incisor and molar; E: Expression of AMELX of mutant mandibular incisor; F: Expression of DSPP of mutant mandibular incisor; G,H: Expression of BrdU of mutant mandibular incisor and molar; I,J: Statistics analysis result of relative expression of AMELX and DSPP; K,L: Statistics analysis result of relative expression of BrdU in mandibular incisor and first molar respectively. (White arrow: IEEs;*: p<0.05,**: p<0.01)
      Figure thumbnail gr4
      Figure 4SHH expression is regulated by WTAP-mediated m6A RNA methylation. A: SHH expression in dental IEEs from control mandibular incisor was detected by immunofluorescence staining; B: SHH expression in dental IEEs from mutant mandibular incisor was detected by immunofluorescence staining; C: Results of real-time PCR showed decreased expression of SHH in dental IEEs of mandibular incisor at P0; D: Recombinant SHH protein rescued differentiation ability of wtap knockdown ALC cells; E: Quantitative analysis of the differentiation ability of wtap knockdown ALC cells and after recombinant SHH protein treatment. F: Levels of m6A in dental epithelial tissues of mandibular first molars were detected by UPLC-MS/MS analysis; G: Dot blot assay showed that levels of m6A in wtap knockdown ALC cells was significant decreased; H: Quantitative analysis of m6A level in ALC cells after wtap knockdown; I: m6A enrichment in shh mRNA in dental germ at E14.5 by m6A-RIP-qPCR; J Quantitative analysis of shh mRNA level after wtap knockdown. (**: p<0.01; ***: p<0.001;****: p<0.0001)

      Wtap deletion in enamel epithelium affects the differentiation of ameloblasts at early stages

      To determine how depletion of Wtap affects enamel development, we further assessed the morphologic differences of ameloblasts between cKO and control mice (Fig. 2). Incisors were selected to observe because it can reflect different stages of amelogenesis, including secretory, transition and maturation phases(
      • Smith C.E.L.
      • Poulter J.A.
      • Antanaviciute A.
      • Kirkham J.
      • Brookes S.J.
      • Inglehearn C.F.
      • et al.
      Amelogenesis Imperfecta; Genes, Proteins, and Pathways.
      ). In control mice, we found morphology of the inner enamel epithelial cells gradually changed from cubic to long columnar in the sagittal section, and the cell polarity gradually became obvious with nucleus far away from basement membrane (Fig. 2A2-4). However, there is no obvious changes in cell morphology of inner enamel epithelial cells in cKO mice (Fig. 2C). The inner enamel epithelium cells of cKO mice showed no increase in length and polarization of these cells were neither observed (Fig. 2C2-4). The cervical loop was significantly thinner, with fewer cells in cKO mice (Fig.2 C1). From the coronal view, more matrix deposited in the junction between inner enamel epithelial cells and odontoblast in wild type incisors (Fig. 2B), while less in cKO incisors (Fig. 2D). Invitro, we also found decreased calcium knots formation in Wtap knock-down ALC cells (
      • Nakata A.
      • Kameda T.
      • Nagai H.
      • Ikegami K.
      • Duan Y.
      • Terada K.
      • et al.
      Establishment and characterization of a spontaneously immortalized mouse ameloblast-lineage cell line.
      ) which confirmed that WTAP is vital for the proliferation and differentiation of ameloblasts (sFig. 4).
      To investigate which stages of ameloblast differentiation has been affected, the genes expression levels of enamel matrix proteins and proteinases, which represent stages of ameloblast cell differentiation, were examined by immunohistochemical staining. We found that AMELX, which is secreted by secretory-stage ameloblasts, was strongly expressed at anterior end of labial side of the incisor in control (Fig. 3A). On the contrary, AMELX expression was significantly decreased in the cKO incisor (Fig. 3E, I). Interestingly, expression of dentin sialophosphoprotein (DSPP) seemed to have no significant difference between wild type and mutant mice (Fig. 3B, F, J). DSPP was reported to expressed transiently in pre-secretory stage ameloblasts, while not expressed in secretory stage. Enamel epithelial cells are post-mitotic cells in pre-secretory-stage ameloblasts. The BrdU staining is present in all G2/S cells, but not post-mitotic cells. In P0 control incisors, BrdU staining was not found at the anterior end of the labial side, but gradually expressed at the posterior region, but was gradually (Fig. 3C). In P0 cKO incisor, we observed a decreased pattern of BrdU staining expression at the posterior region (Fig. 3G, K). In molar, same expression pattern was found in cKO and control mice (Fig. 3D, H, L). Taken together, the in vivo evidence indicates a striking phenotype of abnormal enamel development, resulting from affected differentiation and proliferation of the enamel epithelium to mature ameloblasts at the early stages of enamel development.

