Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Coordinated growth of multiple tissues is fundamental to shaping our body, but the underlying mechanisms remain underexplored. In zebrafish embryos, midline tissues composed of the notochord, floorplate, and hypochord elongate synchronously with their lengths aligned. We show that floorplate and hypochord cells collectively migrate posteriorly along the nascent notochord extracellular matrix as it extends posteriorly, maintaining the tripartite configuration. Fibroblast growth factor-mediated migration in a spatially graded manner causes cell stretching, which triggers Yap-dependent proliferation and controls floorplate and hypochord growth. Supported by mathematical modeling, we further suggest that their growth is fine-tuned by mechanical tethering to the notochord via cadherin 2 at the posterior end. We propose that the notochord instructs and sustains the tripartite formation via leader-follower formation control, a strategy from engineering that spatially organizes multiple agents to coordinate the growth of the midline tissues.

DOI： 10.1126/sciadv.ads2310

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

Congenital heart defects (CHDs) are common birth defects attributed to genetic and environmental factors such as pharmaceuticals and chemicals. Identifying modifiable environmental factors and understanding their impact on heart development is crucial for mitigating chemical-induced CHDs. Given the increasing number of chemical agents, efficient high-throughput systems are essential to evaluate their teratogenic potential during cardiovascular development, which is a major concern for chemical safety. In this study, we developed three transgenic zebrafish reporter lines, myl7: EGFP, kdrl: MRFP and gata1: MKate2, which enable real-time visualization of myocardial and endocardial development and cardiac function based on blood flow. These transgenic embryos were used to investigate the teratogenic effects of chemicals well known to induce heart defects in mammals, including humans. Our real-time imaging revealed that the teratogens induced significant malformations in cardiac morphogenesis, including abnormal heart tube formation, incomplete cardiac looping, and reduced heart chamber size. These teratogens also disrupted the expression of cardiac progenitor markers, suggesting impaired cardiac progenitor development. These defects were detected at the early stages (4-48 hours post-fertilization), suggesting that the stages of progenitor development to heart looing were most susceptible to teratogen exposure, ie the critical period for teratogen-induced heart defects. Functional defects, such as impaired blood flow, were observed using real-time imaging of the gata1-reporter line. This study demonstrates the utilization of transgenic zebrafish embryo models for high-throughput teratogenicity testing, which also allows us to analyze the mechanisms underlying chemical-induced heart defects. Therefore, our zebrafish models would contribute to the identification and reduction of risks associated with CHDs.

DOI： 10.1093/toxsci/kfaf083

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Dorsoventral (DV) patterning is fundamental to vertebrate development, organizing the entire body across different germ layers. Although early DV axis formation, centered on the Spemann-Mangold organizer through the BMP activity gradient, has been extensively studied, the mechanisms shaping DV traits during later development remain largely unexplored. In this review, we highlight recent findings, especially from studies involving the Double anal fin (Da) spontaneous mutant of the small teleost medaka (Oryzias latipes), focusing on the roles of zic1 and zic4 (zic1/zic4) in regulating late DV patterning. These genes establish the dorsal domain of the trunk by converting the initial BMP gradient into distinct on/off spatial compartments within somites and their derivatives, acting as selector genes that define dorsal-specific traits, including myotome structure, body shape, and dorsal fin development. We also discuss how the zic-mediated dorsal domain is established and maintained from embryogenesis through adulthood. Furthermore, we provide evidence that zic-dependent action on the dorsal characteristics is dosage-dependent. We propose that the zic1/zic4-mediated DV patterning mechanism may represent a conserved regulatory framework that has been adapted to support the diverse body plans observed across vertebrate species.

