研究者データベース

中川 真一(ナカガワ シンイチ)
薬学研究院 創薬科学部門 生体機能科学分野
教授

基本情報

所属

  • 薬学研究院 創薬科学部門 生体機能科学分野

職名

  • 教授

学位

  • 理学博士(京都大学)

ホームページURL

科研費研究者番号

  • 50324679

J-Global ID

研究キーワード

  • Xist   エピジェネティックス   核内構造体   パラスペックル   Neat1   神経細胞   ノンコーディングRNA   形態   poly A   ヘテロカリオン   核マトリックス   Gomafu   ロゼット   マウス   グリア   シナプス   ノックアウトマウス   中枢神経系   核スペックル   分化   

研究分野

  • ライフサイエンス / 分子生物学
  • ライフサイエンス / 発生生物学

職歴

  • 2016年05月 - 現在 北海道大学大学院 薬学研究院 教授
  • 2010年04月 - 2016年04月 理化学研究所 准主任研究員
  • 2005年04月 - 2010年03月 理化学研究所 独立主幹研究員
  • 2002年04月 - 2005年03月 理化学研究所 発生再生科学総合研究センター 研究員
  • 2000年04月 - 2002年03月 京都大学 大学院・生命科学研究科 助手
  • 1998年04月 - 2000年03月 ケンブリッジ大学 ポスドク

研究活動情報

論文

  • Shinichi Nakagawa, Rei Yoshimoto, Yuta Nakayama, Ikuko Yamamoto, Shigeyuki Tanaka, Misuzu Kurihara, Yu Suzuki, Takehiko Kobayashi, Hiroko Kozuka-Hata, Masaaki Oyama, Mari Mito, Shintaro Iwasaki, Tomohiro Yamazaki, Tetsuro Hirose, Kimi Araki
    2022年09月02日
  • Akihiro Yamada, Hikaru Toya, Mayuko Tanahashi, Misuzu Kurihara, Mari Mito, Shintaro Iwasaki, Satoshi Kurosaka, Toru Takumi, Archa Fox, Yoshimi Kawamura, Kyoko Miura, Shinichi Nakagawa
    RNA (New York, N.Y.) 28 8 1128 - 1143 2022年08月 [査読有り][通常論文]
     
    Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA NEAT1_2 The molecular mechanisms of paraspeckle formation have been mainly studied using human or mouse cells, and it is not known if the same molecular components are involved in the formation of paraspeckles in other mammalian species. We thus investigated the expression pattern of NEAT1_2 in naked mole-rats (nNEAT1_2), which exhibit extreme longevity and lower susceptibility to cancer. In the intestine, nNEAT1_2 is widely expressed along the entire intestinal epithelium, which is different from the expression of mNeat1_2 that is restricted to the cells of the distal tip in mice. Notably, the expression of FUS, a FET family RNA binding protein, essential for the formation of paraspeckles both in humans and mice, was absent in the distal part of the intestinal epithelium in naked mole-rats. Instead, mRNAs of other FET family proteins EWSR1 and TAF15 were expressed in the distal region. Exogenous expression of these proteins in Fus-deficient murine embryonic fibroblast cells rescued the formation of paraspeckles. These observations suggest that nNEAT1_2 recruits a different set of RNA binding proteins in a cell type-specific manner during the formation of paraspeckles in different organisms.
  • Yan Wu, Pengfei Li, Liu Liu, Andrew J Goodwin, Perry V Halushka, Tetsuro Hirose, Shinichi Nakagawa, Jiliang Zhou, Meng Liu, Hongkuan Fan
    Molecular therapy : the journal of the American Society of Gene Therapy 30 7 2618 - 2632 2022年07月06日 [査読有り][通常論文]
     
    Sepsis-associated encephalopathy (SAE) is characterized by acute and diffuse brain dysfunction and correlates with long-term cognitive impairments with no targeted therapy. We used a mouse model of sepsis-related cognitive impairment to examine the role of lncRNA nuclear enriched abundant transcript 1 (Neat1) in SAE. We observed that Neat1 expression was increased in neuronal cells from septic mice and that it directly interacts with hemoglobin subunit beta (Hbb), preventing its degradation. The Neat1/Hbb axis suppressed postsynaptic density protein 95 (PSD-95) levels and decreased dendritic spine density. Neat1 knockout mice exhibited decreased Hbb levels, which resulted in increased PSD-95 levels, increased neuronal dendritic spine density, and decreased anxiety and memory impairment. Neat1 silencing via the antisense oligonucleotide GapmeR ameliorated anxiety-like behavior and cognitive impairment post-sepsis. In conclusion, we uncovered a previously unknown mechanism of the Neat1/Hbb axis in regulating neuronal dysfunction, which may lead to a novel treatment strategy for SAE.
  • Tatsuya Aonuma, Bruno Moukette, Satoshi Kawaguchi, Nipuni P Barupala, Marisa N Sepúlveda, Kyle Frick, Yaoliang Tang, Maya Guglin, Subha V Raman, Chenleng Cai, Suthat Liangpunsakul, Shinichi Nakagawa, Il-Man Kim
    Circulation. Heart failure 15 4 CIRCHEARTFAILURE121008686  2022年01月10日 [査読有り][通常論文]
     
    BACKGROUND: MicroRNA-150 (miR-150) plays a protective role in heart failure (HF). Long noncoding RNA, myocardial infarction-associated transcript (MIAT) regulates miR-150 function in vitro by direct interaction. Concurrent with miR-150 downregulation, MIAT is upregulated in failing hearts, and gain-of-function single-nucleotide polymorphisms in MIAT are associated with increased risk of myocardial infarction (MI) in humans. Despite the correlative relationship between MIAT and miR-150 in HF, their in vivo functional relationship has never been established, and molecular mechanisms by which these 2 noncoding RNAs regulate cardiac protection remain elusive. METHODS: We use MIAT KO (knockout), Hoxa4 (homeobox a4) KO, MIAT TG (transgenic), and miR-150 TG mice. We also develop DTG (double TG) mice overexpressing MIAT and miR-150. We then use a mouse model of MI followed by cardiac functional, structural, and mechanistic studies by echocardiography, immunohistochemistry, transcriptome profiling, Western blotting, and quantitative real-time reverse transcription-polymerase chain reaction. Moreover, we perform expression analyses in hearts from patients with HF. Lastly, we investigate cardiac fibroblast activation using primary adult human cardiac fibroblasts and in vitro assays to define the conserved MIAT/miR-150/HOXA4 axis. RESULTS: Using novel mouse models, we demonstrate that genetic overexpression of MIAT worsens cardiac remodeling, while genetic deletion of MIAT protects hearts against MI. Importantly, miR-150 overexpression attenuates the detrimental post-MI effects caused by MIAT. Genome-wide transcriptomic analysis of MIAT null mouse hearts identifies Hoxa4 as a novel downstream target of the MIAT/miR-150 axis. Hoxa4 is upregulated in cardiac fibroblasts isolated from ischemic myocardium and subjected to hypoxia/reoxygenation. HOXA4 is also upregulated in patients with HF. Moreover, Hoxa4 deficiency in mice protects the heart from MI. Lastly, protective actions of cardiac fibroblast miR-150 are partially attributed to the direct and functional repression of profibrotic Hoxa4. CONCLUSIONS: Our findings delineate a pivotal functional interaction among MIAT, miR-150, and Hoxa4 as a novel regulatory mechanism pertinent to ischemic HF.
  • Saori Yokoi, Shinichi Nakagawa
    Methods in molecular biology (Clifton, N.J.) 2509 269 - 278 2022年 [査読有り][招待有り]
     
    Long noncoding RNAs (lncRNAs) constitute a large fraction of the transcriptome in mammals, and recent studies have revealed important functions of lncRNAs in a variety of biological processes. However, the fraction of lncRNAs that have been functionally validated is small, and only sequence and expression information are available for most lncRNAs. Here, we describe the procedures for UV-phenol aqueous-phase RNA sequencing (UPA-seq), a method for searching for functional lncRNA candidates among whole genomes based on the assumption that functional lncRNAs exert their functions through associations with proteins.
  • Osamu Takahashi, Mayuko Tanahashi, Saori Yokoi, Mari Kaneko, Kaori Yanaka, Shinichi Nakagawa, Hiroshi Maita
    Genes to cells : devoted to molecular & cellular mechanisms 27 1 43 - 60 2022年01月 [査読有り][通常論文]
     