      Loss of Wtap deregulated SHH expression and affects ameloblasts differentiation

      To investigate the potential mechanism, we probed for Sonic hedgehog (Shh), which is play an important role in generation of ameloblasts from stem cells(
      • Seidel K.
      • Ahn C.P.
      • Lyons D.
      • Nee A.
      • Ting K.
      • Brownell I.
      • et al.
      Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor. Development. Research Support, N.I.H..
      ). In cKO, expression of SHH in ameloblasts of incision was decreased (Fig. 4 A-C). This decreased expression was confirmed by RT-qPCR (Fig. 4C). In 16.5dpc cKO, the tooth embryo develops in the cap stage. We also observed decreased expression of SHH in ameloblasts of molar (sFig. 5A-C). In vitro, recombinant SHH protein also can rescue the differentiation ability of Wtap knockdown ALC cells(Fig. 4D, E). The results indicated that WTAP plays a critical role in regulation of SHH expression in ameloblasts. Previous studies proved that WTAP is an important subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic m6A regulation(
      • Ping X.L.
      • Sun B.F.
      • Wang L.
      • Xiao W.
      • Yang X.
      • Wang W.J.
      • et al.
      Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase.
      ). We found that m6A level was notably reduced in cKO dental epithelial tissue of mandibular first molar (Fig. 4F). And in vitro, results of dot blot assay also showed that m6A level is obviously reduced after WTAP deletion in ALC cells(Fig. 4G, H). M6A-RIP-qPCR proved m6A enrichment in Shh mRNA (Fig. 4I). Futher more, result of mRNA stability assay showed that shh mRNA half-life decreased after wtap knock out in ALC cells(Fig. 4J), which means shh mRNA stability became weaker. Previous studies also found that WTAP exerts a concentration-dependent inhibitory effect on the expression of WT1-sensitive genes that regulate both mitogenic and survival pathways(
      • Small T.W.
      • Bolender Z.
      • Bueno C.
      • O'Neil C.
      • Nong Z.
      • Rushlow W.
      • et al.
      Wilms' tumor 1-associating protein regulates the proliferation of vascular smooth muscle cells.
      ). However, we found that WT1 was hardly expressed in dental epithelial cells and Wt1 conditional knock-out mice displayed normal amelogenesis as well as wild type mice (sFig. 6). It is indicated that WTAP affects enamel development not by inhibiting WT1-sensitive genes pathway. Concluded that WTAP mediated ameloblasts differentiation via affecting SHH signaling through m6A not binding to WT1. Then WTAP may promoted enamel development by regulating the expression of SHH protein via m6A methylation.