DOI： 10.1016/j.cdev.2025.204012

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Tissue elongation is a fundamental morphogenetic process to construct complex embryonic structures. In zebrafish, somites rapidly elongate in both dorsal and ventral directions, transforming from a cuboidal to a V-shape within a few hours of development. Despite its significance, the cellular behaviors that directly lead to somite elongation have not been examined at single-cell resolution. Here, we describe the motion and shapes of all cells composing the dorsal half of the somite in three-dimensional space using lightsheet microscopy. We identified two types of cell movements-in horizontal and dorsal directions-that occur simultaneously within individual cells, creating a complex, twisted flow of cells during somite elongation. Chemical inhibition of Sdf1 signaling disrupted the collective movement in both directions and inhibited somite elongation, suggesting that Sdf1 signaling is crucial for this cell flow. Furthermore, three-dimensional computational modeling suggested that horizontal cell rotation accelerates the perpendicular elongation of the somite along the dorsoventral axis. Together, our study offers novel insights into the role of collective cell migration in tissue morphogenesis, which proceeds dynamically in the three-dimensional space of the embryo.

DOI： 10.1016/j.cdev.2024.203969

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

Limb reduction has occurred multiple times in tetrapod history. Among ratites, wing reductions range from mild vestigialization to complete loss, with emus (Dromaius novaehollandiae) serving as a model for studying the genetic mechanisms behind limb reduction. Here, we explore the developmental mechanisms underlying wing reduction in emu. Our analyses reveal that immobilization resulting from the absence of distal muscles contributes to skeletal shortening, fusion and left-right intraindividual variation. Expression analysis and single cell-RNA sequencing identify muscle progenitors displaying a dual lateral plate mesodermal and myogenic signature. These cells aggregate at the proximal region of wing buds and undergo cell death. We propose that this cell death, linked to the lack of distal muscle masses, underlines the morphological features and variability in skeletal elements due to reduced mechanical loading. Our results demonstrate that differential mobility during embryonic development may drive morphological diversification in vestigial structures.

DOI： 10.1038/s41467-024-52203-x

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

Craniofacial anomalies are one of the most frequent birth defects worldwide and are often caused by genetic and environmental factors such as pharmaceuticals and chemical agents. Although identifying adverse outcome pathways (AOPs) is a central issue for evaluating the teratogenicity, the AOP causing craniofacial anomalies has not been identified. Recently, zebrafish has gained interest as an emerging model for predicting teratogenicity because of high throughput, cost-effectiveness and availability of various tools for examining teratogenic mechanisms. Here, we established zebrafish sox10-EGFP reporter lines to visualize cranial neural crest cells (CNCCs) and have identified the AOPs for craniofacial anomalies. When we exposed the transgenic embryos to teratogens that were reported to cause craniofacial anomalies in mammals, CNCC migration and subsequent morphogenesis of the first pharyngeal arch were impaired at 24 hours post-fertilization. We also found that cell proliferation and apoptosis of the migratory CNCCs were disturbed, which would be key events of the AOP. From these results, we propose that our sox10-EGFP reporter lines serve as a valuable model for detecting craniofacial skeletal abnormalities, from early to late developmental stages. Given that the developmental process of CNCCs around this stage is highly conserved between zebrafish and mammals, our findings can be extrapolated to mammalian craniofacial development and thus help in predicting craniofacial anomalies in human.

DOI： 10.1093/toxsci/kfad078

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

The left-right symmetry breaking of vertebrate embryos requires nodal flow. However, the molecular mechanisms that mediate the asymmetric gene expression regulation under nodal flow remain elusive. Here, we report that heat shock factor 1 (HSF1) is asymmetrically activated in the Kupffer's vesicle of zebrafish embryos in the presence of nodal flow. Deficiency in HSF1 expression caused a significant situs inversus and disrupted gene expression asymmetry of nodal signaling proteins in zebrafish embryos. Further studies demonstrated that HSF1 is a mechanosensitive protein. The mechanical sensation ability of HSF1 is conserved in a variety of mechanical stimuli in different cell types. Moreover, cilia and Ca2+-Akt signaling axis are essential for the activation of HSF1 under mechanical stress in vitro and in vivo. Considering the conserved expression of HSF1 in organisms, these findings unveil a fundamental mechanism of gene expression regulation by mechanical clues during embryonic development and other physiological and pathological transformations.

