The Experts below are selected from a list of 258 Experts worldwide ranked by ideXlab platform
Hidemitsu Harada - One of the best experts on this subject based on the ideXlab platform.
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cell dynamics in cervical loop Epithelium during transition from crown to root implications for hertwig s epithelial root sheath formation
Journal of Periodontal Research, 2013Co-Authors: Masaaki Sakano, Keishi Otsu, Satoshi Fukumoto, Aya Yamada, Hidemitsu HaradaAbstract:Background and Objective Some clinical cases of hypoplastic tooth root are congenital. Because the formation of Hertwig's epithelial root sheath (HERS) is an important event for root development and growth, we have considered that understanding the HERS developmental mechanism contributes to elucidate the causal factors of the disease. To find integrant factors and phenomenon for HERS development and growth, we studied the proliferation and mobility of the cervical loop (CL). Material and Methods We observed the cell movement of CL by the DiI labeling and organ culture system. To examine cell proliferation, we carried out immunostaining of CL and HERS using anti-Ki67 antibody. Cell motility in CL was observed by tooth germ slice organ culture using green fluorescent protein mouse. We also examined the expression of paxillin associated with cell movement. Results Imaging using DiI labeling showed that, at the apex of CL, the Epithelium elongated in tandem with the growth of outer Enamel Epithelium (OEE). Cell proliferation assay using Ki67 immunostaining showed that OEE divided more actively than Inner Enamel Epithelium (IEE) at the onset of HERS formation. Live imaging suggested that mobility of the OEE and cells in the apex of CL were more active than in IEE. The expression of paxillin was observed strongly in OEE and the apex of CL. Conclusion The more active growth and movement of OEE cells contributed to HERS formation after reduction of the growth of IEE. The expression pattern of paxillin was involved in the active movement of OEE and HERS. The results will contribute to understand the HERS formation mechanism and elucidate the cause of anomaly root.
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the epithelial mesenchymal interaction plays a role in the maintenance of the stem cell niche of mouse incisors via fgf10 and fgf9 signaling
The Open Biotechnology Journal, 2008Co-Authors: Tamaki Yokohamatamaki, Satoshi Wakisaka, Naoki Fujiwara, Shunichi Shibata, Hidemitsu HaradaAbstract:The continuous eruption of mouse incisors throughout life is maintained by adult stem cells in the apical end. In these teeth, the continuous expression of Fgf10 in the mesenchyme plays a role in the maintenance of the epithelial stem cell compartment, referred to as the "apical bud." However, little is known about the epithelial signaling that induces and maintains Fgf10 expression. Focusing on the epithelial-mesenchymal interaction during tooth development, we thor- oughly investigated candidates expressed in the apical bud. In situ hybridization and immunostaining showed that Fgf9 mRNA and protein were detected in the basal Epithelium, stellate reticulum, and Inner Enamel Epithelium of the apical bud. Recombinant Fgf9 protein stimulated cell proliferation in cultures of apical end mesenchyme. Furthermore, Fgf9- releasing beads inhibited apoptosis in mesenchymal tissue cultures and maintained the expression of Fgf10. On the other hand, Fgf10-releasing beads induced Fgf9 expression in cultures of apical buds. Taken together, these results suggest that the stem cell niche in growing incisors is maintained by an epithelial mesenchymal interaction via Fgf9 and Fgf10 signal- ing.
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cessation of fgf10 signaling resulting in a defective dental epithelial stem cell compartment leads to the transition from crown to root formation
Development, 2006Co-Authors: Tamaki Yokohamatamaki, Hideyo Ohuchi, Satoshi Wakisaka, Hayato Ohshima, Naoki Fujiwara, Yunosuke Takada, Yasuo Ichimori, Hidemitsu HaradaAbstract:Mouse, rat and human molars begin to form root after the completion of crown formation. In these teeth, fibroblast growth factor (Fgf) 10 disappears in the transitional stage from crown formation to root. By contrast, rodent incisors and vole molars demonstrate continuous growth, owing to the formation and maintenance of a stem cell compartment by the constant expression of Fgf10. To clarify the relationship between root formation and disappearance of Fgf10, we carried out two experiments for the loss and gain of Fgf10 function. First, we examined postnatal growth in the incisors of Fgf10-deficient mice, which have the defect of a dental epithelial stem cell compartment referred to as ;apical bud', after implantation under the kidney capsule. The growth at the labial side in the mutant mice mimics the development of limited-growth teeth. 5'-Bromo-2'-deoxyuridine (BrdU) labeling and cytokeratin (CK) 14 and Notch2 immunostaining suggested that the inhibition of Inner Enamel Epithelium growth and the more-active proliferation of the outer Enamel Epithelium and/or stellate reticulum result in Hertwig's epithelial root sheath formation. Second, we examined the effects of Fgf10 overexpression in the transitional stage of molar germs, which led to the formation of apical bud involving in the inhibition of HERS formation. Taken together, these results suggest that the disappearance of Fgf10 signaling leads to the transition from crown to root formation, owing to the loss of a dental epithelial stem cell compartment.