      Discussion

      Enamel development involves various stages that are tightly controlled by several key molecules of major signaling pathways expressed in epithelial and mesenchymal cells. Subtle alterations during this complicated process could lead to severe enamel defects in structure, color and shape. For a long time, it was believed that all the genetic information was stored in the sequence of DNA. However, the differences in tooth morphology between monozygotic twins indicated that other mechanisms exert their effect on gene translation
      • Duncan H.F.
      • Smith A.J.
      • Fleming G.J.
      • Cooper P.R.
      Histone deacetylase inhibitors induced differentiation and accelerated mineralization of pulp-derived cells. J Endod. Comparative Study Research Support.
      ,
      • Townsend G.
      • Hughes T.
      • Luciano M.
      • Bockmann M.
      • Brook A.
      Genetic and environmental influences on human dental variation: a critical evaluation of studies involving twins.
      . These mechanisms are called epigenetics, which are alterations in gene expression without changes in the DNA sequence. Several studies proved epigenetics are important in the regulation of tooth development and tooth regeneration
      • Townsend G.
      • Bockmann M.
      • Hughes T.
      • Brook A.
      Genetic, environmental and epigenetic influences on variation in human tooth number, size and shape. Odontology.
      ,
      • Hughes T.
      • Bockmann M.
      • Mihailidis S.
      • Bennett C.
      • Harris A.
      • Seow W.K.
      • et al.
      Genetic, epigenetic, and environmental influences on dentofacial structures and oral health: ongoing studies of Australian twins and their families.
      . Modulations in microRNA, lncRNA, DNA methylation and chromatin modifications are proved important regulatory mechanisms during tooth development(
      • Yoshioka H.
      • Minamizaki T.
      • Yoshiko Y.
      The dynamics of DNA methylation and hydroxymethylation during amelogenesis.
      • Fan Y.
      • Zhou Y.
      • Zhou X.
      • Sun F.
      • Gao B.
      • Wan M.
      • et al.
      MicroRNA 224 Regulates Ion Transporter Expression in Ameloblasts To Coordinate Enamel Mineralization.
      • Le M.H.
      • Warotayanont R.
      • Stahl J.
      • Den Besten P.K.
      • Nakano Y.
      Amelogenin Exon4 Forms a Novel miRNA That Directs Ameloblast and Osteoblast Differentiation.
      • Kamiunten T.
      • Ideno H.
      • Shimada A.
      • Nakamura Y.
      • Kimura H.
      • Nakashima K.
      • et al.
      Coordinated expression of H3K9 histone methyltransferases during tooth development in mice.
      • Yin K.
      • Hacia J.G.
      • Zhong Z.
      • Paine M.L.
      Genome-wide analysis of miRNA and mRNA transcriptomes during amelogenesis.
      • Jia Q.
      • Jiang W.
      • Ni L.
      Down-regulated non-coding RNA (lncRNA-ANCR) promotes osteogenic differentiation of periodontal ligament stem cells.
      • Babajko S.
      • Meary F.
      • Petit S.
      • Fernandes I.
      • Berdal A.
      Transcriptional regulation of MSX1 natural antisense transcript. Cells Tissues Organs.
      ). However, RNA modification in regulation of tooth development remains unclear. Here, we used the epithelial cell special marker, keratin 14, to establish the genetic mice lineage to conditionally delete Wtap from the initiation of enamel formation. Loss of Wtap leads to a severe amelogenesis imperfecta-like phenotype with smaller tooth size, thinner enamel, suggesting the functional importance of WTAP in ameloblasts. WTAP was initially identified as a nuclear protein that specifically interacts with WT1 in the development of mouse embryo(
      • Fukusumi Y.
      • Naruse C.
      • Asano M.
      Wtap is required for differentiation of endoderm and mesoderm in the mouse embryo.
      ). However, WT1 is not required for enamel formation in our study. Deletion of Wt1 in mice didn’t generate the same phenotype of Wtap cKO mice. Hence, we performed dot blot assay proved that m6A level is obviously reduced after Wtap deletion. The results indicate that WTAP mediated m6A plays an important role in amelogenesis.
      The process of amelogenesis include four defined stages: presecretory, secretory, transition and maturation. Hu et al. illustrated the changing ameloblast morphologies throughout amelogenesis as viewed histologically(
      • Hu J.C.
      • Chun Y.H.
      • Al Hazzazzi T.
      • Simmer J.P.
      Enamel formation and amelogenesis imperfecta. Cells Tissues Organs. Research Support, N.I.H..
      ). However, in cKO mice, we observed shorter and unpolarized ameloblasts. The cervical loop was significantly thinner. The changing ameloblast morphologies from the root to the cut end cannot be found. The results indicated that WTAP plays a crucial role in maturation of ameloblast. Previous studies proved that SHH and FGF signaling are required from the initiation stage of tooth development onwards. In E11.5 mouse, the molar begin to forming, a group of FGF8-positive cells form a rosette-like structure and move towards to a SHH-positive cell center(
      • Prochazka J.
      • Prochazkova M.
      • Du W.
      • Spoutil F.
      • Tureckova J.
      • Hoch R.
      • et al.
      Migration of Founder Epithelial Cells Drives Proper Molar Tooth Positioning and Morphogenesis. Dev Cell. Research Support, N.I.H..
      ). Moreover, inhibition of SHH signaling was result in the abnormality of the growth and invagination of dental epithelium(
      • Prochazka J.
      • Prochazkova M.
      • Du W.
      • Spoutil F.
      • Tureckova J.
      • Hoch R.
      • et al.
      Migration of Founder Epithelial Cells Drives Proper Molar Tooth Positioning and Morphogenesis. Dev Cell. Research Support, N.I.H..
      ). It is also believed that SHH is indispensable for the development of cytoskeleton in ameloblasts, which is critical to maintaining epithelial cell polarity and intercellular communication(
      • Gritli-Linde A.
      • Bei M.
      • Maas R.
      • Zhang X.M.
      • Linde A.
      • McMahon A.P.
      Shh signaling within the dental epithelium is necessary for cell proliferation, growth and polarization. Development. Research Support, Non-U.S. Gov't Research Support, U.S. Gov't.
      ). In cKO mice, expression of SHH in ameloblasts was decreased, indicating that WTAP modulates ameloblast maturation by regulating the expression of SHH.
      Previous studies have shown that WTAP mediated m6A could influence many biological processes. m6A-rip-qPCR also verified that m6A enrichment in shh mRNA in dental tissue. Furthermore, incubation of recombinant SHH partially rescued the capacity of mineralization ability in Wtap silenced ameloblasts. Based on these evidences, WTAP was believed to regulate Shh expression by maintaining the level of m6A modification. It will be of interest to explore the precise regulating mechanism in the future. Mice lacking WTAP die between embryonic day 6.5-10.5 and show dramatic defects in endoderm and mesoderm formation
      • Horiuchi K.
      • Umetani M.
      • Minami T.
      • Okayama H.
      • Takada S.
      • Yamamoto M.
      • et al.
      Wilms' tumor 1-associating protein regulates G2/M transition through stabilization of cyclin A2 mRNA.
      ,
      • Fukusumi Y.
      • Naruse C.
      • Asano M.
      Wtap is required for differentiation of endoderm and mesoderm in the mouse embryo.
      . The cKO mice in our study die between postnatal day 1-2. It limits our observation of the tooth phenotype in the matured mice at 8 weeks old or later. Inducible gene knockout mice should be used in the future study.
      Here, we presented some findings demonstrating the importance of m6A in amelogenesis. Firstly, conditional deletion of Wtap in ameloblasts led to defective amelogenesis, demonstrating an essential role of WTAP-mediated m6A modification in ameloblast maturation. Secondly, m6A-rip-qPCR assays revealed that deletion of Wtap resulted in downregulation of Shh. In conclusion, WTAP plays a critical role in amelogenesis and may regulate the maturation of ameloblasts by modulating expression of SHH and downstream molecules.