    Genomes of higher eukaryotes encode many uncharacterized proteins, and the functions of these proteins cannot be predicted from the primary sequences due to a lack of conserved functional domains. In this study, we focused on a poorly characterized protein UGS148 that is highly expressed in a specialized cell type called tanycytes that line the ventral wall of the third ventricle in the hypothalamus. Immunostaining of UGS148 revealed the fine morphology of tanycytes with highly branched apical ER membranes. Immunoprecipitation revealed that UGS148 associated with mitochondrial ATPase at least in vitro, and ER and mitochondrial signals occasionally overlapped in tanycytes. Mutant mice lacking UGS148 did not exhibit overt phenotypes, suggesting that UGS148 was not essential in mice reared under normal laboratory conditions. We also found that RNA probes that were predicted to uniquely detect UGS148 mRNA cross-reacted with uncharacterized RNAs, highlighting the importance of experimental validation of the specificity of probes during the hybridization-based study of RNA localization.
  • Mi Kyung Park, Li Zhang, Kyung-Won Min, Jung-Hyun Cho, Chih-Chen Yeh, Hyesu Moon, Daniel Hormaechea-Agulla, Hyejin Mun, Seungbeom Ko, Ji Won Lee, Sonali Jathar, Aubrey S Smith, Yixin Yao, Nguyen Thu Giang, Hong Ha Vu, Victoria C Yan, Mary C Bridges, Antonis Kourtidis, Florian Muller, Jeong Ho Chang, Su Jung Song, Shinichi Nakagawa, Tetsuro Hirose, Je-Hyun Yoon, Min Sup Song
    Cell metabolism 33 12 2380 - 2397 2021年12月07日 [査読有り][通常論文]
     
    Accelerated glycolysis is the main metabolic change observed in cancer, but the underlying molecular mechanisms and their role in cancer progression remain poorly understood. Here, we show that the deletion of the long noncoding RNA (lncRNA) Neat1 in MMTV-PyVT mice profoundly impairs tumor initiation, growth, and metastasis, specifically switching off the penultimate step of glycolysis. Mechanistically, NEAT1 directly binds and forms a scaffold bridge for the assembly of PGK1/PGAM1/ENO1 complexes and thereby promotes substrate channeling for high and efficient glycolysis. Notably, NEAT1 is upregulated in cancer patients and correlates with high levels of these complexes, and genetic and pharmacological blockade of penultimate glycolysis ablates NEAT1-dependent tumorigenesis. Finally, we demonstrate that Pinin mediates glucose-stimulated nuclear export of NEAT1, through which it exerts isoform-specific and paraspeckle-independent functions. These findings establish a direct role for NEAT1 in regulating tumor metabolism, provide new insights into the Warburg effect, and identify potential targets for therapy.
  • Yoshinori Nishimoto, Shinichi Nakagawa, Hideyuki Okano
    Neurochemistry international 150 105175 - 105175 2021年09月02日 [査読有り][通常論文]
     
    Amyotrophic lateral sclerosis (ALS) is a representative neurological disease that is known to devastate entire motor neurons within a period of just a few years. Discoveries of the specific pathologies of relevant RNA-binding proteins, including TAR DNA-binding protein-43 (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS), and the causative genes of both familial and sporadic ALS have provided crucial information that could lead to a cure. In recent ALS research the GGGGCC-repeat expansion in the C9orf72 gene was identified as one of the most important pathological findings, suggesting the significance of both nuclear dysfunction due to dipeptide repeat proteins (DPRs) and RNA toxicity (such as pathological alterations of non-coding RNAs). In research on model animals carrying ALS-related molecules, the determination of whether a factor is protective or toxic has been controversial. Herein, we review the findings regarding NEAT1 RNA and C9orf72 GGGGCC repeats associated with ALS, from the viewpoint of conversion from the protective stage in the nucleus in early-phase ALS to late-phase induction of cell death. This review will provide insights for the development of RNA effectors as novel ALS treatments.
  • Keita Yamamoto, Susumu Goyama, Shuhei Asada, Takeshi Fujino, Taishi Yonezawa, Naru Sato, Reina Takeda, Akiho Tsuchiya, Tomofusa Fukuyama, Yosuke Tanaka, Akihiko Yokoyama, Hikaru Toya, Ayana Kon, Yasuhito Nannya, Rena Onoguchi-Mizutani, Shinichi Nakagawa, Tetsuro Hirose, Seishi Ogawa, Nobuyoshi Akimitsu, Toshio Kitamura
    Cell reports 36 8 109576 - 109576 2021年08月24日 [査読有り][通常論文]
     
    Paraspeckles are membraneless organelles formed through liquid-liquid phase separation and consist of multiple proteins and RNAs, including NONO, SFPQ, and NEAT1. The role of paraspeckles and the component NONO in hematopoiesis remains unknown. In this study, we show histone modifier ASXL1 is involved in paraspeckle formation. ASXL1 forms phase-separated droplets, upregulates NEAT1 expression, and increases NONO-NEAT1 interactions through the C-terminal intrinsically disordered region (IDR). In contrast, a pathogenic ASXL mutant (ASXL1-MT) lacking IDR does not support the interaction of paraspeckle components. Furthermore, paraspeckles are disrupted and Nono localization is abnormal in the cytoplasm of hematopoietic stem and progenitor cells (HSPCs) derived from ASXL1-MT knockin mice. Nono depletion and the forced expression of cytoplasmic NONO impair the repopulating potential of HSPCs, as does ASXL1-MT. Our study indicates a link between ASXL1 and paraspeckle components in the maintenance of normal hematopoiesis.
  • Tomohiro Yamazaki, Tetsuya Yamamoto, Hyura Yoshino, Sylvie Souquere, Shinichi Nakagawa, Gerard Pierron, Tetsuro Hirose
    The EMBO journal 40 12 e107270  2021年06月15日 [査読有り][通常論文]
     
    Paraspeckles are constructed by NEAT1_2 architectural long noncoding RNAs. Their characteristic cylindrical shapes, with highly ordered internal organization, distinguish them from typical liquid-liquid phase-separated condensates. We experimentally and theoretically investigated how the shape and organization of paraspeckles are determined. We identified the NEAT1_2 RNA domains responsible for shell localization of the NEAT1_2 ends, which determine the characteristic internal organization. Using the soft matter physics, we then applied a theoretical framework to understand the principles that determine NEAT1_2 organization as well as shape, number, and size of paraspeckles. By treating paraspeckles as amphipathic block copolymer micelles, we could explain and predict the experimentally observed behaviors of paraspeckles upon NEAT1_2 domain deletions or transcriptional modulation. Thus, we propose that paraspeckles are block copolymer micelles assembled through a type of microphase separation, micellization. This work provides an experiment-based theoretical framework for the concept that ribonucleoprotein complexes (RNPs) can act as block copolymers to form RNA-scaffolding biomolecular condensates with optimal sizes and structures in cells.
  • Shinichi Nakagawa, Tomohiro Yamazaki, Taro Mannen, Tetsuro Hirose
    Mammalian genome : official journal of the International Mammalian Genome Society 33 2 382 - 401 2021年06月03日 [査読有り][通常論文]
     
    Long noncoding RNAs (lncRNAs) have long been collectively and passively defined as transcripts that do not encode proteins. However, extensive functional studies performed over the last decade have enabled the classification of lncRNAs into multiple categories according to their functions and/or molecular properties. Architectual RNAs (arcRNAs) are a group of lncRNAs that serve as architectural components of submicron-scale cellular bodies or nonmembranous organelles, which are composed of specific sets of proteins and nucleic acids involved in particular molecular processes. In this review, we focus on arcRNAs that function in the nucleus, which provide a structural basis for the formation of nuclear bodies, nonmembranous organelles in the cell nucleus. We will summarize the current list of arcRNAs and proteins associated with classic and more recently discovered nuclear bodies and discuss general rules that govern the formation of nuclear bodies, emphasizing weak multivalent interactions mediated by innately flexible biomolecules.
  • Michio Sato, Tsuyoshi Kadomatsu, Keishi Miyata, Junco S Warren, Zhe Tian, Shunshun Zhu, Haruki Horiguchi, Aman Makaju, Anna Bakhtina, Jun Morinaga, Taichi Sugizaki, Kaname Hirashima, Kumiko Yoshinobu, Mai Imasaka, Masatake Araki, Yoshihiro Komohara, Tomohiko Wakayama, Shinichi Nakagawa, Sarah Franklin, Koichi Node, Kimi Araki, Yuichi Oike
    Nature communications 12 1 2529 - 2529 2021年05月05日 [査読有り][通常論文]
     
