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DOI： 10.1016/j.bbrc.2023.09.014.

Language：English   Publisher：Communications Biology  

Postnatal cell fate is postulated to be primarily determined by the local tissue microenvironment. Here, we find that Mediator 1 (Med1) dependent epigenetic mechanisms dictate tissue-specific lineage commitment and progression of dental epithelia. Deletion of Med1, a key component of the Mediator complex linking enhancer activities to gene transcription, provokes a tissue extrinsic lineage shift, causing hair generation in incisors. Med1 deficiency gives rise to unusual hair growth via primitive cellular aggregates. Mechanistically, we find that MED1 establishes super-enhancers that control enamel lineage transcription factors in dental stem cells and their progenies. However, Med1 deficiency reshapes the enhancer landscape and causes a switch from the dental transcriptional program towards hair and epidermis on incisors in vivo, and in dental epithelial stem cells in vitro. Med1 loss also provokes an increase in the number and size of enhancers. Interestingly, control dental epithelia already exhibit enhancers for hair and epidermal key transcription factors; these transform into super-enhancers upon Med1 loss suggesting that these epigenetic mechanisms cause the shift towards epidermal and hair lineages. Thus, we propose a role for Med1 in safeguarding lineage specific enhancers, highlight the central role of enhancer accessibility in lineage reprogramming and provide insights into ectodermal regeneration.

DOI： 10.1038/s42003-023-05105-5

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DOI： 10.1038/s42003-023-05105-5.

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Biological Chemistry  

Lipid rafts are membrane microdomains rich in cholesterol, sphingolipids, glycosylphosphatidylinositol-anchored proteins (GPI-APs), and receptors. These lipid raft components are localized at the plasma membrane and are essential for signal transmission and organogenesis. However, few reports have been published on the specific effects of lipid rafts on tooth development. Using microarray and single-cell RNA sequencing methods, we found that a GPI-AP, lymphocyte antigen-6/Plaur domain-containing 1 (Lypd1), was specifically expressed in preodontoblasts. Depletion of Lypd1 in tooth germ using an ex vivo organ culture system and in mouse dental pulp (mDP) cells resulted in the inhibition of odontoblast differentiation. Activation of bone morphogenetic protein (BMP) signaling by BMP2 treatment in mDP cells promoted odontoblast differentiation via phosphorylation of Smad1/5/8, while this BMP2-mediated odontoblast differentiation was inhibited by depletion of Lypd1. Furthermore, we created a deletion construct of the C terminus containing the omega site in LYPD1; this site is necessary for localizing GPI-APs to the plasma membrane and lipid rafts. We identified that this site is essential for odontoblast differentiation and morphological change of mDP cells. These findings demonstrated that LYPD1 is a novel marker of preodontoblasts in the developing tooth; in addition, they suggest that LYPD1 is important for tooth development and that it plays a pivotal role in odontoblast differentiation by regulating Smad1/5/8 phosphorylation through its effect as a GPI-AP in lipid rafts.

DOI： 10.1016/j.jbc.2023.104638

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：FASEB Journal  

Enamel is formed by the repetitive secretion of a tooth-specific extracellular matrix and its decomposition. Calcification of the enamel matrix via hydroxyapatite (HAP) maturation requires pH cycling to be tightly regulated through the neutralization of protons released during HAP synthesis. We found that Gpr115, which responds to changes in extracellular pH, plays an important role in enamel formation. Gpr115-deficient mice show partial enamel hypomineralization, suggesting that other pH-responsive molecules may be involved. In this study, we focused on the role of Gpr111/Adgrf2, a duplicate gene of Gpr115, in tooth development. Gpr111 was highly expressed in mature ameloblasts. Gpr111-KO mice showed enamel hypomineralization. Dysplasia of enamel rods and high carbon content seen in Gpr111-deficient mice suggested the presence of residual enamel matrices in enamel. Depletion of Gpr111 in dental epithelial cells induced the expression of ameloblast-specific protease, kallikrein-related peptidase 4 (Klk4), suggesting that Gpr111 may act as a suppressor of Klk4 expression. Moreover, reduction of extracellular pH to 6.8 suppressed the expression of Gpr111, while the converse increased Klk4 expression. Such induction of Klk4 was synergistically enhanced by Gpr111 knockdown, suggesting that proper enamel mineralization may be linked to the modulation of Klk4 expression by Gpr111. Furthermore, our in vitro suppression of Gpr111 and Gpr115 expression indicated that their suppressive effect on calcification was additive. These results suggest that both Gpr111 and Gpr115 respond to extracellular pH, contribute to the expression of proteolytic enzymes, and regulate the pH cycle, thereby playing important roles in enamel formation.