      Experimental Procedures

      Animal and ethics statement.

      Wtap floxed mice, K14 Cre mice were gifts from Pro. Minghan Tong’s lab (CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology). All mice were C57BL/6 genetic background and were bred under specific-pathogen free conditions. Wtap f/+; K14 Cre mice mated with Wtap f/f mice were utilized to obtain Wtap f/f; K14 Cre mice embryos. Primers for genotyping were listed in supplementary table 1. This study was approved by the Ethical Review Committee of the Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine (Shanghai, China).

      Histochemical and Immunohistochemical analysis.

      Heads from embryonic or postnatal mice were dissected in phosphate-buffered saline (PBS),and fixed in 4% paraformaldehyde-PBS solution overnight. The tissues were then dehydrated through graded ethanol, embedded in paraffin wax and sectioned by 4 μm. Hematoxylin and Eosin staining was the first step for observation. Immunofluorescence was performed to identify the expression pattern of key signaling molecule. Firstly, slides were boiled in sodium citrate buffer (10 μM, pH 6.0) for 15 min. When it were cooled in room temperature, PBS with 0.1% Triton X-100 was used to wash the slides. Next, 10% donkey serum and 0.1% Triton X-100 in PBS were used to block the nonspecific antigen for 60 min at room temperature. Then, the slides were incubated with the primary antibodies (WTAP, 1:200, Santa Cruz; Amelx, 1:100, Amelx, Santa Cruz; SHH, Bio-Techne, 1:100) in blocking buffer overnight at 4 °C. On the following day, after three times (10 min/times) wash in PBS with 0.1% Triton X-100, Alexa Fluor 488-/594-conjugated donkey secondary antibody (Jackson Immuno Research Laboratories; 1:500) were then added on the slides. Incubate at room temperature for 60 min, the slides were washed in PBS, rinsed quickly in pure ethanol, mounted in Prolong Gold Antifade medium with DAPI (Molecular Probes). Finally, the results were analyzed by fluorescence microscopy (Olympus).

      Masson staining.

      The prepared sections were deparaffinized, hydrated and washed. The staining procedures were operated according to the manufacturer protocol (Co.,Ltd.Maixin, MST-8003, Fuzhou, China). Compound dye solution supplied in the kit was used to nuclear staining for 5 min. Next, the slides were washed by clean water. Followed by phosphato-molybdic acid staining(5 min) and immersed in 2% aniline blue solution for 5 min. Then, the slides were differentiated with 1% differentiation solution for 40 sec, washed in water, dehydrated by graded ethanol and mounted with neutral gum. Enamel tissues in the microscopic performance of Masson staining usually were red, dentin and bone were blue and muscles were purple. All of the pictures in our study were taken by Nikon camera.

      BrdU labeling.

      BrdU was injured into the pregrant mouse or newborn mouse (50 μg/g of body weight) 2 hours prior to sacrificing the mouse. Samples were embedded as described above. After hydrated through graded ethanol, the sections were incubated in 0.1%PBST for 30 minutes and incubated in 2M HCl solution for 30 minutes under 37 °C, followed neutralization by boric acid for 10 minutes at room temperature. The sections were blocked with normal donkey serum at room temperature for 60 minutes before incubation in BrdU antibody (1:200, sigma Aldrich) overnight at 4 °C. Sections were washed in PBST three times. Second antibody donkey anti mouse were diluted 1:500 for 60 minutes at room temperature.

      Cell culture.