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stratum intermedium lineage diverges from ameloblast lineage via notch signaling
Biochemical and Biophysical Research Communications, 2006Co-Authors: Hidemitsu Harada, Tamaki Yokohamatamaki, Hayato Ohshima, Yasuo Ichimori, Shintarou Kawano, Kenichi Katsube, Satoshi WakisakaAbstract:The stratum intermedium develops as flattened cell layers on the proximal side of the ameloblast layer during tooth development. However, little information is available regarding the origin and the role. In this study, we indicate that some stratum intermedium cells originate from the Inner Enamel Epithelium (IEE) in rat incisor organ cultures using DiI as a tracer. Immunohistochemical and in situ hybridization studies showed that the stratum intermedium cells express the Notch1 protein and Hes1 mRNAs, while the IEE and ameloblasts express the Jagged1. Further, we examined the role of Notch signaling using the dental epithelial cell line HAT-7. Recombinant Jagged1 protein enhanced the appearance of stratum intermedium cells in HAT-7 cultures and neutralization with an anti-Jagged1 antibody inhibited these effects. Additionally, overexpression of the Notch1 internal domain increased the number of stratum intermedium cells. We hypothesize that the stratum intermedium lineage differentiates from the ameloblast lineage via Notch signaling.
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cell dynamics in the growth and differentiation of dental Epithelium during tooth development stratum intermedium cells originated from Inner Enamel Epithelium
Journal of Hard Tissue Biology, 2005Co-Authors: Hidemitsu HaradaAbstract:The stratum intermedium develops as flattened cell layer on the proximal side of the ameloblast layer during the bell stage of tooth development. Stratum intermedium cells strongly express alkaline phosphatase (ALP) activity and have been considered to play a complementary role in the Enamel mineralization, however the origin and/or the role of these cells have not been elucidated. In the present study, we focused on the lineage of stratum intermedium cells in continuously growing rodent incisors and analyzed it by using DiI tracers experiment and using the incisors organ culture. The results indicated that some stratum intermedium cells were originated from the Inner Enamel Epithelium. Immunohistochemical and in situ hybridization studies showed that the stratum intermedium cells expressed the Notch-1, Notch-2, and Hes1, while the Inner Enamel Epithelium and ameloblasts expressed their ligands Jagged-1. Furthermore, we examined th role of Notch signaling in the development of the stratum intermedium cells by use of the dental epithelial cell line, HAT-7. Recombinant Jagged1 protein enhanced the appearance of the stratum intermedium cells in HAT-7.On the other hand, anti-sense Notch1 decreased the number of stratum intermedium cells.Taken together, we propose a hypothesis that the lineage of the stratum intermedium differentiates from the ameloblasts lineage through Notch signaling.
Hidetaka Sakai - One of the best experts on this subject based on the ideXlab platform.
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Itm2a Expression in the Developing Mouse First Lower Molar, and the Subcellular Localization of Itm2a in Mouse Dental Epithelial Cells
2016Co-Authors: Makiko Kihara, Tamotsu Kiyoshima, Hiroko Wada, Kana Hasegawa, Kengo Nagata, Hiroaki Fujiwara, Hirotaka Someya, Ichiro Takahashi, Hidetaka SakaiAbstract:Itm2a is a type II transmembrane protein with a BRICHOS domain. We investigated the temporospatial mRNA and protein expression patterns of Itm2a in the developing lower first molar, and examined the subcellular localization of Itm2a in murine dental epithelial (mDE6) cells. From the initiation to the bud stage, the in situ and protein signals of Itm2a were not detected in either the dental epithelial or mesenchymal cells surrounding the tooth bud. However, at the bell stage, these signals of Itm2a were primarily observed in the Inner Enamel Epithelium of the Enamel organ. After the initiation of the matrix formation, strong signals were detected in ameloblasts and odontoblasts. Itm2a showed a punctate pattern in the cytoplasm of the mDE6 cells. The perinuclear-localized Itm2a displayed a frequent overlap with the Golgi apparatus marker, GM130. A tiny amount of Itm2a was colocalized with lysosomes and endoplasmic reticulum. Minimal or no overlap between the Itm2a-EGFP signals with the other organelle markers for endoplasmic reticulum, lysosome and mitochondria used in this study noted in the cytoplasm. These findings suggest that Itm2a may play a role in cell differentiation during odontogenesis, rather than during the initiation of tooth germ formation, and may be related to the targeting of protein