DOI： 10.1096/fj.202202053R

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Biochemical and Biophysical Research Communications  

Iroquois homeobox (Irx) genes are TALE-class homeobox genes that are evolutionarily conserved across species and have multiple critical cellular functions in fundamental tissue development processes. Previous studies have shown that Irxs genes are expressed during tooth development. However, the precise roles of genes in teeth remain unclear. Here, we demonstrated for the first time that Irx3 is an essential molecule for the proliferation and differentiation of odontoblasts. Using cDNA synthesized from postnatal day 1 (P1) tooth germs, we examined the expression of all Irx genes (Irx1-Irx6) by RT-PCR and found that all genes except Irx4 were expressed in the tooth tissue. Irx1-Irx3 a were expressed in the dental epithelial cell line M3H1 cells, while Irx3 and Irx5 were expressed in the dental mesenchymal cell line mDP cells. Only Irx3 was expressed in both undifferentiated cell lines. Immunostaining also revealed the presence of IRX3 in the dental epithelial cells and mesenchymal condensation. Inhibition of endogenous Irx3 by siRNA blocks the proliferation and differentiation of mDP cells. Wnt3a, Wnt5a, and Bmp4 are factors involved in odontoblast differentiation and were highly expressed in mDP cells by quantitative PCR analysis. Interestingly, the expression of Wnt5a (but not Wnt3a or Bmp4) was suppressed by Irx3 siRNA. These results suggest that Irx3 plays an essential role in part through the regulation of Wnt5a expression during odontoblast proliferation and differentiation.

DOI： 10.1016/j.bbrc.2023.02.004

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Language：English   Publisher：Springer  

Recent advances in regenerative technology have made the regeneration of various organs using pluripotent stem cells possible. However, a simpler screening method for evaluating regenerated organs is required to apply this technology to clinical regenerative medicine in the future. We have developed a simple evaluation method using a mouse tooth germ culture model of organs formed by epithelial–mesenchymal interactions. In this study, we successfully established a simple method that controls tissue development in a temperature-dependent manner using a mouse tooth germ ex vivo culture model. We observed that the development of the cultured tooth germ could be delayed by low-temperature culture and resumed by the subsequent culture at 37 °C. Furthermore, the optimal temperature for the long-term preservation of tooth germ was 25 °C, a subnormothermic temperature that maintains the expression of stem cell markers. We also found that subnormothermic temperature induces the expression of cold shock proteins, such as cold-inducible RNA-binding protein, RNA-binding motif protein 3, and serine and arginine rich splicing factor 5. This study provides a simple screening method to help establish the development of regenerative tissue technology using a tooth organ culture model. Our findings may be potentially useful for making advances in the field of regenerative medicine.

DOI： 10.1038/s41598-023-29629-2

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1038/s41598-023-29629-2.

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Journal of Oral Biosciences  

Objectives: Epithelial-mesenchymal interactions are extremely important in tooth development and essential for ameloblast differentiation, especially during tooth formation. We aimed to identify the type of mesenchymal cells important in ameloblast differentiation. Methods: We used two types of cell culture systems with chambers and found that a subset of debtal mesenchimal cells is important for the differentiatiuon of dental spithelial cells into ameloblasts. Further, we induced dental pulp stem cell-like cells from dental pulp stem cells using the small molecule compound BIO ( a GSK-3 inhibitor IX) to clarify the mechanism involved in ameloblast differentiation induced by dental pulp stem cells. Results: The BIO-induced dental pulp cells promoted the expression of mesenchymal stem cell markers Oct3/4 and Bcrp1. Furthermore, we used artificial dental pulp stem cells induced by BIO to identify the molecules expressed in dental pulp stem cells required for ameloblast differentiation. Panx3 expression was induced in the dental pulp stem cell through interaction with the dental epithelial cells. In addition, ATP release from cells increased in Panx3-expressing cells. We also confirmed that ATP stimulation is accepted in dental epithelial cells. Conclusions: These results showed that the Panx3 expressed in dental pulp stem cells is important for ameloblast differentiation and that ATP release by Panx3 may play a role in epithelial–mesenchymal interaction.