      Ameloblast-lineage cell (ALC) were gifts from Pro. Wantao Chen’s lab (Shanghai Key Laboratory of Stomatology). Extracting method was described in previous study(
      • Nakata A.
      • Kameda T.
      • Nagai H.
      • Ikegami K.
      • Duan Y.
      • Terada K.
      • et al.
      Establishment and characterization of a spontaneously immortalized mouse ameloblast-lineage cell line.
      ). ALC cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplied with 10% FBS in 37 °C, 5% CO2. Cells was plated in 6-well plate about 70% confluent. Change to fresh culture media containing 8ug/mL polybrene. Incubate cells at 37 °C, 5% CO2 overnight. Change to fresh media 6 hours after infection. Infected cells were selected by culture medium with 2 μg/ml puromycin 24 hours after infection.

      Plko.1-shRNA construction.

      Plko.1-shRNA plasmic was constructed according to the manufacturer (http:www.addgene.org/tools/protocols/plko). Sequence of oligos were listed in supplementary table 2.

      Real-time PCR and m6A-rip-qPCR.

      Total RNA for dot blot was isolated from enamel organ and ALC cells was extratd with Trizol reagent (Invitrogen), then mRNA was extracted using GenElute mRNA miniprep (Sigma-Aldrich). Sequence of primers were listed in supplementary table 3.

      Western blotting.

      Cells were harvested and lysed in SDS lysis, protein concentration from lysis supernatant was determined by the Bradford method. 40 μg protein of each sample were separated by SDS-PAGE under reducing conditions and transferred to PVDF membrane for 45 minutes at 130V. Membranes were saturated with 5% skimmed milk, and incubated with antibodies (WTAP, 1:1000, Sant Cruz; GAPDH, 1:10000, Santa Cruz) overnight at 4 °C. After washing with TBST (supplemented with 0.1% Tween-20), the membranes were incubated with peroxidase-conjugated IgG second antibody and rinsed with TBST, and finally developed using the ECL Western Blotting Analysis System (Shanghai Yeasen CO. Ltd, China).

      Crystal violet staining and alizarin red staining.

      ALC cells were cultured in mineralization-inducing media containing 100 mmol/L ascorbic acid, 10 nmol/L dexamethasone and 2 mmol/L b-glycerophosphate. After incubation for 3 weeks, cells were fixed with 4% paraformaldehyde and stained with 1% Alizarin Red solution for 30 minutes at room temperature.

      m6A dot blot assay

      GenElute mRNA Miniprep Kit (sigma) was used to extract mRNA from mandibular tissue and ALC cells according to manufacturer’s protocols. At embryonic stages E14.5, mice enamel organ was harvest. To ensure sufficient concentration of mRNA, the enamel organ of 20 biological replicates were pooled for each sample. Dots (50 ng mRNA per 1.5 μL dot) were applied to an Amersham Hybond-N+ membrane (GE Healthcare). After complete drying the mRNA sample, a UV Stratalinker 2400 was used to crosslink mRNA to the membrane by running the auto-crosslink program at 3000kJ. After three times washing in PBST(0.1% Tween-20 in PBS), the membrane was blocked in 5% skim milk in PBST for 2 hours. Then repeating three times PBST wash, the mRNA crosslinked membrane was incubated with primary anti-m6A antibody (212B11, Synaptic Systems) at 1:1000 dilution for 2 hours at room temperature. Repeating three washes in PBST, HRP-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch) was used to incubate for 1 hour at room temperature. Finally, after three washes, the membrane was visualized. Additionally, before incubation with antibodies, the membrane was stained with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2) to confirm equal mRNA loading. Next, quantified m6A levels were normalized to amount of mRNA loaded. For each time point, three biological samples in technical duplicates were used.

      UPLC-MS/MS analysis of m6A levels

      Nuclease P1 (1U; Sigma) in 20 μl of buffer which contained 10 mM of NH4Ac (pH 5.3) was used to digest mRNA at 42 oC for 4 h. Then 100 mM NH4HCO3 and alkaline phosphatase (0.5 U) were then added to about 50-100 ng purified mRNA for another incubation at 37 °C for 4 h. Next, the supernatant of digested sample was collected by centrifugation (4 °C, 13 000 rpm, 20 min) and then injected into UPLC-MS/MS. UPLC (SHIMADZU) equipped with ZORBAX SB-Aq column (Agilent) was used to separate the nucleosides. Then, Triple Quad 5500 (AB SCIEX) in positive ion multiple reaction-monitoring (MRM) mode was used to detect the nucleosides. According to nucleoside-to-base ion mass transitions, the modifications were quantified: m/z 268.0-136.0 for A, and m/z 282.0-150.1 for m6A. Pure nucleosides were used to generate standard curves. Then, the concentrations of A and m6A in the sample were calculated. Finally, the percentage of total unmodified A represents the level of m6A.

      mRNA stability assay

      5×105 ALC cells with stably expressed shWtap or shNegative control were seeded into 6-well plates. After 24 hours, cells were treated with 5 μg/ml actinomycin D and collected at indicated time points. The total RNA was extracted by TRIzol protocol (Takara) and analyzed by RT-PCR. Half-life of mRNA were analysized by graph pad 8.3.0 according to previously published paper.