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Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) expression and possible function in mouse tooth germ development
Journal of Molecular Histology, 2016Co-Authors: Kana Hasegawa, Hiroko Wada, Hidetaka Sakai, Kengo Nagata, Hiroaki Fujiwara, Naohisa Wada, Hirotaka Someya, Yurie Mikami, Tamotsu KiyoshimaAbstract:Abnormal expression of Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is involved in the pathogenesis of FSHD. FRG1 is also important for the normal muscular and vascular development. Our previous study showed that FRG1 is one of the highly expressed genes in the mandible on embryonic day 10.5 (E10.5) than on E12.0. In this study, we investigated the temporospatial expression pattern of FRG1 mRNA and protein during the development of the mouse lower first molar, and also evaluated the subcellular localization of the FRG1 protein in mouse dental epithelial (mDE6) cells. The FRG1 expression was identified in the dental epithelial and mesenchymal cells at the initiation and bud stages. It was detected in the Inner Enamel Epithelium at the cap and early bell stages. At the late bell and root formation stages, these signals were detected in ameloblasts and odontoblasts during the formation of Enamel and dentin matrices, respectively. The FRG1 protein was localized in the cytoplasm in the mouse tooth germ in vivo, while FRG1 was detected predominantly in the nucleus and faintly in the cytoplasm in mDE6 cells in vitro. In mDE6 cells treated with bone morphogenetic protein 4 (BMP4), the protein expression of FRG1 increased in cytoplasm, suggesting that FRG1 may translocate to the cytoplasm. These findings suggest that FRG1 is involved in the morphogenesis of the tooth germ, as well as in the formation of Enamel and dentin matrices and that FRG1 may play a role in the odontogenesis in the mouse following BMP4 stimulation.
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itm2a expression in the developing mouse first lower molar and the subcellular localization of itm2a in mouse dental epithelial cells
PLOS ONE, 2014Co-Authors: Makiko Kihara, Tamotsu Kiyoshima, Hiroko Wada, Kana Hasegawa, Kengo Nagata, Hiroaki Fujiwara, Hirotaka Someya, Ichiro Takahashi, Hidetaka SakaiAbstract:Itm2a is a type II transmembrane protein with a BRICHOS domain. We investigated the temporospatial mRNA and protein expression patterns of Itm2a in the developing lower first molar, and examined the subcellular localization of Itm2a in murine dental epithelial (mDE6) cells. From the initiation to the bud stage, the in situ and protein signals of Itm2a were not detected in either the dental epithelial or mesenchymal cells surrounding the tooth bud. However, at the bell stage, these signals of Itm2a were primarily observed in the Inner Enamel Epithelium of the Enamel organ. After the initiation of the matrix formation, strong signals were detected in ameloblasts and odontoblasts. Itm2a showed a punctate pattern in the cytoplasm of the mDE6 cells. The perinuclear-localized Itm2a displayed a frequent overlap with the Golgi apparatus marker, GM130. A tiny amount of Itm2a was colocalized with lysosomes and endoplasmic reticulum. Minimal or no overlap between the Itm2a-EGFP signals with the other organelle markers for endoplasmic reticulum, lysosome and mitochondria used in this study noted in the cytoplasm. These findings suggest that Itm2a may play a role in cell differentiation during odontogenesis, rather than during the initiation of tooth germ formation, and may be related to the targeting of proteins associated with Enamel and dentin matrices in the secretory pathway.
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The Itm2a protein expression in the tooth germ on E15–E18.
2014Co-Authors: Makiko Kihara, Tamotsu Kiyoshima, Hiroko Wada, Kana Hasegawa, Kengo Nagata, Hiroaki Fujiwara, Hirotaka Someya, Ichiro Takahashi, Hidetaka SakaiAbstract:A. At the cap stage (E15), the Inner Enamel Epithelium (black arrows) exhibited the expression of the Itm2a protein, but the signal was weak. A strong signal was observed in the outer Enamel Epithelium (red arrows) and dental sac (blue arrowheads). B. At the cap stage (E16), the Inner Enamel Epithelium (black arrows) exhibited a strong signal for the Itm2a protein, as did the outer Enamel Epithelium (red arrows). C & D. At the early bell stage (E17–18), the Itm2a protein was detected in the outer Enamel Epithelium cells (red arrows), the Inner Enamel Epithelium cells (black arrows) and dental sac (blue arrowheads). E & F. The yellow- and green-boxed areas in B are respectively shown at a higher magnification. G. The boxed areas in D are shown at a higher magnification. Li; lingual side, Bu; buccal side. Scale bars; 100 µm (A, B), 200 µm (C, D), 50 µm (E–G).