DOI： 10.1016/j.job.2022.10.002

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Language：English   Publisher：(一社)歯科基礎医学会  

チャンバー内細胞培養系を用いて歯原性間葉系細胞のサブセットが歯原性上皮細胞からエナメル芽細胞への分化に重要であることを発見した。歯髄細胞からGSK3β阻害剤BIOを用いて歯髄幹細胞様細胞を誘導し、歯髄幹細胞により誘導されるエナメル芽細胞分化のメカニズムについて検討した。BIOで誘導された歯髄細胞は間葉系幹細胞マーカーOct3/4およびBcrp1を高発現した。BIOで誘導された人工歯髄幹細胞を用いて、歯髄幹細胞に発現するエナメル芽細胞分化に必要な分子を同定した。歯原性上皮細胞との相互作用を介して歯髄幹細胞でPanx3発現が誘導された。さらにPanx3発現細胞でATP遊離が増加した。ATP刺激は歯原性上皮細胞に受容された。以上の結果から、歯髄幹細胞に発現するPanx3がエナメル芽細胞分化に重要で、Panx3によるATP遊離が上皮間葉系相互作用に寄与すると考えられた。

Language：English   Publisher：American Journal of Orthodontics and Dentofacial Orthopedics  

Introduction: We evaluated the effects of secondary bone grafting (SBG) on oral health–related and generic health–related quality of life (OHRQOL and HRQOL, respectively) in preadolescent orthodontic patients with alveolar bone defects. Methods: We divided 101 orthodontic patients aged 8-10 years into 3 groups: 39 general orthodontic patients, 18 patients with orofacial clefts who did not require SBG, and 44 patients with alveolar defects who required SBG using particulate cancellous bone and marrow obtained from the iliac crest. The participants completed the self-report Child Perceptions Questionnaire (CPQ) and Paediatric Quality of Life Inventory (version 4.0) for OHRQOL and HRQOL, respectively, and their scores were assessed. The quality of life (QOL) of patients who required SBG was examined before, 1 month, and 6 months after SBG. The relationships between OHRQOL or HRQOL and potential patient factors were also evaluated. Results: Physical HRQOL subscale scores worsened 1 month after SBG, whereas the total OHRQOL and HRQOL scores before and after SBG showed no significant changes. OHRQOL and HRQOL showed no significant differences among the 3 groups before SBG. The presence of oronasal fistula was associated with poorer OHRQOL in patients with cleft lip and/or palate. Conclusions: SBG and orthodontic treatment had a relatively small impact on the QOL of the preadolescent children in this study. Understanding the influence of SBG and patient factors on QOL would enable better treatment and care for these patients.

DOI： 10.1016/j.ajodo.2022.08.012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Dental Research  