      Data availability

      All data are contained within the article.

      Acknowledgments

      Work is sponsored by grants from the National Natural Science Foundation of China (32000571), Shanghai Sailing Program (20YF1423100) and Biological clinical sample project of Shanghai Ninth people’s hospital(YBKB201903).
      We thank Dr. Minghan Tong for reagents and mice and the members of the Tong’s laboratory for useful discussions.

      References

        • Zeichner-David M.
        • Diekwisch T.
        • Fincham A.
        • Lau E.
        • MacDougall M.
        • Moradian-Oldak J.
        • et al.
        Control of ameloblast differentiation.
        Int J Dev Biol. Research Support, U.S. Gov't, P.H.S. 1995; 39 (Review Feb) (Epub 1995/02/01): 69-92
        • Shibata S.
        • Suzuki S.
        • Tengan T.
        • Yamashita Y.
        A histochemical study of apoptosis in the reduced ameloblasts of erupting mouse molars.
        Arch Oral Biol. Jul. 1995; 40 (Epub 1995/07/01): 677-680
        • Bartlett J.D.
        Dental enamel development: proteinases and their enamel matrix substrates.
        ISRN Dent. Review Sep 16. 2013; 2013 (Epub 2013/10/26)684607
        • Desrosiers R.C.
        • Friderici K.H.
        • Rottman F.M.
        Characterization of Novikoff hepatoma mRNA methylation and heterogeneity in the methylated 5' terminus. Biochemistry. Research Support, U.S. Gov't.
        P.H.S. Oct 7 1975; 14 (Epub 1975/10/07): 4367-4374
        • Patil D.P.
        • Chen C.K.
        • Pickering B.F.
        • Chow A.
        • Jackson C.
        • Guttman M.
        • et al.
        m(6)A RNA methylation promotes XIST-mediated transcriptional repression.
        Nature. Research Support, Non-U.S. Gov't Research Support, N.I.H. 2016; 537 (Extramural Sep 15) (Epub 2016/09/08): 369-373
        • Zhao B.S.
        • Roundtree I.A.
        • He C.
        Post-transcriptional gene regulation by mRNA modifications.
        Nat Rev Mol Cell Biol. Review Jan. 2017; 18 (Epub 2016/11/04): 31-42
        • Wang X.
        • Lu Z.
        • Gomez A.
        • Hon G.C.
        • Yue Y.
        • Han D.
        • et al.
        N6-methyladenosine-dependent regulation of messenger RNA stability. Nature. Research Support, N.I.H..
        Extramural Research Support, U.S. Gov't, Non-P.H.S. Jan 2. 2014; 505 (Epub 2013/11/29): 117-120
        • Xiao W.
        • Adhikari S.
        • Dahal U.
        • Chen Y.S.
        • Hao Y.J.
        • Sun B.F.
        • et al.
        Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing.
        Mol Cell. Research Support, Non-U.S. Gov't Feb 18. 2016; 61 (Epub 2016/02/16): 507-519
        • Wang X.
        • Zhao B.S.
        • Roundtree I.A.
        • Lu Z.
        • Han D.
        • Ma H.
        • et al.
        N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency. Cell. Research Support, Non-U.S. Gov't Research Support, U.S. Gov't.
        Non-P.H.S. Jun 4. 2015; 161 (Epub 2015/06/06): 1388-1399
        • Zhou J.
        • Wan J.
        • Gao X.
        • Zhang X.
        • Jaffrey S.R.
        • Qian S.B.
        Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature. Research Support, N.I.H..
        Extramural Research Support, U.S. Gov't, Non-P.H.S. Oct 22 2015; 526 (Epub 2015/10/13): 591-594
        • Geula S.
        • Moshitch-Moshkovitz S.
        • Dominissini D.
        • Mansour A.A.
        • Kol N.
        • Salmon-Divon M.
        • et al.
        Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation.
        Science. Research Support, Non-U.S. Gov't Feb 27. 2015; 347 (Epub 2015/01/09): 1002-1006
        • Zhao B.S.
        • Wang X.
        • Beadell A.V.
        • Lu Z.
        • Shi H.
        • Kuuspalu A.
        • et al.
        m(6)A-dependent maternal mRNA clearance facilitates zebrafish maternal-to-zygotic transition. Nature. Research Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov′t Research Support, U.S. Gov′t.
        Non-P.H.S. Feb 23. 2017; 542 (Epub 2017/02/14): 475-478
      1. Lence T, Akhtar J, Bayer M, Schmid K, Spindler L, Ho CH, et al. m(6)A modulates neuronal functions and sex determination in Drosophila. Nature. Research Support, Non-U.S. Gov′t Dec 8 2016;540(7632):242-247. Epub 2016/12/06.