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in situ expression of the mitochondrial atpase6 gene in the developing tooth germ of the mouse lower first molar
Journal of Molecular Histology, 2011Co-Authors: Jun-ya Honda, Ieyoshi Kobayashi, Tamotsu Kiyoshima, Hiroko Wada, Kengo Nagata, Hiroaki Fujiwara, Yukiko Ookuma, Ichiro Takahashi, Maho Shiotsuka, Hidetaka SakaiAbstract:We previously performed cDNA subtraction between the mouse mandibles on embryonic day 10.5 (E10.5) in the pre-initiation stage of the odontogenesis and E12.0 in the late initiation stage to identify genes expressed at its beginning. Adenosine triphosphate synthase subunit a (Atpase6) is one of the highly expressed genes in the E12.0 mandible including tooth germs. In situ hybridization was conducted using the mouse mandibular first molar from E10.5 to E18.0 to determine the precise expression patterns of Atpase6 mRNA in the developing tooth germ. Atpase6 mRNA was strongly expressed in the presumptive dental Epithelium and the underlying mesenchyme at E10.5, and in the thickened dental Epithelium at E12.0 and E13.0. Strong in situ signals were observed in the Epithelium at E14.0, and in the Enamel organ excluded the area of the primary Enamel knot at E15.0. Atpase6 was strongly expressed in the Inner Enamel Epithelium, the adjacent stratum intermedium, and the outer Enamel Epithelium in the cervical loops from E16.0 to E18.0. In addition, strong Atpase6 signals were coincidently demonstrated in various developing cranio-facial organs. These results suggest that Atpase6 participates in the high energy-utilizing functions of the cells related to the initiation and the development of the tooth germ as well as those of the other cranio-facial organs.
Hayato Ohshima - One of the best experts on this subject based on the ideXlab platform.
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msx2 prevents stratified squamous Epithelium formation in the Enamel organ
Journal of Dental Research, 2018Co-Authors: Mitsushiro Nakatomi, Hiroko Idayonemochi, C Nakatomi, Kotaro Saito, Shinichi Kenmotsu, Richard L Maas, Hayato OhshimaAbstract:Tooth Enamel is manufactured by the Inner Enamel Epithelium of the multilayered Enamel organ. Msx2 loss-of-function mutation in a mouse model causes an abnormal accumulation of epithelial cells in ...
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Expression of Panx3 in tooth germ.
2017Co-Authors: Tsutomu Iwamoto, Yoshihiko Yamada, Keigo Yoshizaki, Takashi Nakamura, Hayato Ohshima, Masahiro Saito, Masaki Ishikawa, Asuna Sugimoto, Hiroko Ida-yonemochi, Satoshi FukumotoAbstract:(A) RT-PCR analysis (upper three panels) and northern blotting analysis (lower three panels) using RNA from postnatal day 1 (P1) mouse tissues (molar, incisor, brain, lung, heart, liver, skin, kidney, and bone). (B) RT-PCR analysis using the dental Epithelium (DE) and mesenchyme (DM), dissected from P1 mouse tooth germ. (C) Immnostaining with anti-Panx3 antibody (red) and DAPI nuclear staining (blue). (D) Light microscopy images of semi-thin sections stained with methylene blue for immunoelectron microscopy. Panx3 is present in the preodontoblasts (pre-od) but not in the odontoblasts (od). (E) Immunoelectron microscopy images of the Panx3 protein in preodontoblasts from P1 incisors showing labeling in the preodontoblasts (upper panels) and at the cell-cell contact sites (lower panels). dp; dental papilla, iee; Inner Enamel Epithelium, si; stratum intermedium, BM; basement membrane.
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patterns of morphological variation in Enamel dentin junction and outer Enamel surface of human molars
Journal of Anatomy, 2014Co-Authors: Wataru Morita, Hayato Ohshima, Wataru Yano, Tomohito Nagaoka, Mikiko Abe, Masato NakatsukasaAbstract:Tooth crown patterning is governed by the growth and folding of the Inner Enamel Epithelium (IEE) and the following Enamel deposition forms outer Enamel surface (OES). We hypothesized that overall dental crown shape and covariation structure are determined by processes that configurate shape at the Enamel–dentine junction (EDJ), the developmental vestige of IEE. This this hypothesis was tested by comparing patterns of morphological variation between EDJ and OES in human permanent maxillary first molar (UM1) and deciduous second molar (um2). Using geometric morphometric methods, we described morphological variation and covariation between EDJ and OES, and evaluated the strength of two components of phenotypic variability, canalization and morphological integration, in addition to the relevant evolutionary flexibility, i.e. the ability to respond to selective pressure. The strength of covariation between EDJ and OES was greater in um2 than in UM1, and the way that multiple traits covary between EDJ and OES was different between these teeth. The variability analyses showed that EDJ had less shape variation and a higher level of morphological integration than OES, which indicated that canalization and morphological integration acted as developmental constraints. These tendencies were greater in UM1 than in um2. On the other hand, EDJ and OES had a comparable level of evolvability in these teeth. Amelogenesis could play a significant role in tooth shape and covariation structure, and its influence was not constant among teeth, which may be responsible for the differences in the rate and/or period of Enamel formation.