    In the past decade, many long noncoding RNAs (lncRNAs) have been identified and their in vitro functions defined, although in some cases their functions in vivo remain less clear. Moreover, unlike nuclear lncRNAs, the roles of cytoplasmic lncRNAs are less defined. Here, using a gene trapping approach in mouse embryonic stem cells, we identify Caren (short for cardiomyocyte-enriched noncoding transcript), a cytoplasmic lncRNA abundantly expressed in cardiomyocytes. Caren maintains cardiac function under pathological stress by inactivating the ataxia telangiectasia mutated (ATM)-DNA damage response (DDR) pathway and activating mitochondrial bioenergetics. The presence of Caren transcripts does not alter expression of nearby (cis) genes but rather decreases translation of an mRNA transcribed from a distant gene encoding histidine triad nucleotide-binding protein 1 (Hint1), which activates the ATM-DDR pathway and reduces mitochondrial respiratory capacity in cardiomyocytes. Therefore, the cytoplasmic lncRNA Caren functions in cardioprotection by regulating translation of a distant gene and maintaining cardiomyocyte homeostasis.
  • Rei Yoshimoto, Jagat K Chhipi-Shrestha, Tilman Schneider-Poetsch, Masaaki Furuno, A Maxwell Burroughs, Shohei Noma, Harukazu Suzuki, Yoshihide Hayashizaki, Akila Mayeda, Shinichi Nakagawa, Daisuke Kaida, Shintaro Iwasaki, Minoru Yoshida
    Cell chemical biology 28 9 1356 - 1365 2021年03月23日 [査読有り][通常論文]
     
    RNA splicing, a highly conserved process in eukaryotic gene expression, is seen as a promising target for anticancer agents. Splicing is associated with other RNA processing steps, such as transcription and nuclear export; however, our understanding of the interaction between splicing and other RNA regulatory mechanisms remains incomplete. Moreover, the impact of chemical splicing inhibition on long non-coding RNAs (lncRNAs) has been poorly understood. Here, we demonstrate that spliceostatin A (SSA), a chemical splicing modulator that binds to the SF3B subcomplex of the U2 small nuclear ribonucleoprotein particle (snRNP), limits U1 snRNP availability in splicing, resulting in premature cleavage and polyadenylation of MALAT1, a nuclear lncRNA, as well as protein-coding mRNAs. Therefore, truncated transcripts are exported into the cytoplasm and translated, resulting in aberrant protein products. Our work demonstrates that active recycling of the splicing machinery maintains homeostasis of RNA processing beyond intron excision.
  • Liu Yang, Jianxin Deng, Wenxia Ma, Aijun Qiao, Shiyue Xu, Yang Yu, Chan Boriboun, Xiang Kang, Dunzheng Han, Patrick Ernst, Lufang Zhou, Jiawei Shi, Eric Zhang, Tao-Sheng Li, Hongyu Qiu, Shinichi Nakagawa, Seth Blackshaw, Jianyi Zhang, Gangjian Qin
    Theranostics 11 16 7995 - 8007 2021年 [査読有り][通常論文]
     
    Rationale: The conserved long non-coding RNA (lncRNA) myocardial infarction associate transcript (Miat) was identified for its multiple single-nucleotide polymorphisms that are strongly associated with susceptibility to MI, but its role in cardiovascular biology remains elusive. Here we investigated whether Miat regulates cardiac response to pathological hypertrophic stimuli. Methods: Both an angiotensin II (Ang II) infusion model and a transverse aortic constriction (TAC) model were used in adult WT and Miat-null knockout (Miat-KO) mice to induce pathological cardiac hypertrophy. Heart structure and function were evaluated by echocardiography and histological assessments. Gene expression in the heart was evaluated by RNA sequencing (RNA-seq), quantitative real-time RT-PCR (qRT-PCR), and Western blotting. Primary WT and Miat-KO mouse cardiomyocytes were isolated and used in Ca2+ transient and contractility measurements. Results: Continuous Ang II infusion for 4 weeks induced concentric hypertrophy in WT mice, but to a lesser extent in Miat-KO mice. Surgical TAC for 6 weeks resulted in decreased systolic function and heart failure in WT mice but not in Miat-KO mice. In both models, Miat-KO mice displayed reduced heart-weight to tibia-length ratio, cardiomyocyte cross-sectional area, cardiomyocyte apoptosis, and cardiac interstitial fibrosis and a better-preserved capillary density, as compared to WT mice. In addition, Ang II treatment led to significantly reduced mRNA and protein expression of the Ca2+ cycling genes Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and ryanodine receptor 2 (RyR2) and a dramatic increase in global RNA splicing events in the left ventricle (LV) of WT mice, and these changes were largely blunted in Miat-KO mice. Consistently, cardiomyocytes isolated from Miat-KO mice demonstrated more efficient Ca2+ cycling and greater contractility. Conclusions: Ablation of Miat attenuates pathological hypertrophy and heart failure, in part, by enhancing cardiomyocyte contractility.
  • Olof Gidlöf, Kerstin Bader, Selvi Celik, Mario Grossi, Shinichi Nakagawa, Tetsuro Hirose, Bernhard Metzler, Björn Olde, David Erlinge
    Cell death & disease 11 8 677 - 677 2020年08月13日 [査読有り][通常論文]
     
    While restoration of coronary blood flow to the ischemic heart is the most effective strategy for reducing infarct size, reperfusion injury represents a significant limiting factor on clinical outcomes in myocardial infarction patients. Ischemic preconditioning (IPC) has been shown to inhibit reperfusion injury and represents an attractive model for studying cardioprotective signal transduction pathways. Long non-coding RNAs (lncRNAs) are a structurally and functionally heterogenous class of RNA transcripts with unknown roles in IPC-induced cardioprotection. Through microarray-based expression profiling of 31,423 lncRNAs in cardiac tissue from IPC mice, we identified the nuclear transcript Neat1 to be rapidly and robustly decreased in response to IPC. siRNA-mediated knock down of Neat1 reduced apoptosis and necrosis in murine cardiomyocytes (CM) and human iPS-derived CMs in response to prolonged hypoxia and hypoxia-reoxygenation, assessed with Annexin V/propidium iodide-staining, a Caspase 3/7 activity assay, LDH release, and western blot for cleaved Caspase 3. Mechanistically, Neat1 was shown to regulate processing of pro-apoptotic microRNA-22 (miR-22) in murine and human CM nuclei using a luciferase reporter assay. Hypoxia-induced downregulation of Neat1 was shown to result in accumulation of unprocessed pri-miRNA and decreased availability of biologically active miRNA, including miR-22. Addition of exogenous synthetic miR-22 reversed the protective effect of Neat1 knock down in human iPS-CM. In conclusion, we have identified the nuclear lncRNA Neat1 as part of a conserved oxygen-sensitive feedback mechanism by regulation of miRNA processing and a potential target in cardioprotection.
  • Michail S Kukharsky, Natalia N Ninkina, Haiyan An, Vsevolod Telezhkin, Wenbin Wei, Camille Rabesahala de Meritens, Johnathan Cooper-Knock, Shinichi Nakagawa, Tetsuro Hirose, Vladimir L Buchman, Tatyana A Shelkovnikova
    Translational psychiatry 10 1 171 - 171 2020年05月28日 [査読有り][通常論文]
     