Organ development is dictated by the regulation of genes preferentially expressed in tissues or cell types. Gene expression profiling and identification of specific genes in organs can provide insights into organogenesis. Therefore, genome-wide analysis is a powerful tool for clarifying the mechanisms of development during organogenesis as well as tooth development. Single-cell RNA sequencing (scRNA-seq) is a suitable tool for unraveling the gene expression profile of dental cells. Using scRNA-seq, we can obtain a large pool of information on gene expression; however, identification of functional genes, which are key molecules for tooth development, via this approach remains challenging. In the present study, we performed cap analysis of gene expression sequence (CAGE-seq) using mouse tooth germ to identify the genes preferentially expressed in teeth. The CAGE-seq counts short reads at the 5′-end of transcripts; therefore, this method can quantify the amount of transcripts without bias related to the transcript length. We hypothesized that this CAGE data set would be of great help for further understanding a gene expression profile through scRNA-seq. We aimed to identify the important genes involved in tooth development via bioinformatics analyses, using a combination of scRNA-seq and CAGE-seq. We obtained the scRNA-seq data set of 12,212 cells from postnatal day 1 mouse molars and the CAGE-seq data set from postnatal day 1 molars. scRNA-seq analysis revealed the spatiotemporal expression of cell type–specific genes, and CAGE-seq helped determine whether these genes are preferentially expressed in tooth or ubiquitously. Furthermore, we identified candidate genes as novel tooth-enriched and dental cell type–specific markers. Our results show that the integration of scRNA-seq and CAGE-seq highlights the genes important for tooth development among numerous gene expression profiles. These findings should contribute to resolving the mechanism of tooth development and establishing the basis for tooth regeneration in the future.

DOI： 10.1177/00220345211049785

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Cellular Physiology  

Cell- and tissue-specific extracellular matrix (ECM) composition plays an important role in organ development, including teeth, by regulating cell behaviors, such as cell proliferation and differentiation. Here, we demonstrate for the first time that von Willebrand factor D and epidermal growth factor (EGF) domains (Vwde), a previously uncharacterized ECM protein, is specifically expressed in teeth and regulates cell proliferation and differentiation in inner enamel epithelial cells (IEEs) and enamel formation. We identified the Vwde as a novel ECM protein through bioinformatics using the NCBI expressed sequence tag database for mice. Vwde complementary DNA encodes 1773 amino acids containing a signal peptide, a von Willebrand factor type D domain, and tandem calcium-binding EGF-like domains. Real-time polymerase chain reaction demonstrated that Vwde is highly expressed in tooth tissue but not in other tissues including the brain, lung, heart, liver, kidney, and bone. In situ hybridization revealed that the IEEs expressed Vwde messenger RNA in developing teeth. Immunostaining showed that VWDE was localized at the proximal and the distal ends of the pericellular regions of the IEEs. Vwde was induced during the differentiation of mouse dental epithelium-derived M3H1 cells. Vwde-transfected M3H1 cells secreted VWDE protein into the culture medium and inhibited cell proliferation, whereas ameloblastic differentiation was promoted. Furthermore, Vwde increased the phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B and strongly induced the expression of the intercellular junction protein, N-cadherin (Ncad). Interestingly, the suppression of endogenous Vwde inhibited the expression of Ncad. Finally, we created Vwde-knockout mice using the CRISPR-Cas9 system. Vwde-null mice showed low mineral density, rough surface, and cracks in the enamel, indicating the enamel hypoplasia phenotype. Our findings suggest that Vwde assembling the matrix underneath the IEEs is essential for Ncad expression and enamel formation.

DOI： 10.1002/jcp.30667

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer  

Development of chemotherapy has led to a high survival rate of cancer patients; however, the severe side effects of anticancer drugs, including organ hypoplasia, persist. To assume the side effect of anticancer drugs, we established a new ex vivo screening model and described a method for suppressing side effects. Cyclophosphamide (CPA) is a commonly used anticancer drug and causes severe side effects in developing organs with intensive proliferation, including the teeth and hair. Using the organ culture model, we found that treatment with CPA disturbed the growth of tooth germs by inducing DNA damage, apoptosis and suppressing cellular proliferation and differentiation. Furthermore, low temperature suppressed CPA-mediated inhibition of organ development. Our ex vivo and in vitro analysis revealed that low temperature impeded Rb phosphorylation and caused cell cycle arrest at the G1 phase during CPA treatment. This can prevent the CPA-mediated cell damage of DNA replication caused by the cross-linking reaction of CPA. Our findings suggest that the side effects of anticancer drugs on organ development can be avoided by maintaining the internal environment under low temperature.