DOI： 10.1016/j.isci.2023.107864

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

Anterior and posterior paired appendages of vertebrates are notable examples of heterochrony in the relative timing of their development. In teleosts, posterior paired appendages (pelvic fin buds) emerge much later than their anterior paired appendages (pectoral fin buds). Pelvic fin buds of zebrafish (Danio rerio) appear at 3 weeks post-fertilization (wpf) during the larva-to-juvenile transition (metamorphosis), whereas pectoral fin buds arise from the lateral plate mesoderm on the yolk surface at the embryonic stage. Here we explored the mechanism by which presumptive pelvic fin cells maintain their fate, which is determined at the embryonic stage, until the onset of metamorphosis. Expression analysis revealed that transcripts of pitx1, one of the key factors for the development of posterior paired appendages, became briefly detectable in the posterior lateral plate mesoderm at early embryonic stages. Further analysis indicated that the pelvic fin-specific pitx1 enhancer was in the poised state at the larval stage and is activated at the juvenile stage. We discuss the implications of these findings for the heterochronic development of pelvic fin buds.

DOI： 10.3389/fcell.2023.1170691

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Cellular protrusions are fundamental structures for a wide variety of cellular behaviors, such as cell migration, cell-cell interaction, and signal reception. Visualization of cellular protrusions in living cells can be achieved by labeling of cytoskeletal actin with genetically encoded fluorescent probes. Here, we describe a detailed experimental procedure to visualize cellular protrusions in medaka embryos, which consists of the following steps: preparation of Actin-Chromobody-GFP and α-bungarotoxin mRNAs for actin labeling and immobilization of the embryo, respectively; microinjection of the mRNAs into embryos in a mosaic fashion to sparsely label individual cells; removal of the hard chorion, which hampers observation; and visualization of cellular protrusions in the embryo with a confocal microscope. Overall, our protocol provides a simple method to reveal cellular protrusions in vivo by confocal microscopy.

DOI： 10.21769/BioProtoc.4710

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The dorsal axial muscles, or epaxial muscles, are a fundamental structure covering the spinal cord and vertebrae, as well as mobilizing the vertebrate trunk. To date, mechanisms underlying the morphogenetic process shaping the epaxial myotome are largely unknown. To address this, we used the medaka zic1/zic4-enhancer mutant Double anal fin (Da), which exhibits ventralized dorsal trunk structures resulting in impaired epaxial myotome morphology and incomplete coverage over the neural tube. In wild type, dorsal dermomyotome (DM) cells reduce their proliferative activity after somitogenesis. Subsequently, a subset of DM cells, which does not differentiate into the myotome population, begins to form unique large protrusions extending dorsally to guide the epaxial myotome dorsally. In Da, by contrast, DM cells maintain the high proliferative activity and mainly form small protrusions. By combining RNA- and ChIP-sequencing analyses, we revealed direct targets of Zic1, which are specifically expressed in dorsal somites and involved in various aspects of development, such as cell migration, extracellular matrix organization, and cell-cell communication. Among these, we identified wnt11 as a crucial factor regulating both cell proliferation and protrusive activity of DM cells. We propose that dorsal extension of the epaxial myotome is guided by a non-myogenic subpopulation of DM cells and that wnt11 empowers the DM cells to drive the coverage of the neural tube by the epaxial myotome.

DOI： 10.7554/eLife.71845

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

Kupffer's vesicle (KV) in the teleost embryo is a fluid-filled vesicle surrounded by a layer of epithelial cells with rotating primary cilia. KV transiently acts as the left-right organizer and degenerates after the establishment of left-right asymmetric gene expression. Previous labelling experiments in zebrafish embryos indicated that descendants of KV-epithelial cells are incorporated into mesodermal tissues after the collapse of KV. However, the overall picture of their differentiation potency had been unclear due to the lack of suitable genetic tools and molecular analyses. In the present study, we established a novel zebrafish transgenic line with a promoter of dand5, in which all KV-epithelial cells and their descendants are specifically labelled until the larval stage. We found that KV-epithelial cells undergo epithelial-mesenchymal transition upon KV collapse and infiltrate into adjacent mesodermal progenitors, the presomitic mesoderm and chordoneural hinge. Once incorporated, the descendants of KV-epithelial cells expressed distinct mesodermal differentiation markers and contributed to the mature populations such as the axial muscles and notochordal sheath through normal developmental process. These results indicate that differentiated KV-epithelial cells possess unique plasticity in that they are reemployed into mesodermal lineages through transdifferentiation after they complete their initial role in KV.