    NEAT1 is a highly and ubiquitously expressed long non-coding RNA (lncRNA) which serves as an important regulator of cellular stress response. However, the physiological role of NEAT1 in the central nervous system (CNS) is still poorly understood. In the current study, we addressed this by characterising the CNS function of the Neat1 knockout mouse model (Neat1-/- mice), using a combination of behavioural phenotyping, electrophysiology and expression analysis. RNAscope® in situ hybridisation revealed that in wild-type mice, Neat1 is expressed across the CNS regions, with high expression in glial cells and low expression in neurons. Loss of Neat1 in mice results in an inadequate reaction to physiological stress manifested as hyperlocomotion and panic escape response. In addition, Neat1-/- mice display deficits in social interaction and rhythmic patterns of activity but retain normal motor function and memory. Neat1-/- mice do not present with neuronal loss, overt neuroinflammation or gross synaptic dysfunction in the brain. However, cultured Neat1-/- neurons are characterised by hyperexcitability and dysregulated calcium homoeostasis, and stress-induced neuronal activity is also augmented in Neat1-/- mice in vivo. Gene expression analysis showed that Neat1 may act as a weak positive regulator of multiple genes in the brain. Furthermore, loss of Neat1 affects alternative splicing of genes important for the CNS function and implicated in neurological diseases. Overall, our data suggest that Neat1 is involved in stress signalling in the brain and fine-tunes the CNS functions to enable adaptive behaviour in response to physiological stress.
  • Saori Yokoi, Kiyoshi Naruse, Yasuhiro Kamei, Satoshi Ansai, Masato Kinoshita, Mari Mito, Shintaro Iwasaki, Shuntaro Inoue, Teruhiro Okuyama, Shinichi Nakagawa, Larry J Young, Hideaki Takeuchi
    Proceedings of the National Academy of Sciences of the United States of America 117 9 4802 - 4808 2020年03月03日 [査読有り][通常論文]
     
    Oxytocin is a central neuromodulator required for facilitating mate preferences for familiar individuals in a monogamous rodent (prairie vole), irrespective of sex. While the role of oxytocin in mate choice is only understood in a few monogamous species, its function in nonmonogamous species, comprising the vast majority of vertebrate species, remains unclear. To address this issue, we evaluated the involvement of an oxytocin homolog (isotocin, referred herein as oxt) in mate choice in medaka fish (Oryzias latipes). Female medaka prefer to choose familiar mates, whereas male medaka court indiscriminately, irrespective of familiarity. We generated mutants of the oxt ligand (oxt) and receptor genes (oxtr1 and oxtr2) and revealed that the oxt-oxtr1 signaling pathway was essential for eliciting female mate preference for familiar males. This pathway was also required for unrestricted and indiscriminate mating strategy in males. That is, either oxt or oxtr1 mutation in males decreased the number of courtship displays toward novel females, but not toward familiar females. Further, males with these mutations exhibited enhanced mate-guarding behaviors toward familiar females, but not toward novel females. In addition, RNA-sequencing (seq) analysis revealed that the transcription of genes involved in gamma-amino butyric acid metabolism as well as those encoding ion-transport ATPase are up-regulated in both oxt and oxtr1 mutants only in female medaka, potentially explaining the sex difference of the mutant phenotype. Our findings provide genetic evidence that oxt-oxtr1 signaling plays a role in the mate choice for familiar individuals in a sex-specific manner in medaka fish.
  • Isobe M, Toya H, Mito M, Chiba T, Asahara H, Hirose T, Nakagawa S
    RNA (New York, N.Y.) 26 3 251 - 264 2020年03月 [査読有り][通常論文]
     
    Neat1 is a long noncoding RNA (lncRNA) that serves as an architectural component of the nuclear bodies known as paraspeckles. Two isoforms of Neat1, the short isoform Neat1_1 and the long isoform Neat1_2, are generated from the same gene locus by alternative 3' processing. Neat1_1 is the most abundant and the best conserved isoform expressed in various cell types, whereas Neat1_2 is expressed in a small population of particular cell types, including the tip cells of the intestinal epithelium. To investigate the physiological significance of isoform switching, we created mutant mice that solely expressed Neat1_2 by deleting the upstream polyadenylation (poly-A) signal (PAS) required for the production of Neat1_1. We observed the loss of Neat1_1 and strong up-regulation of Neat1_2 in various tissues and cells and the subsequent hyperformation of paraspeckles, especially in cells that normally express Neat1_2. However, the mutant mice were born at the expected Mendelian ratios and did not exhibit obvious external and histological abnormalities. These observations suggested that the hyperformation of paraspeckles does not interfere with the development and growth of these animals under normal laboratory conditions.
  • Tomohiro Yamazaki, Shinichi Nakagawa, Tetsuro Hirose
    Cold Spring Harbor symposia on quantitative biology 84 227 - 237 2020年02月04日 [査読有り][通常論文]
     
    Long noncoding RNAs (lncRNAs) are fundamental regulators of various cellular processes. A subset of lncRNAs, termed architectural RNAs (arcRNAs), function in the formation and maintenance of phase-separated membraneless organelles in multiple eukaryotic species. These membraneless organelles represent an important type of compartmentalization in the crowded cellular environment and have several distinct features. The NEAT1_2 lncRNA is a well-characterized arcRNA that functions as an essential scaffold of paraspeckle nuclear bodies. Here, we describe the biogenesis of paraspeckles on arcRNAs through phase separation, focusing on the specific functions of multiple NEAT1_2 RNA domains and their partner RNA-binding proteins. Finally, we present an updated model of paraspeckle formation and discuss future perspectives of research into arcRNA-instructed architectures of phase-separated nuclear bodies.
  • Maita H, Nakagawa S
    Wiley interdisciplinary reviews. RNA 11 1 e1574  2020年01月 [査読有り][通常論文]
     
    Phosphorylation of the RNA polymerase II C-terminal domain (Pol II CTD) has important roles in the kinetic coupling of splicing with transcription, which is essential for many genes to maintain correct splicing patterns. However, because of the extensively repeated low complexity sequences of Pol II CTD, it was unclear how phosphorylation-dependent molecular interactions were able to provide sufficient specificity to spatiotemporally partition various cotranscriptional events. Here we try to view the molecular mechanisms governing cotranscriptional splicing from the role of phase separation based on recent studies showing the ability of Pol II CTD to form droplets. This article is categorized under:   RNA Processing > Splicing Regulation/Alternative Splicing   RNA Processing > Splicing Mechanisms   RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.
  • Adriaens C, Rambow F, Bervoets G, Silla T, Mito M, Chiba T, Asahara H, Hirose T, Nakagawa S, Jensen TH, Marine JC
    RNA (New York, N.Y.) 25 12 1681 - 1695 2019年12月 [査読有り][通常論文]
  • Gurumurthy CB, Sato M, Nakamura A, Inui M, Kawano N, Islam MA, Ogiwara S, Takabayashi S, Matsuyama M, Nakagawa S, Miura H, Ohtsuka M
    Nature protocols 14 8 2452 - 2482 2019年08月 [査読有り][通常論文]
     
    Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps. GONAD involves injection of CRISPR components (Cas9 mRNA and guide RNA (gRNA)) into the oviducts of pregnant females 1.5 d post conception, followed by in vivo electroporation to deliver the components into the zygotes in situ. Using GONAD, we demonstrated that target genes can be disrupted and analyzed at different stages of mouse embryonic development. Subsequently, we developed improved GONAD (i-GONAD) by delivering CRISPR ribonucleoproteins (RNPs; Cas9 protein or Cpf1 protein and gRNA) into day-0.7 pregnant mice, which made it suitable for routine generation of knockout and large-deletion mouse models. i-GONAD can also generate knock-in models containing up to 1-kb inserts when single-stranded DNA (ssDNA) repair templates are supplied. i-GONAD offers other advantages: it does not require vasectomized males and pseudo-pregnant females, the females used for i-GONAD are not sacrificed and can be used for other experiments, it can be easily adopted in laboratories lacking sophisticated microinjection equipment, and can be implemented by researchers skilled in small-animal surgery but lacking embryo-handling skills. Here, we provide a step-by-step protocol for establishing the i-GONAD method. The protocol takes ∼6 weeks to generate the founder mice.
  • Shinoda S, Kitagawa S, Nakagawa S, Wei FY, Tomizawa K, Araki K, Araki M, Suzuki T, Suzuki T
    Nucleic acids research 47 16 8734 - 8745 2019年07月 [査読有り][通常論文]
     
    Post-transcriptional modifications in mitochondrial tRNAs (mt-tRNAs) play critical roles in mitochondrial protein synthesis, which produces respiratory chain complexes. In this study, we took advantage of mass spectrometric analysis to map 5-methylcytidine (m5C) at positions 48-50 in eight mouse and six human mt-tRNAs. We also confirmed the absence of m5C in mt-tRNAs isolated from Nsun2 knockout (KO) mice, as well as from NSUN2 KO human culture cells. In addition, we successfully reconstituted m5C at positions 48-50 of mt-tRNA in vitro with NSUN2 protein in the presence of S-adenosylmethionine. Although NSUN2 is predominantly localized to the nucleus and introduces m5C into cytoplasmic tRNAs and mRNAs, structured illumination microscopy clearly revealed NSUN2 foci inside mitochondria. These observations provide novel insights into the role of NSUN2 in the physiology and pathology of mitochondrial functions.
  • Hirose T, Yamazaki T, Nakagawa S
    Wiley interdisciplinary reviews. RNA 10 6 e1545  2019年05月 [査読有り][通常論文]
     