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cessation of fgf10 signaling resulting in a defective dental epithelial stem cell compartment leads to the transition from crown to root formation
Development, 2006Co-Authors: Tamaki Yokohamatamaki, Hideyo Ohuchi, Satoshi Wakisaka, Hayato Ohshima, Naoki Fujiwara, Yunosuke Takada, Yasuo Ichimori, Hidemitsu HaradaAbstract:Mouse, rat and human molars begin to form root after the completion of crown formation. In these teeth, fibroblast growth factor (Fgf) 10 disappears in the transitional stage from crown formation to root. By contrast, rodent incisors and vole molars demonstrate continuous growth, owing to the formation and maintenance of a stem cell compartment by the constant expression of Fgf10. To clarify the relationship between root formation and disappearance of Fgf10, we carried out two experiments for the loss and gain of Fgf10 function. First, we examined postnatal growth in the incisors of Fgf10-deficient mice, which have the defect of a dental epithelial stem cell compartment referred to as ;apical bud', after implantation under the kidney capsule. The growth at the labial side in the mutant mice mimics the development of limited-growth teeth. 5'-Bromo-2'-deoxyuridine (BrdU) labeling and cytokeratin (CK) 14 and Notch2 immunostaining suggested that the inhibition of Inner Enamel Epithelium growth and the more-active proliferation of the outer Enamel Epithelium and/or stellate reticulum result in Hertwig's epithelial root sheath formation. Second, we examined the effects of Fgf10 overexpression in the transitional stage of molar germs, which led to the formation of apical bud involving in the inhibition of HERS formation. Taken together, these results suggest that the disappearance of Fgf10 signaling leads to the transition from crown to root formation, owing to the loss of a dental epithelial stem cell compartment.
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stratum intermedium lineage diverges from ameloblast lineage via notch signaling
Biochemical and Biophysical Research Communications, 2006Co-Authors: Hidemitsu Harada, Tamaki Yokohamatamaki, Hayato Ohshima, Yasuo Ichimori, Shintarou Kawano, Kenichi Katsube, Satoshi WakisakaAbstract:The stratum intermedium develops as flattened cell layers on the proximal side of the ameloblast layer during tooth development. However, little information is available regarding the origin and the role. In this study, we indicate that some stratum intermedium cells originate from the Inner Enamel Epithelium (IEE) in rat incisor organ cultures using DiI as a tracer. Immunohistochemical and in situ hybridization studies showed that the stratum intermedium cells express the Notch1 protein and Hes1 mRNAs, while the IEE and ameloblasts express the Jagged1. Further, we examined the role of Notch signaling using the dental epithelial cell line HAT-7. Recombinant Jagged1 protein enhanced the appearance of stratum intermedium cells in HAT-7 cultures and neutralization with an anti-Jagged1 antibody inhibited these effects. Additionally, overexpression of the Notch1 internal domain increased the number of stratum intermedium cells. We hypothesize that the stratum intermedium lineage differentiates from the ameloblast lineage via Notch signaling.
Aya Yamada - One of the best experts on this subject based on the ideXlab platform.