DOI： 10.1111/dgd.12774

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

The medaka contains seven zic genes, two of which, zic1 and zic4, have been studied extensively. The analyses are mainly based on the double anal fin (Da) mutant, which was isolated from the wild. Da is an enhancer mutant of zic1/zic4, and the expression of zic1/zic4 is specifically lost in the dorsal half of the somites, which leads to a mirror-image duplication of the ventral half across the lateral midline from larva to adult. The studies of medaka Da give us important insights into the function of zic1/zic4 in mesodermal tissues and also the mechanism of dorsoventral patterning in the vertebrate trunk region occurring during late development, which is a long-standing mystery in developmental biology. In this chapter, we introduce genomic organization of medaka zic genes and discuss their function, mainly focusing on zic1 and zic4 in dorsoventral patterning of the trunk region and possible connections to human congenital disorders.

DOI： 10.1007/978-981-10-7311-3_8

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Teleost fish exhibit remarkable diversity in morphology, such as fins and coloration, particularly on the dorsal side. These structures are evolutionary adaptive because their back is highly visible to other individuals. However, owing to the late phenotypic appearance (from larva to adult) and lack of appropriate mutants, the genetic mechanisms that regulate these dorsoventrally asymmetric external patterns are largely unknown. To address this, we have analyzed the spontaneous medaka mutant Double anal fin (Da), which exhibits a mirror-image duplication of the ventral half across the lateral midline from larva to adult. Da is an enhancer mutant for zic1 and zic4 in which their expression in dorsal somites is lost. We show that the dorsoventral polarity in Da somites is lost and then demonstrate using transplantation techniques that somites and their derived tissues globally determine the multiple dorsal-specific characteristics of the body (fin morphology and pigmentation) from embryo to adult. Intriguingly, the zic1/zic4 expression in the wild type persists throughout life in the dorsal parts of somite derivatives, i.e. the myotome, dermis and vertebrae, forming a broad dorsal domain in the trunk. Comparative analysis further implies a central role for zic1/zic4 in morphological diversification of the teleost body. Taken together, we propose that the teleost trunk consists of dorsal/ventral developmental modules and that zic1/zic4 in somites function as selector genes in the dorsal module to regulate multiple dorsal morphologies.

DOI： 10.1242/dev.088567

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

The vertebrate mineralized skeleton is known to have first emerged as an exoskeleton that extensively covered the fossil jawless fish. The evolutionary origin of this exoskeleton has long been attributed to the emergence of the neural crest, but experimental evaluation for this is still poor. Here we determine the embryonic origin of scales and fin rays of medaka (teleost trunk exoskeletons) by applying long-term cell labelling methods, and demonstrate that both tissues are mesodermal in origin. Neural crest cells, however, fail to contribute to these tissues. This result suggests that the trunk neural crest has no skeletogenic capability in fish, instead highlighting the dominant role of the mesoderm in the evolution of the trunk skeleton. This further implies that the role of the neural crest in skeletogenesis has been predominant in the cephalic region from the early stage of vertebrate evolution.