    Long noncoding RNAs (lncRNAs) are extremely diverse and have various significant physiological functions. lncRNAs generally associate with specific sets of RNA-binding proteins (RBPs) to form functional ribonucleoprotein (RNP) complexes. NEAT1 is a highly abundant lncRNA in the mammalian cell nucleus that associates with specific RBPs to form NEAT1 RNPs. Intriguingly, cellular NEAT1 RNPs are extraordinarily large and can be detected using an optical microscope. These gigantic RNPs, so-called paraspeckles, are a type of membraneless nuclear body. Paraspeckles contain approximately 50 NEAT1 RNA molecules together with characteristic RBPs possessing aggregation-prone prion-like domains. Paraspeckle formation proceeds on the nascent NEAT1 transcript in conjunction with NEAT1 biogenesis, which exhibits various features that differ from those exhibited by mRNA biogenesis, including a lack of introns, noncanonical 3' end formation, and nuclear retention. These unique features may be required for the mechanism of paraspeckle formation. NEAT1 possesses three distinct RNA domains (A, B, and C), which function in stabilization (A), isoform switching (B), and paraspeckle assembly (C). In particular, the central C domain contains smaller subdomains that are high-affinity binding sites for the essential paraspeckle proteins (NONO and SFPQ) that subsequently polymerize along NEAT1. Subsequent recruitment of additional essential PSPs (FUS and RBM14) induces liquid-liquid phase separation to build a massive paraspeckle structure. Thus, the molecular anatomy of the NEAT1 arcRNA provides an ideal model to understand how lncRNAs form the functional RNP machinery. This article is characterized under: RNA Export and Localization > Nuclear Export/Import RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.
  • Gast M, Rauch B, Haghikia A, Nakagawa S, Haas J, Stroux A, Schmidt D, Schumann P, Weiss S, Jensen L, Kratzer A, Kraenkel N, Müller C, Börnigen D, Hirose T, Blankenberg S, Escher F, Kühl A, Kuss A, Meder B, Landmesser U, Zeller T, Poller W
    Cardiovascular research 2019年03月 [査読有り][通常論文]
  • Mari Mito, Mitsutaka Kadota, Shinichi Nakagawa, Shintaro Iwasaki
    Journal of visualized experiments : JoVE 143 2019年01月23日 [査読有り][通常論文]
     
    Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.
  • Katsel P, Roussos P, Fam P, Khan S, Tan W, Hirose T, Nakagawa S, Pletnikov MV, Haroutunian V
    NPJ schizophrenia 5 1 3  2019年01月 [査読有り][通常論文]
  • Taiwa Komatsu, Saori Yokoi, Koichi Fujii, Mari Mito, Yusuke Kimura, Shintaro Iwasaki, Shinichi Nakagawa
    RNA (New York, N.Y.) 24 12 1785 - 1802 2018年12月 [査読有り][通常論文]
     
    While a large number of long noncoding RNAs (lncRNAs) are transcribed from the genome of higher eukaryotes, systematic prediction of their functionality has been challenging due to the lack of conserved sequence motifs or structures. Assuming that some lncRNAs function as large ribonucleoprotein complexes and thus are easily crosslinked to proteins upon UV irradiation, we performed RNA-seq analyses of RNAs recovered from the aqueous phase after UV irradiation and phenol-chloroform extraction (UPA-seq). As expected, the numbers of UPA-seq reads mapped to known functional lncRNAs were remarkably reduced upon UV irradiation. Comparison with ENCODE eCLIP data revealed that lncRNAs that exhibited greater decreases upon UV irradiation preferentially associated with proteins containing prion-like domains (PrLDs). Fluorescent in situ hybridization (FISH) analyses revealed the nuclear localization of novel functional lncRNA candidates, including one that accumulated at the site of transcription. We propose that UPA-seq provides a useful tool for the selection of lncRNA candidates to be analyzed in depth in subsequent functional studies.
  • Kim J, Piao HL, Kim BJ, Yao F, Han Z, Wang Y, Xiao Z, Siverly AN, Lawhon SE, Ton BN, Lee H, Zhou Z, Gan B, Nakagawa S, Ellis MJ, Liang H, Hung MC, You MJ, Sun Y, Ma L
    Nature genetics 50 12 1705 - 1715 2018年12月 [査読有り][通常論文]
  • Nakagawa S, Yamazaki T, Hirose T
    Open biology 8 10 2018年10月 [査読有り][通常論文]
     
    Paraspeckles are nuclear bodies built on an architectural long noncoding RNA, NEAT1, and a series of studies have revealed their molecular components, fine internal structures and cellular and physiological functions. Emerging lines of evidence suggest that paraspeckle formation is elicited by phase separation of associating RNA-binding proteins containing intrinsically disordered regions, which induce ordered arrangement of paraspeckle components along NEAT1. In this review, we will summarize the history of paraspeckle research over the last couple of decades, especially focusing on the function and structure of the nuclear bodies. We also discuss the future directions of research on long noncoding RNAs that form 'RNP milieux', large and flexible phase-separated ribonucleoprotein complexes.
  • Ahmed ASI, Dong K, Liu J, Wen T, Yu L, Xu F, Kang X, Osman I, Hu G, Bunting KM, Crethers D, Gao H, Zhang W, Liu Y, Wen K, Agarwal G, Hirose T, Nakagawa S, Vazdarjanova A, Zhou J
    Proceedings of the National Academy of Sciences of the United States of America 115 37 E8660 - E8667 2018年09月 [査読有り][通常論文]
  • Gast M, Rauch BH, Nakagawa S, Haghikia A, Jasina A, Haas J, Nath N, Jensen L, Stroux A, Böhm A, Friebel J, Rauch U, Skurk C, Blankenberg S, Zeller T, Prasanth KV, Meder B, Kuss A, Landmesser U, Poller W
    Cardiovascular research 2018年08月 [査読有り][通常論文]
  • Tomohiro Yamazaki, Sylvie Souquere, Takeshi Chujo, Simon Kobelke, Yee Seng Chong, Archa H. Fox, Charles S. Bond, Shinichi Nakagawa, Gerard Pierron, Tetsuro Hirose
    Molecular Cell 70 6 1038 - 1053.e7 2018年06月21日 [査読有り][通常論文]
     
    A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation. The NEAT1 architectural noncoding RNA has a modular domain structure with distinct functions required for paraspeckle formation. Among the functional domains, the middle domain is composed of three distinct, functionally redundant, subdomains that recruit NONO dimers to initiate assembly of the phase-separated paraspeckle structure.
  • Huan Lin, Kenjyo Miyauchi, Tai Harada, Ryo Okita, Eri Takeshita, Hirofumi Komaki, Kaoru Fujioka, Hideki Yagasaki, Yu-Ichi Goto, Kaori Yanaka, Shinichi Nakagawa, Yuriko Sakaguchi, Tsutomu Suzuki
    Nature communications 9 1 1875 - 1875 2018年05月14日 [査読有り][通常論文]
     
    It has been generally thought that tRNA modifications are stable and static, and their frequencies are rarely regulated. N6-threonylcarbamoyladenosine (t6A) occurs at position 37 of five mitochondrial (mt-)tRNA species. We show that YRDC and OSGEPL1 are responsible for t6A37 formation, utilizing L-threonine, ATP, and CO2/bicarbonate as substrates. OSGEPL1-knockout cells exhibit respiratory defects and reduced mitochondrial translation. We find low level of t6A37 in mutant mt-tRNA isolated from the MERRF-like patient's cells, indicating that lack of t6A37 results in pathological consequences. Kinetic measurements of t6A37 formation reveal that the Km value of CO2/bicarbonate is extremely high (31 mM), suggesting that CO2/bicarbonate is a rate-limiting factor for t6A37 formation. Consistent with this, we observe a low frequency of t6A37 in mt-tRNAs isolated from human cells cultured without bicarbonate. These findings indicate that t6A37 is regulated by sensing intracellular CO2/bicarbonate concentration, implying that mitochondrial translation is modulated in a codon-specific manner under physiological conditions.
  • Archa H. Fox, Shinichi Nakagawa, Tetsuro Hirose, Charles S. Bond
    Trends in Biochemical Sciences 43 2 124 - 135 2018年02月01日 [査読有り][通常論文]
     