        • Little N.A.
        • Hastie N.D.
        • Davies R.C.
        Identification of WTAP, a novel Wilms' tumour 1-associating protein.
        Hum Mol Genet. Sep 22 2000; 9 (Epub 2000/09/26): 2231-2239
        • Horiuchi K.
        • Umetani M.
        • Minami T.
        • Okayama H.
        • Takada S.
        • Yamamoto M.
        • et al.
        Wilms' tumor 1-associating protein regulates G2/M transition through stabilization of cyclin A2 mRNA.
        Proc Natl Acad Sci U S A. Research Support, Non-U.S. Gov't Nov 14. 2006; 103 (Epub 2006/11/08): 17278-17283
        • Fukusumi Y.
        • Naruse C.
        • Asano M.
        Wtap is required for differentiation of endoderm and mesoderm in the mouse embryo.
        Dev Dyn. Research Support, Non-U.S. Gov't Mar. 2008; 237 (Epub 2008/01/29): 618-629
        • Ping X.L.
        • Sun B.F.
        • Wang L.
        • Xiao W.
        • Yang X.
        • Wang W.J.
        • et al.
        Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase.
        Cell Res. Research Support, Non-U.S. Gov't Feb. 2014; 24 (Epub 2014/01/11): 177-189
        • Chen Y.
        • Peng C.
        • Chen J.
        • Chen D.
        • Yang B.
        • He B.
        • et al.
        WTAP facilitates progression of hepatocellular carcinoma via m6A-HuR-dependent epigenetic silencing of ETS1.
        Mol Cancer. Research Support, Non-U.S. Gov't. Aug 22 2019; 18 (Epub 2019/08/24): 127
        • Bertero A.
        • Brown S.
        • Madrigal P.
        • Osnato A.
        • Ortmann D.
        • Yiangou L.
        • et al.
        The SMAD2/3 interactome reveals that TGFbeta controls m(6)A mRNA methylation in pluripotency.
        Nature. Research Support, Non-U.S. Gov't Mar 8. 2018; 555 (Epub 2018/03/01): 256-259
        • Jia G.X.
        • Lin Z.
        • Yan R.G.
        • Wang G.W.
        • Zhang X.N.
        • Li C.
        • et al.
        WTAP Function in Sertoli Cells Is Essential for Sustaining the Spermatogonial Stem Cell Niche.
        Stem Cell Reports. Research Support, Non-U.S. Gov't. Oct 13 2020; 15 (Epub 2020/10/15): 968-982
        • Smith C.E.L.
        • Poulter J.A.
        • Antanaviciute A.
        • Kirkham J.
        • Brookes S.J.
        • Inglehearn C.F.
        • et al.
        Amelogenesis Imperfecta; Genes, Proteins, and Pathways.
        Front Physiol. Review. 2017; 8 (Epub 2017/07/12): 435
        • Nakata A.
        • Kameda T.
        • Nagai H.
        • Ikegami K.
        • Duan Y.
        • Terada K.
        • et al.
        Establishment and characterization of a spontaneously immortalized mouse ameloblast-lineage cell line.
        Biochem Biophys Res Commun. Research Support, Non-U.S. Gov't. Sep 5 2003; 308 (Epub 2003/08/21): 834-839
        • Seidel K.
        • Ahn C.P.
        • Lyons D.
        • Nee A.
        • Ting K.
        • Brownell I.
        • et al.
        Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor. Development. Research Support, N.I.H..
        ExtramuralResearch Support, Non-U.S. Gov't Nov. 2010; 137 (Epub 2010/10/28): 3753-3761
        • Small T.W.
        • Bolender Z.
        • Bueno C.
        • O'Neil C.
        • Nong Z.
        • Rushlow W.
        • et al.
        Wilms' tumor 1-associating protein regulates the proliferation of vascular smooth muscle cells.
        Circ Res. Research Support, Non-U.S. Gov't. Dec 8 2006; 99 (Epub 2006/11/11): 1338-1346
        • Duncan H.F.
        • Smith A.J.
        • Fleming G.J.
        • Cooper P.R.
        Histone deacetylase inhibitors induced differentiation and accelerated mineralization of pulp-derived cells. J Endod. Comparative Study Research Support.
        Non-U.S. Gov't Mar. 2012; 38 (Epub 2012/02/22): 339-345
        • Townsend G.
        • Hughes T.
        • Luciano M.
        • Bockmann M.
        • Brook A.
        Genetic and environmental influences on human dental variation: a critical evaluation of studies involving twins.