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single cell rna sequencing from mouse incisor reveals dental epithelial cell type specific genes
Frontiers in Cell and Developmental Biology, 2020Co-Authors: Yuta Chiba, Aya Yamada, Kan Saito, Daniel Martin, Erich T Boger, Craig Rhodes, Keigo Yoshizaki, Takashi Nakamura, Robert J Morell, Yoshihiko YamadaAbstract:Dental epithelial stem cells give rise to four types of dental epithelial cells: Inner Enamel Epithelium (IEE), outer Enamel Epithelium (OEE), stratum intermedium (SI), and stellate reticulum (SR). IEE cells further differentiate into Enamel-forming ameloblasts, which play distinct roles, and are essential for Enamel formation. These are conventionally classified by their shape, although their transcriptome and biological roles are yet to be fully understood. Here, we aimed to use single-cell RNA sequencing to clarify the heterogeneity of dental epithelial cell types. Unbiased clustering of 6,260 single cells from incisors of postnatal day 7 mice classified them into two clusters of ameloblast, IEE/OEE, SI/SR, and two mesenchymal populations. Secretory-stage ameloblasts expressed Amel and Enam were divided into Dspp + and Ambn + ameloblasts. Pseudo-time analysis indicated Dspp + ameloblasts differentiate into Ambn + ameloblasts. Further, Dspp and Ambn could be stage-specific markers of ameloblasts. Gene ontology analysis of each cluster indicated potent roles of cell types: OEE in the regulation of tooth size and SR in the transport of nutrients. Subsequently, we identified novel dental epithelial cell marker genes, namely Pttg1, Atf3, Cldn10, and Krt15. The results not only provided a resource of transcriptome data in dental cells but also contributed to the molecular analyses of Enamel formation.
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Expression patterns of PKP1 and ZO-1 in P1 incisors.
2016Co-Authors: Kanako Miyazaki, Aya Yamada, Kan Saito, Keigo Yoshizaki, Masaki Ishikawa, Chieko Arai, Han Xue, Keita Funada, Naoto Haruyama, Yoshihiko YamadaAbstract:A, PKP1 (green), ZO-1 (green), and AMBN (red) expression in cervical loop of P1 incisors at pre-secretory and secretory stages, as determined by immunocytochemistry. Nuclei were stained with DAPI (blue). iee, Inner Enamel Epithelium; oee, outer Enamel Epithelium; sr, stellate reticulum; si, stratum intermedium; dp, dental papilla. B, CLDE cells were cultured after transfection with control or Pkp1 siRNA in the presence of Ca2+ for 48 h, then the expressions of AMBN, ZO-1, Desmoplakin, PKP1, and β-catenin were assessed by western blotting. GAPDH was used as a loading control. C, CLDE cells were cultured after transfection with control or Pkp1 siRNA in the presence of Ca2+ for 48 h. Differential interference contrast image (left) and immunolabeling of keratin 14 (green; center panel and enlarged in right panel) are shown.
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PKP1 expressed in developing tooth germs.
2016Co-Authors: Kanako Miyazaki, Aya Yamada, Kan Saito, Keigo Yoshizaki, Masaki Ishikawa, Chieko Arai, Han Xue, Keita Funada, Naoto Haruyama, Yoshihiko YamadaAbstract:A, Differentially expressed genes identified by microarray analysis of teeth as compared to whole embryos at E14. Highlighted plot indicates Pkp1. Red and blue plots represent up- and down-regulated genes, respectively. B, qRT-PCR analysis of Pkp1 expression in teeth, skin, lungs, livers, kidneys, hearts, eyes, and brains of E14.5 embryos after normalization to Gapdh mRNA expression. C, qRT-PCR analysis of Pkp1 expression in teeth obtained from E11 to P7 a normalization to Gapdh mRNA expression. D, PKP1 (green) expression in E13, E14, E16, and P1 mice, as detected by immunocytochemistry. Broken lines represent basement membrane of teeth. Enlarged images are shown below each panel. de, dental Epithelium; dm, dental mesenchyme; iee, Inner Enamel Epithelium; ek, Enamel knot; sr, stellate reticulum; si, stratum intermedium; dp, dental papilla.
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Expression of collagen IV, laminin β1γ1, and ameloblastin in incisors from WT and Itgβ1CKO mice.
2015Co-Authors: Kan Saito, Aya Yamada, Keigo Yoshizaki, Takashi Nakamura, Emiko Fukumoto, Kenji Yuasa, Tsutomu Iwamoto, Masahiro Saito, Satoshi FukumotoAbstract:A. The lower incisor of a wild type (WT) mouse was analyzed using immunostaining with anti-collagen IV and anti-laminin β1γ1 antibodies. Laminin β1γ1 was expressed in the basal membrane, papillary layer, and immature ameloblasts. B, C. The lower incisors of WT and Itgβ1 conditional knockout (CKO) mouse were immunostained by anti-ameloblastin and anti-laminin β1γ1 antibodies. Ameloblastin was detected in the incisor of a WT but not a Itgβ1CKO mouse. Laminin β1γ1 was detected in Itgβ1CKO (asterisk) but not WT incisors. iee, Inner Enamel Epithelium; am, ameloblasts; od, odontoblasts.
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Analysis of fibronectin expression during tooth development.