    Long noncoding RNA (lncRNA) molecules are some of the newest and least understood players in gene regulation. Hence, we need good model systems with well-defined RNA and protein components. One such system is paraspeckles – protein-rich nuclear organelles built around a specific lncRNA scaffold. New discoveries show how paraspeckles are formed through multiple RNA–protein and protein–protein interactions, some of which involve extensive polymerization, and others with multivalent interactions driving phase separation. Once formed, paraspeckles influence gene regulation through sequestration of component proteins and RNAs, with subsequent depletion in other compartments. Here we focus on the dual aspects of paraspeckle structure and function, revealing an emerging role for these dynamic bodies in a multitude of cellular settings.
  • Mari Mito, Mitsutaka Kadota, Kaori Tanaka, Yasuhide Furuta, Kuniya Abe, Shintaro Iwasaki, Shinichi Nakagawa
    Scientific reports 8 1 1143 - 1143 2018年01月18日 [査読有り][通常論文]
     
    The nervous system of higher eukaryotes is composed of numerous types of neurons and glia that together orchestrate complex neuronal responses. However, this complex pool of cells typically poses analytical challenges in investigating gene expression profiles and their epigenetic basis for specific cell types. Here, we developed a novel method that enables cell type-specific analyses of epigenetic modifications using tandem chromatin immunoprecipitation sequencing (tChIP-Seq). FLAG-tagged histone H2B, a constitutive chromatin component, was first expressed in Camk2a-positive pyramidal cortical neurons and used to purify chromatin in a cell type-specific manner. Subsequent chromatin immunoprecipitation using antibodies against H3K4me3-a chromatin modification mainly associated with active promoters-allowed us to survey the histone modifications in Camk2a-positive neurons. Indeed, tChIP-Seq identified hundreds of H3K4me3 modifications in promoter regions located upstream of genes associated with neuronal functions and genes with unknown functions in cortical neurons. tChIP-Seq provides a versatile approach to investigating the epigenetic modifications of particular cell types in vivo.
  • Stephano S. Mello, Carolyn Sinow, Nitin Raj, Pawel K. Mazur, Kathryn Bieging-Rolett, Daniela Kenzelmann Broz, Jamie F. Conklin Imam, Hannes Vogel, Laura D. Wood, Julien Sage, Tetsuro Hirose, Shinichi Nakagawa, John Rinn, Laura D. Attardi
    GENES & DEVELOPMENT 31 11 1095 - 1108 2017年06月 [査読有り][通常論文]
     
    The p53 gene is mutated in over half of all cancers, reflecting its critical role as a tumor suppressor. Although p53 is a transcriptional activator that induces myriad target genes, those p53-inducible genes most critical for tumor suppression remain elusive. Here, we leveraged p53 ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) and RNA-seq (RNA sequencing) data sets to identify new p53 target genes, focusing on the noncoding genome. We identify Neat1, a noncoding RNA (ncRNA) constituent of paraspeckles, as a p53 target gene broadly induced by mouse and human p53 in different cell types and by diverse stress signals. Using fibroblasts derived from Neat1(-/-) mice, we examined the functional role of Neat1 in the p53 pathway. We found that Neat1 is dispensable for cell cycle arrest and apoptosis in response to genotoxic stress. In sharp contrast, Neat1 plays a crucial role in suppressing transformation in response to oncogenic signals. Neat1 deficiency enhances transformation in oncogene-expressing fibroblasts and promotes the development of premalignant pancreatic intraepithelial neoplasias (PanINs) and cystic lesions in Kras(G12D)-expressing mice. Neat1 loss provokes global changes in gene expression, suggesting a mechanism by which its deficiency promotes neoplasia. Collectively, these findings identify Neat1 as a p53-regulated large intergenic ncRNA (lincRNA) with a key role in suppressing transformation and cancer initiation, providing fundamental new insight into p53-mediated tumor suppression.
  • Takeshi Chujo, Tomohiro Yamazaki, Tetsuya Kawaguchi, Satoshi Kurosaka, Toru Takumi, Shinichi Nakagawa, Tetsuro Hirose
    EMBO JOURNAL 36 10 1447 - 1462 2017年05月 [査読有り][通常論文]
     
    NEAT1_2 long noncoding RNA (lncRNA) is the molecular scaffold of paraspeckle nuclear bodies. Here, we report an improved RNA extraction method: extensive needle shearing or heating of cell lysate in RNA extraction reagent improved NEAT1_2 extraction by 20-fold (a property we term "semi-extractability"), whereas using a conventional method NEAT1_2 was trapped in the protein phase. The improved extraction method enabled us to estimate that approximately 50 NEAT1_2 molecules are present in a single paraspeckle. Another architectural lncRNA, IGS16, also exhibited similar semi-extractability. A comparison of RNA-seq data from needle-sheared and control samples revealed the existence of multiple semi-extractable RNAs, many of which were localized in sub-nuclear granule-like structures. The semi-extractability of NEAT1_2 correlated with its association with paraspeckle proteins and required the prion-like domain of the RNA-binding protein FUS. This observation suggests that tenacious RNA-protein and protein-protein interactions, which drive nuclear body formation, are responsible for semi-extractability. Our findings provide a foundation for the discovery of the architectural RNAs that constitute nuclear bodies.
  • Poller W, Dimmeler S, Heymans S, Zeller T, Haas J, Karakas M, Leistner DM, Jakob P, Nakagawa S, Blankenberg S, Engelhardt S, Thum T, Weber C, Meder B, Hajjar R, andmesser U
    European heart journal 2017年04月 [査読有り][通常論文]
  • Xiaona Chen, Liangqiang He, Yu Zhao, Yuying Li, Suyang Zhang, Kun Sun, Karl So, Fengyuan Chen, Liang Zhou, Leina Lu, Lijun Wang, Xihua Zhu, Xichen Bao, Miguel A. Esteban, Shinichi Nakagawa, Kannanganattu V. Prasanth, Zhenguo Wu, Hao Sun, Huating Wang
    CELL DISCOVERY 3 17002  2017年03月 [査読有り][通常論文]
     
    Malat1 is one of the most abundant long non-coding RNAs in various cell types; its exact cellular function is still a matter of intense investigation. In this study we characterized the function of Malat1 in skeletal muscle cells and muscle regeneration. Utilizing both in vitro and in vivo assays, we demonstrate that Malat1 has a role in regulating gene expression during myogenic differentiation of myoblast cells. Specifically, we found that knockdown of Malat1 accelerates the myogenic differentiation in cultured cells. Consistently, Malat1 knockout mice display enhanced muscle regeneration after injury and deletion of Malat1 in dystrophic mdx mice also improves the muscle regeneration. Mechanistically, in the proliferating myoblasts, Malat1 recruits Suv39h1 to MyoD-binding loci, causing trimethylation of histone 3 lysine 9 (H3K9me3), which suppresses the target gene expression. Upon differentiation, the pro-myogenic miR-181a is increased and targets the nuclear Malat1 transcripts for degradation through Ago2-dependent nuclear RNA-induced silencing complex machinery; the Malat1 decrease subsequently leads to the destabilization of Suv39h1/HP1 beta/HDAC1-repressive complex and displacement by a Set7-containing activating complex, which allows MyoD trans-activation to occur. Together, our findings identify a regulatory axis of miR-181a-Malat1-MyoD/Suv39h1 in myogenesis and uncover a previously unknown molecular mechanism of Malat1 action in gene regulation.
  • Mathieu Quesnel-Vallieres, Zahra Dargaei, Manuel Irimia, Thomas Gonatopoulos-Pournatzis, Joanna Y. Ip, Mingkun Wu, Timothy Sterne-Weiler, Shinichi Nakagawa, Melanie A. Woodin, Benjamin J. Blencowe, Sabine P. Cordes
    MOLECULAR CELL 64 6 1023 - 1034 2016年12月 [査読有り][通常論文]
     