DOI： 10.1038/s41598-022-06945-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Cellular Physiology  

Tissue-specific basic helix-loop-helix (bHLH) transcription factors play an important role in cellular differentiation. We recently identified AmeloD as a tooth-specific bHLH transcription factor. However, the role of AmeloD in cellular differentiation has not been investigated. The aim of this study was to elucidate the role of AmeloD in dental epithelial cell differentiation. We found that AmeloD-knockout (AmeloD-KO) mice developed an abnormal structure and altered ion composition of enamel in molars, suggesting that AmeloD-KO mice developed enamel hypoplasia. In molars of AmeloD-KO mice, the transcription factor Sox21 encoding SRY-Box transcription factor 21 and ameloblast differentiation marker genes were significantly downregulated. Furthermore, overexpression of AmeloD in the dental epithelial cell line M3H1 upregulated Sox21 and ameloblast differentiation marker genes, indicating that AmeloD is critical for ameloblast differentiation. Our study demonstrated that AmeloD is an important transcription factor in amelogenesis for promoting ameloblast differentiation. This study provides new insights into the mechanisms of amelogenesis.

DOI： 10.1002/jcp.30639

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Bone  

Amelogenesis consists of secretory, transition, maturation, and post-maturation stages, and the morphological changes of ameloblasts at each stage are closely related to their function. p130 Crk-associated substrate (Cas) is a scaffold protein that modulates essential cellular processes, including cell adhesion, cytoskeletal changes, and polarization. The expression of p130Cas was observed from the secretory stage to the maturation stage in ameloblasts. Epithelial cell-specific p130Cas-deficient (p130Cas^Δepi-) mice exhibited enamel hypomineralization with chalk-like white mandibular incisors in young mice and attrition in aged mouse molars. A micro-computed tomography analysis and Vickers micro-hardness testing showed thinner enamel, lower enamel mineral density and hardness in p130Cas^Δepi- mice in comparison to p130Cas^flox/flox mice. Scanning electron microscopy, and an energy dispersive X-ray spectroscopy analysis indicated the disturbance of the enamel rod structure and lower Ca and P contents in p130Cas^Δepi- mice, respectively. The disorganized arrangement of ameloblasts, especially in the maturation stage, was observed in p130Cas^Δepi- mice. Furthermore, expression levels of enamel matrix proteins, such as amelogenin and ameloblastin in the secretory stage, and functional markers, such as alkaline phosphatase and iron accumulation, and Na⁺/Ca²⁺+K⁺-exchanger in the maturation stage were reduced in p130Cas^Δepi- mice. These findings suggest that p130Cas plays important roles in amelogenesis (197 words).

DOI： 10.1016/j.bone.2021.116210

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DOI： 10.1002/jcp.30389

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DOI： 10.3389/fphys.2021.748574

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DOI： 10.7717/peerj.11297

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DOI： 10.1016/j.pdj.2020.09.003

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DOI： 10.3390/ijms21238952

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DOI： 10.1074/jbc.RA120.014281

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DOI： 10.3389/fcell.2020.595593

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DOI： 10.3389/fcell.2020.00841

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DOI： 10.1016/j.isci.2020.101329

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DOI： 10.1074/jbc.RA118.005298

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DOI： 10.1177/0022034518808254

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DOI： 10.1074/jbc.RA118.003373

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DOI： 10.1038/s41598-017-18089-0

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DOI： 10.1177/0022034517719872

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DOI： 10.1074/jbc.M117.780866

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DOI： 10.1371/journal.pone.0177557

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DOI： 10.1038/srep45181

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DOI： 10.1038/srep23670.

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DOI： 10.1371/journal.pone.0152206.

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DOI： 10.1074/jbc.M115.674663.

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DOI： 10.1371/journal.pone.0121667

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DOI： 10.1523/JNEUROSCI.2045-11.2012

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DOI： 10.1177/0022034511424408

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DOI： 10.1016/j.bbrc.2010.12.116

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DOI： 10.1016/j.archoralbio.2010.03.011

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DOI： 10.1016/j.jpedsurg.2009.07.070

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DOI： 10.1074/jbc.M109.033464

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DOI： 10.1074/jbc.M710308200

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DOI： 10.1007/s00383-007-1977-8

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DOI： 10.1597/06-069.1

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