2015Co-Authors: Kan Saito, Aya Yamada, Keigo Yoshizaki, Takashi Nakamura, Emiko Fukumoto, Kenji Yuasa, Tsutomu Iwamoto, Masahiro Saito, Satoshi FukumotoAbstract:A. Immunofluorescence analysis of fibronectin expression using an anti-fibronectin antibody in sagittal sections from 6-week-old mouse incisors. The incisors were separated into the apical bud (AB), presecretory (PS), secretory (S), early maturation (EM), and late maturation (LM) stages. B. Immunofluorescence and immunohistochemical results are shown at a higher magnification. Fibronectin was expressed in the basal lamina at the S stage and in the papillary layer at the LM stage. C. Fibronectin mRNA was detected using in situ hybridization in the basal lamina, pre-odontoblasts, pre-ameloblasts from PS to S (PS-S) and LM ameloblasts, with lower expression seen during the EM stage. D. The expression levels of fibronectin and amelogenin were investigated by real-time PCR using PS, S, EM, and LM dental epithelial cells separated from the incisor. E. The expression of fibronectin in dental epithelial (DE) and dental mesenchymal (DM) cells isolated from the molars of P1 and P7 mice (n = 5) was investigated by real-time PCR. Amelogenin mRNA expression increased in DE cells, while fibronectin mRNA expression decreased with differentiation. iee, Inner Enamel Epithelium; dp, dental pulp; si, stratum intermedia; am, ameloblasts; pam, pre-ameloblasts; od, odontoblasts; pod, pre-odontoblasts; pl, papillary layer.
Satoshi Wakisaka - One of the best experts on this subject based on the ideXlab platform.
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the epithelial mesenchymal interaction plays a role in the maintenance of the stem cell niche of mouse incisors via fgf10 and fgf9 signaling
The Open Biotechnology Journal, 2008Co-Authors: Tamaki Yokohamatamaki, Satoshi Wakisaka, Naoki Fujiwara, Shunichi Shibata, Hidemitsu HaradaAbstract:The continuous eruption of mouse incisors throughout life is maintained by adult stem cells in the apical end. In these teeth, the continuous expression of Fgf10 in the mesenchyme plays a role in the maintenance of the epithelial stem cell compartment, referred to as the "apical bud." However, little is known about the epithelial signaling that induces and maintains Fgf10 expression. Focusing on the epithelial-mesenchymal interaction during tooth development, we thor- oughly investigated candidates expressed in the apical bud. In situ hybridization and immunostaining showed that Fgf9 mRNA and protein were detected in the basal Epithelium, stellate reticulum, and Inner Enamel Epithelium of the apical bud. Recombinant Fgf9 protein stimulated cell proliferation in cultures of apical end mesenchyme. Furthermore, Fgf9- releasing beads inhibited apoptosis in mesenchymal tissue cultures and maintained the expression of Fgf10. On the other hand, Fgf10-releasing beads induced Fgf9 expression in cultures of apical buds. Taken together, these results suggest that the stem cell niche in growing incisors is maintained by an epithelial mesenchymal interaction via Fgf9 and Fgf10 signal- ing.
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cessation of fgf10 signaling resulting in a defective dental epithelial stem cell compartment leads to the transition from crown to root formation
Development, 2006Co-Authors: Tamaki Yokohamatamaki, Hideyo Ohuchi, Satoshi Wakisaka, Hayato Ohshima, Naoki Fujiwara, Yunosuke Takada, Yasuo Ichimori, Hidemitsu HaradaAbstract:Mouse, rat and human molars begin to form root after the completion of crown formation. In these teeth, fibroblast growth factor (Fgf) 10 disappears in the transitional stage from crown formation to root. By contrast, rodent incisors and vole molars demonstrate continuous growth, owing to the formation and maintenance of a stem cell compartment by the constant expression of Fgf10. To clarify the relationship between root formation and disappearance of Fgf10, we carried out two experiments for the loss and gain of Fgf10 function. First, we examined postnatal growth in the incisors of Fgf10-deficient mice, which have the defect of a dental epithelial stem cell compartment referred to as ;apical bud', after implantation under the kidney capsule. The growth at the labial side in the mutant mice mimics the development of limited-growth teeth. 5'-Bromo-2'-deoxyuridine (BrdU) labeling and cytokeratin (CK) 14 and Notch2 immunostaining suggested that the inhibition of Inner Enamel Epithelium growth and the more-active proliferation of the outer Enamel Epithelium and/or stellate reticulum result in Hertwig's epithelial root sheath formation. Second, we examined the effects of Fgf10 overexpression in the transitional stage of molar germs, which led to the formation of apical bud involving in the inhibition of HERS formation. Taken together, these results suggest that the disappearance of Fgf10 signaling leads to the transition from crown to root formation, owing to the loss of a dental epithelial stem cell compartment.