    A key challenge in understanding and ultimately treating autism is to identify common molecular mechanisms underlying this genetically heterogeneous disorder. Transcriptomic profiling of autistic brains has revealed correlated misregulation of the neuronal splicing regulator nSR100/SRRM4 and its target microexon splicing program in more than one-third of analyzed individuals. To investigate whether nSR100 misregulation is causally linked to autism, we generated mutant mice with reduced levels of this protein and its target splicing program. Remarkably, these mice display multiple autistic-like features, including altered social behaviors, synaptic density, and signaling. Moreover, increased neuronal activity, which is often associated with autism, results in a rapid decrease in nSR100 and splicing of microexons that significantly overlap those misregulated in autistic brains. Collectively, our results provide evidence that misregulation of an nSR100-dependent splicing network controlled by changes in neuronal activity is causally linked to a substantial fraction of autism cases.
  • Takehisa Sakaguchi, Yuko Hasegawa, Neil Brockdorff, Ken Tsutsui, Kimiko M. Tsutsui, Takashi Sado, Shinichi Nakagawa
    DEVELOPMENTAL CELL 39 1 11 - 12 2016年10月 [査読有り][通常論文]
  • Jason A. West, Mari Mito, Satoshi Kurosaka, Toru Takumi, Chiharu Tanegashima, Takeshi Chujo, Kaori Yanaka, Robert E. Kingston, Tetsuro Hirose, Charles Bond, Archa Fox, Shinichi Nakagawa
    JOURNAL OF CELL BIOLOGY 214 7 817 - 830 2016年09月 [査読有り][通常論文]
     
    Paraspeckles are nuclear bodies built on the long noncoding RNA Neat1, which regulates a variety of physiological processes including cancer progression and corpus luteum formation. To obtain further insight into the molecular basis of the function of paraspeckles, we performed fine structural analyses of these nuclear bodies using structural illumination microscopy. Notably, paraspeckle proteins are found within different layers along the radially arranged bundles of Neat1 transcripts, forming a characteristic core-shell spheroidal structure. In cells lacking the RNA binding protein Fus, paraspeckle spheroids are disassembled into smaller particles containing Neat1, which are diffusely distributed in the nucleoplasm. Sequencing analysis of RNAs purified from paraspeckles revealed that AG-rich transcripts associate with Neat1, which are distributed along the shell of the paraspeckle spheroids. We propose that paraspeckles sequester core components inside the spheroids, whereas the outer surface associates with other components in the nucleoplasm to fulfill their function.
  • Aparna Anantharaman, Mahdieh Jadaliha, Vidisha Tripathi, Shinichi Nakagawa, Tetsuro Hirose, Michael F. Jantsch, Supriya G. Prasanth, Kannanganattu V. Prasanth
    SCIENTIFIC REPORTS 6 34043  2016年09月 [査読有り][通常論文]
     
    Paraspeckles are sub-nuclear domains that are nucleated by long noncoding RNA Neat1. While interaction of protein components of paraspeckles and Neat1 is understood, there is limited information on the interaction of non-structural RNA components with paraspeckles. Here, by varying paraspeckle number and size, we investigate how paraspeckles influence the nuclear organization of their nonstructural RNA component Ctn RNA. Our results show that Ctn RNA remains nuclear-retained in the absence of intact paraspeckles, suggesting that they do not regulate nuclear retention of Ctn RNA. In the absence of Neat1, Ctn RNA continues to interact with paraspeckle protein NonO to form residual nuclear foci. In addition, in the absence of Neat1-nucleated paraspeckles, a subset of Ctn RNA localizes to the perinucleolar regions. Concomitant with increase in number of paraspeckles, transcriptional reactivation resulted in increased number of paraspeckle-localized Ctn RNA foci. Similar to Neat1, proteasome inhibition altered the localization of Ctn RNA, where it formed enlarged paraspeckle-like foci. Super-resolution structured illumination microscopic analyses revealed that in paraspeckles, Ctn RNA partially co-localized with Neat1, and displayed a more heterogeneous intra-paraspeckle localization. Collectively, these results show that while paraspeckles do not influence nuclear retention of Ctn RNA, they modulate its intranuclear compartmentalization.
  • Carmen Adriaens, Laura Standaert, Jasmine Barra, MathilDe latil, Annelien Verfaillie, Peter Kalev, Bram Boeckx, Paul W. G. Wijnhoven, Enrico Radaelli, William Vermi, Eleonora Leucci, Gaelle Lapouge, Benjamin Beck, Joost van den Oord, Shinichi Nakagawa, Tetsuro Hirose, Anna A. Sablina, Diether Lambrechts, Stein Aerts, Cedric Blanpain, Jean-Christophe Marine
    NATURE MEDICINE 22 8 861 - + 2016年08月 [査読有り][通常論文]
     
    In a search for mediators of the p53 tumor suppressor pathway, which induces pleiotropic and often antagonistic cellular responses, we identified the long noncoding RNA (lncRNA) NEAT1. NEAT1 is an essential architectural component of paraspeckle nuclear bodies, whose pathophysiological relevance remains unclear. Activation of p53, pharmacologically or by oncogene-induced replication stress, stimulated the formation of paraspeckles in mouse and human cells. Silencing Neat1 expression in mice, which prevents paraspeckle formation, sensitized preneoplastic cells to DNA-damage-induced cell death and impaired skin tumorigenesis. We provide mechanistic evidence that NEAT1 promotes ATR signaling in response to replication stress and is thereby engaged in a negative feedback loop that attenuates oncogene-dependent activation of p53. NEAT1 targeting in established human cancer cell lines induced synthetic lethality with genotoxic chemotherapeutics, including PARP inhibitors, and nongenotoxic activation of p53. This study establishes a key genetic link between NEAT1 paraspeckles, p53 biology and tumorigenesis and identifies NEAT1 as a promising target to enhance sensitivity of cancer cells to both chemotherapy and p53 reactivation therapy.
  • Joanna Y. Ip, Masamitsu Sone, Chieko Nashiki, Qun Pan, Kiyoyuki Kitaichi, Kaori Yanaka, Takaya Abe, Keizo Takao, Tsuyoshi Miyakawa, Benjamin J. Blencowe, Shinichi Nakagawa
    SCIENTIFIC REPORTS 6 27204  2016年06月 [査読有り][通常論文]
     
    The long noncoding RNA Gomafu/MIAT/Rncr2 is thought to function in retinal cell specification, stem cell differentiation and the control of alternative splicing. To further investigate physiological functions of Gomafu, we created mouse knockout (KO) model that completely lacks the Gomafu gene. The KO mice did not exhibit any developmental deficits. However, behavioral tests revealed that the KO mice are hyperactive. This hyperactive behavior was enhanced when the KO mice were treated with the psychostimulant methamphetamine, which was associated with an increase in dopamine release in the nucleus accumbens. RNA sequencing analyses identified a small number of genes affected by the deficiency of Gomafu, a subset of which are known to have important neurobiological functions. These observations suggest that Gomafu modifies mouse behavior thorough a mild modulation of gene expression and/or alternative splicing of target genes.
  • Mari Mito, Tetsuya Kawaguchi, Tetsuro Hirose, Shinichi Nakagawa
    METHODS 98 158 - 165 2016年04月 [査読有り][通常論文]
     
    A number of non-membranous cellular bodies have been identified in higher eukaryotes, and these bodies contain a specific set of proteins and RNAs that are used to fulfill their functions. The size of these RNA-containing cellular bodies is usually on a submicron scale, making it difficult to observe fine structures using optical microscopy due to the diffraction limitation of visible light. Recently, microscope companies have released super-resolution microscopes that were developed using different principles, enabling the observation of sub-micron structures not resolvable in conventional fluorescent microscopy. Here, we describe multi-color fluorescent in situ hybridization techniques optimized for the simultaneous detection of RNA and proteins using super-resolution microscopy, namely structured illumination microscopy (SIM). (C) 2015 The Authors. Published by Elsevier Inc.
  • Martina Gast, Blanche Schroen, Antje Voigt, Jan Haas, Uwe Kuehl, Dirk Lassner, Carsten Skurk, Felicitas Escher, Xiaomin Wang, Adelheid Kratzer, Katharina Michalik, Anna Papageorgiou, Tim Peters, Madlen Loebel, Sabrina Wilk, Nadine Althof, Kannanganattu V. Prasanth, Hugo Katus, Benjamin Meder, Shinichi Nakagawa, Carmen Scheibenbogen, Heinz-Peter Schultheiss, Ulf Landmesser, Stefanie Dimmeler, Stephane Heymans, Wolfgang Poller
    JOURNAL OF MOLECULAR CELL BIOLOGY 8 2 178 - 181 2016年04月 [査読有り][通常論文]
  • Xinying Zong, Shinichi Nakagawa, Susan M. Freier, Jingyi Fei, Taekjip Ha, Supriya G. Prasanth, Kannanganattu V. Prasanth
    NUCLEIC ACIDS RESEARCH 44 6 2898 - 2908 2016年04月 [査読有り][通常論文]
     