DOI： 10.1038/ncomms2643

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

Teleosts have an asymmetrical caudal fin skeleton formed by the upward bending of the caudal-most portion of the body axis, the ural region. This homocercal type of caudal fin ensures powerful and complex locomotion and is regarded as one of the most important innovations for teleosts during adaptive radiation in an aquatic environment. However, the mechanisms that create asymmetric caudal fin remain largely unknown. The spontaneous medaka (teleost fish) mutant, Double anal fin (Da), exhibits a unique symmetrical caudal skeleton that resembles the diphycercal type seen in Polypterus and Coelacanth. We performed a detailed analysis of the Da mutant to obtain molecular insight into caudal fin morphogenesis. We first demonstrate that a large transposon, inserted into the enhancer region of the zic1 and zic4 genes (zic1/zic4) in Da, is associated with the mesoderm-specific loss of their transcription. We then show that zic1/zic4 are strongly expressed in the dorsal part of the ural mesenchyme and thereby induce asymmetric caudal fin development in wild-type embryos, whereas their expression is lost in Da. Comparative analysis further indicates that the dorsal mesoderm expression of zic1/zic4 is conserved in teleosts, highlighting the crucial role of zic1/zic4 in caudal fin morphogenesis.

DOI： 10.1016/j.cub.2012.01.063

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Event date： 2023.7

Language：English   Presentation type：Poster presentation  

Venue：Sendai International Center, Miyagi   Country：Japan  

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Event date： 2023.7

Language：English   Presentation type：Poster presentation  

Venue：Tohoku University, Miyagi   Country：Japan  

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Event date： 2022.10

Language：English   Presentation type：Poster presentation  

Venue：Salgados Palace, Algarve   Country：Portugal  

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Event date： 2022.9

Language：English   Presentation type：Poster presentation  

Venue：National Cerebral and Cardiovascular Center, Osaka   Country：Japan  

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Event date： 2022.5 - 2022.6

Language：English   Presentation type：Oral presentation (general)  

Venue：Kanazawa Bunka Hall, Ishikawa   Country：Japan  

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Event date： 2021.12

Language：Japanese   Presentation type：Poster presentation  

Venue：パシフィコ横浜   Country：Japan  

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Event date： 2020.5

Language：English   Presentation type：Oral presentation (general)  

Venue：Kumamoto   Country：Japan  

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Event date： 2020.3 - 2020.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Universidad Peruana Cayetano Heredia (UPCH), Lima   Country：Peru  

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Event date： 2019.7

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：東京慈恵会医科大学   Country：Japan  

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Event date： 2019.5

Language：English   Presentation type：Oral presentation (general)  

Venue：International House Osaka, Osaka   Country：Japan  

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Event date： 2017.7

Language：English   Presentation type：Oral presentation (general)  

Venue：Peter Giligan Centre for Research and Learning   Country：Canada  

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Event date： 2012.9

Language：English   Presentation type：Poster presentation  

Venue：Kyoto University, Kyoto   Country：Japan  

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Event date： 2012.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：大阪大学   Country：Japan  

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Event date： 2012.7

Language：English   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：National University of Singapore   Country：Singapore  

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Event date： 2011.5

Language：English   Presentation type：Oral presentation (general)  

Venue：Okinawa Convention Center, Okinawa   Country：Japan  

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Event date： 2010.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：八王子セミナーハウス   Country：Japan  

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Event date： 2010.8

Language：English   Presentation type：Poster presentation  

Venue：Albuquerque Convention Center, New Mexico   Country：United States  

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Event date： 2010.6

Language：English   Presentation type：Poster presentation  

Venue：Kyoto International Conference Center, Kyoto   Country：Japan  

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Event date： 2009.9

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：静岡グランシップ   Country：Japan  

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Event date： 2009.5

Language：English   Presentation type：Poster presentation  

Venue：Toki Messe, Niigata   Country：Japan  

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Event date： 2009.1

Language：Japanese   Presentation type：Poster presentation  

Venue：東京大学   Country：Japan  

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Event date： 2008.9

Language：Japanese   Presentation type：Poster presentation  

Venue：岡崎カンファレンスセンター   Country：Japan  

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Event date： 2008.5

Language：English   Presentation type：Poster presentation  

Venue：Tokushima Arts Foundation for Culture, Tokushima   Country：Japan  

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Language：English   Presentation type：Poster presentation  

Venue：RIKEN Center for Biosystems Dynamics Research (BDR), Hyogo   Country：Japan  

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Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Pacifico Yokohama, Kanagawa   Country：Japan  

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Award type：Award from international society, conference, symposium, etc.  Country：Japan

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Country：Japan

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Country：Japan

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