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stratum intermedium lineage diverges from ameloblast lineage via notch signaling
Biochemical and Biophysical Research Communications, 2006Co-Authors: Hidemitsu Harada, Tamaki Yokohamatamaki, Hayato Ohshima, Yasuo Ichimori, Shintarou Kawano, Kenichi Katsube, Satoshi WakisakaAbstract:The stratum intermedium develops as flattened cell layers on the proximal side of the ameloblast layer during tooth development. However, little information is available regarding the origin and the role. In this study, we indicate that some stratum intermedium cells originate from the Inner Enamel Epithelium (IEE) in rat incisor organ cultures using DiI as a tracer. Immunohistochemical and in situ hybridization studies showed that the stratum intermedium cells express the Notch1 protein and Hes1 mRNAs, while the IEE and ameloblasts express the Jagged1. Further, we examined the role of Notch signaling using the dental epithelial cell line HAT-7. Recombinant Jagged1 protein enhanced the appearance of stratum intermedium cells in HAT-7 cultures and neutralization with an anti-Jagged1 antibody inhibited these effects. Additionally, overexpression of the Notch1 internal domain increased the number of stratum intermedium cells. We hypothesize that the stratum intermedium lineage differentiates from the ameloblast lineage via Notch signaling.
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expression of cytokeratin 14 in ameloblast lineage cells of the developing tooth of rat both in vivo and in vitro
Archives of Oral Biology, 1996Co-Authors: Makoto J Tabata, Satoshi Wakisaka, Tatsushi Matsumura, Jiguang Liu, Kojiro KurisuAbstract:In the search for a cell marker useful for studying tooth development, immunohistochemical studies using antibodies against cytokeratin 14 (K14), c-Met/hepatocyte growth factor receptor and amelogenin were carried out in the developing tooth of the newborn rat and in primary cultured cells of the ameloblast lineage, including Inner Enamel Epithelium cells, preameloblasts and ameloblasts, prepared from the mandibular incisors of postnatal 7-day-old rats. The appearance of K14 was cell- and differentiation-stage specific, i.e. there was a weak expression signal within Inner Enamel epithelial cells that were in the proliferating stage, and there were strong signals within preameloblasts and ameloblasts that were in the post-proliferating and amelogenesis stages, respectively. In the culture system, c-Met appeared in all cells, whereas K14 and amelogenin appeared mainly in clustered cells that were considered to be in the post-proliferating stage. K14 was detected earlier than amelogenin, and it was also confirmed by immunofluorostaining that c-Met, K14 and amelogenin were coexpressed in ameloblasts. These findings indicate that K14 is a good new marker for ameloblast-lineage cells during rat tooth development both in vivo and in vitro.
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hepatocyte growth factor is involved in the morphogenesis of tooth germ in murine molars
Development, 1996Co-Authors: Makoto J Tabata, Satoshi Wakisaka, Tatsushi Matsumura, Jiguang Liu, Kenji Kim, Kazuo Yamashita, J Kato, Masahiro Iwamoto, Kunio Matsumoto, Toshikazu NakamuraAbstract:The patterns of gene expression for hepatocyte growth factor (HGF) and its receptor, c-Met, were revealed in the tooth germ of rat mandibular molars using RT-PCR. In situ hybridization demonstrated that the HGF gene was expressed only in the cells of the dental papilla of the tooth germ in vivo. The characteristic temporospatial distribution of HGF and c-Met during germ development was revealed using immunohistochemical studies in vivo. In order to demonstrate the functional role played by HGF in tooth development, HGF translation arrest by antisense phosphorothioate oligodeoxynucleotide (ODN) was carried out in vitro. In the control experiment, explants of tooth germs from embryonic 14 day mice were cultured in a modification of Trowell's system under serum-free and chemically defined conditions for two weeks. Other explants were cultured with 15mer antisense or sense ODN targeted to the HGF mRNA. Both the control and the sense-treated explants showed normal histological structure, as observed in vivo. On the other hand, antisense-treated explants exhibited an abnormal structure in which the Enamel organs were surrounded by a thin layer of dentin and dental papilla, appearing 'inside-out' compared to the control and sense-treated explants, although the cytodifferentiation of ameloblasts and odontoblasts was not inhibited. The explants treated with recombinant human HGF combined with antisense ODN showed normal development, indicating that exogenous HGF rescued the explants from the abnormal structure caused by antisense ODN. The findings of a BrdU incorporation experiment suggested that the imbalance between the proliferation activity of the Inner Enamel Epithelium and that of the dental papilla caused by HGF translation arrest results in the abnormal structure of the tooth germ. These results indicate that HGF is involved in the morphogenesis of the murine molar.