        Arch Oral Biol. Research Support, Non-U.S. Gov'tReview Dec. 2009; 54 (Epub 2008/08/22): S45-51
        • Townsend G.
        • Bockmann M.
        • Hughes T.
        • Brook A.
        Genetic, environmental and epigenetic influences on variation in human tooth number, size and shape. Odontology.
        Research Support, Non-U.S. Gov'tReview Jan. 2012; 100 (Epub 2011/12/06): 1-9
        • Hughes T.
        • Bockmann M.
        • Mihailidis S.
        • Bennett C.
        • Harris A.
        • Seow W.K.
        • et al.
        Genetic, epigenetic, and environmental influences on dentofacial structures and oral health: ongoing studies of Australian twins and their families.
        Twin Res Hum Genet. Research Support, Non-U.S. Gov'tTwin Study Feb. 2013; 16 (Epub 2013/02/12): 43-51
        • Yoshioka H.
        • Minamizaki T.
        • Yoshiko Y.
        The dynamics of DNA methylation and hydroxymethylation during amelogenesis.
        Histochem Cell Biol. Research Support, Non-U.S. Gov't Nov. 2015; 144 (Epub 2015/07/26): 471-478
        • Fan Y.
        • Zhou Y.
        • Zhou X.
        • Sun F.
        • Gao B.
        • Wan M.
        • et al.
        MicroRNA 224 Regulates Ion Transporter Expression in Ameloblasts To Coordinate Enamel Mineralization.
        Mol Cell Biol. Research Support, Non-U.S. Gov't Aug. 2015; 35 (Epub 2015/06/10): 2875-2890
        • Le M.H.
        • Warotayanont R.
        • Stahl J.
        • Den Besten P.K.
        • Nakano Y.
        Amelogenin Exon4 Forms a Novel miRNA That Directs Ameloblast and Osteoblast Differentiation.
        J Dent Res. Research Support, Non-U.S. Gov't Apr. 2016; 95 (Epub 2015/12/31): 423-429
        • Kamiunten T.
        • Ideno H.
        • Shimada A.
        • Nakamura Y.
        • Kimura H.
        • Nakashima K.
        • et al.
        Coordinated expression of H3K9 histone methyltransferases during tooth development in mice.
        Histochem Cell Biol. Research Support, Non-U.S. Gov't Mar. 2015; 143 (Epub 2014/10/09): 259-266
        • Yin K.
        • Hacia J.G.
        • Zhong Z.
        • Paine M.L.
        Genome-wide analysis of miRNA and mRNA transcriptomes during amelogenesis.
        BMC Genomics. Research Support, N.I.H., Extramural Nov 19. 2014; 15 (Epub 2014/11/20): 998
        • Jia Q.
        • Jiang W.
        • Ni L.
        Down-regulated non-coding RNA (lncRNA-ANCR) promotes osteogenic differentiation of periodontal ligament stem cells.
        Arch Oral Biol. Research Support, Non-U.S. Gov't Feb. 2015; 60 (Epub 2014/12/03): 234-241
        • Babajko S.
        • Meary F.
        • Petit S.
        • Fernandes I.
        • Berdal A.
        Transcriptional regulation of MSX1 natural antisense transcript. Cells Tissues Organs.
        Research Support, Non-U.S. Gov't. 2011; 194 (Epub 2011/06/01): 151-155
        • Hu J.C.
        • Chun Y.H.
        • Al Hazzazzi T.
        • Simmer J.P.
        Enamel formation and amelogenesis imperfecta. Cells Tissues Organs. Research Support, N.I.H..
        Extramural Review. 2007; 186 (Epub 2007/07/14): 78-85
        • Prochazka J.
        • Prochazkova M.
        • Du W.
        • Spoutil F.
        • Tureckova J.
        • Hoch R.
        • et al.
        Migration of Founder Epithelial Cells Drives Proper Molar Tooth Positioning and Morphogenesis. Dev Cell. Research Support, N.I.H..
        Extramural Research Support, Non-U.S. Gov′t. Dec 21 2015; 35 (Epub 2015/12/26): 713-724
        • Gritli-Linde A.
        • Bei M.
        • Maas R.
        • Zhang X.M.
        • Linde A.
        • McMahon A.P.
        Shh signaling within the dental epithelium is necessary for cell proliferation, growth and polarization. Development. Research Support, Non-U.S. Gov't Research Support, U.S. Gov't.
        P.H.S. Dec. 2002; 129 (Epub 2002/10/31): 5323-5337