    The RNase P-mediated endonucleolytic cleavage plays a crucial role in the 3' end processing and cellular accumulation of MALAT1, a nuclear-retained long noncoding RNA that promotes malignancy. The regulation of this cleavage event is largely undetermined. Here we characterize a broadly expressed natural antisense transcript at the MALAT1 locus, designated as TALAM1, that positively regulates MALAT1 levels by promoting the 3' end cleavage and maturation of MALAT1 RNA. TALAM1 RNA preferentially localizes at the site of transcription, and also interacts with MALAT1 RNA. Depletion of TALAM1 leads to defects in the 3' end cleavage reaction and compromises cellular accumulation of MALAT1. Conversely, overexpression of TALAM1 facilitates the cleavage reaction in trans. Interestingly, TALAM1 is also positively regulated by MALAT1 at the level of both transcription and RNA stability. Together, our data demonstrate a novel feed-forward positive regulatory loop that is established to maintain the high cellular levels of MALAT1, and also unravel the existence of sense-antisense mediated regulatory mechanism for cellular lncRNAs that display RNase P-mediated 3' end processing.
  • Tim Peters, Steffie Hermans-Beijnsberger, Abdelaziz Beqqali, Nicole Bitsch, Shinichi Nakagawa, Kannanganattu V. Prasanth, Leon J. de Windt, Ralph J. van Oort, Stephane Heymans, Blanche Schroen
    PLOS ONE 11 2 e0150236  2016年02月 [査読有り][通常論文]
     
    Background Long non-coding RNAs (lncRNAs) are a class of RNA molecules with diverse regulatory functions during embryonic development, normal life, and disease in higher organisms. However, research on the role of lncRNAs in cardiovascular diseases and in particular heart failure is still in its infancy. The exceptionally well conserved nuclear lncRNA Metastasis associated in lung adenocarcinoma transcript 1 (Malat-1) is a regulator of mRNA splicing and highly expressed in the heart. Malat-1 modulates hypoxia-induced vessel growth, activates ERK/MAPK signaling, and scavenges the anti-hypertrophic microRNA-133. We therefore hypothesized that Malat-1 may act as regulator of cardiac hypertrophy and failure during cardiac pressure overload induced by thoracic aortic constriction (TAC) in mice. Results Absence of Malat-1 did not affect cardiac hypertrophy upon pressure overload: Heart weight to tibia length ratio significantly increased in WT mice (sham: 5.78 +/- 0.55, TAC 9.79 +/- 1.82 g/mm; p<0.001) but to a similar extend also in Malat-1 knockout (KO) mice (sham: 6.21 +/- 1.12, TAC 8.91 +/- 1.74 g/mm; p<0.01) with no significant difference between genotypes. As expected, TAC significantly reduced left ventricular fractional shortening in WT (sham: 38.81 +/- 6.53%, TAC: 23.14 +/- 11.99%; p<0.01) but to a comparable degree also in KO mice (sham: 37.01 +/- 4.19%, TAC: 25.98 +/- 9.75%; p<0.05). Histological hallmarks of myocardial remodeling, such as cardiomyocyte hypertrophy, increased interstitial fibrosis, reduced capillary density, and immune cell infiltration, did not differ significantly between WT and KO mice after TAC. In line, the absence of Malat-1 did not significantly affect angiotensin II-induced cardiac hypertrophy, dysfunction, and overall remodeling. Above that, pressure overload by TAC significantly induced mRNA levels of the hypertrophy marker genes Nppa, Nppb and Acta1, to a similar extend in both genotypes. Alternative splicing of Ndrg2 after TAC was apparent in WT (isoform ratio; sham: 2.97 +/- 0.26, TAC 1.57 +/- 0.40; p<0.0001) and KO mice (sham: 3.64 +/- 0.37; TAC: 2.24 +/- 0.76; p<0.0001) and interestingly differed between genotypes both at baseline and after pressure overload (p<0.05 each). Conclusion These findings confirm a role for the lncRNA Malat-1 in mRNA splicing. However, no critical role for Malat-1 was found in pressure overload-induced heart failure in mice, despite its reported role in vascularization, ERK/MAPK signaling, and regulation of miR-133.
  • Rei Yoshimoto, Akila Mayeda, Minoru Yoshida, Shinichi Nakagawa
    BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 1859 1 192 - 199 2016年01月 [査読有り][通常論文]
     
    A recent massive parallel sequencing analysis has shown the fact that more than 80% of the human genome is transcribed into RNA. Among many kinds of the non-protein coding RNAs, we focus on the metastasis associated lung adenocarcinoma transcript 1 (MALAT1) that is a long non-coding RNA upregulated in metastatic carcinoma cells. Two molecular functions of MALAT1 have been proposed, one is the control of alternative splicing and the other is the transcriptional regulation. In this review, we document the molecular characteristics and functions of MALAT1 and shed light on the implication in the molecular pathology of various cancers. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. (C) 2015 Elsevier B.V. All rights reserved.
  • Hirose T, Nakagawa S
    Biochimica et biophysica acta 1859 1 1 - 2 2016年01月 [査読有り][通常論文]
  • Kentaro Ishida, Kenjyo Miyauchi, Yuko Kimura, Mari Mito, Shunpei Okada, Tsutomu Suzuki, Shinichi Nakagawa
    GENES TO CELLS 20 11 887 - 901 2015年11月 [査読有り][通常論文]
     
    Short interspersed elements (SINEs) comprise a significant portion of mammalian genomes and regulate gene expression through a variety of mechanisms. Here, we show that Myodonta clade-specific 4.5S RNA(H) (4.5SH), an abundant nuclear noncoding RNA that is highly homologous to the retrotransposon SINE B1, controls the expression of reporter gene that contains the antisense insertion of SINE B1 via nuclear retention. The depletion of endogenous 4.5SH with antisense oligonucleotides neutralizes the nuclear retention and changes the subcellular distribution of the reporter transcripts containing the antisense SINE B1 insertion. Importantly, endogenous transcripts with antisense SINE B1 were increased in the cytoplasm after knockdown of 4.5SH, leading to a decrease in cellular growth. We propose a tentative hypothesis that the amplification of the 4.5SH cluster in specific rodent species might delineate their evolutionary direction via the regulation of genes containing the antisense insertion of SINE B1.
  • Yoshimoto R, Mayeda A, Yoshida M, Nakagawa S
    Biochimica et biophysica acta 1859 1 192 - 199 2015年10月 [査読有り][通常論文]
  • Norishige Yamada, Yuko Hasegawa, Minghui Yue, Tomofumi Hamada, Shinichi Nakagawa, Yuya Ogawa
    PLoS Genetics 11 8 e1005430  2015年08月01日 [査読有り][通常論文]
     
    To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U.
  • Nakagawa S
    Biochimica et biophysica acta 1859 1 177 - 183 2015年06月 [査読有り][通常論文]
  • Yuko Hasegawa, Shinichi Nakagawa
    Long Noncoding RNAs Structures and Functions 133 - 149 2015年01月01日 [査読有り][通常論文]
     
    Recent studies have revealed the functional signifi cance of long noncoding RNA (lncRNA) in various biological processes including epigenetic gene regulations. Genomic imprinting is one of the epigenetic processes related to lncRNA in mammals, which controls parent-of-origin-specific gene expression essential for normal development. To date, over 100 genes have been recognized as imprinted genes, the majority of them form clusters on the genome. Each of these imprinting clusters contains DNA regulatory elements called imprinting control regions (ICRs), which are frequently located near the lncRNA genes. In some cases, genetically modifi ed mice and human patients exhibiting imprinting disorder show aberrant expression of these lncRNAs, suggesting a close relationship between genomic imprinting and lncRNAs. DNA methylation and histone modifi cations are the principal molecular mechanisms of epigenetic gene regulation, and recent progress has uncovered diversities and complexities of the lncRNA actions in these processes. In this chapter, we summarize research on genomic imprinting by focusing on the role of lncRNAs to provide insight into lncRNA-mediated regulation of gene expression.
  • Yasushi Okada, Shinichi Nakagawa
    Methods in molecular biology (Clifton, N.J.) 1262 21 - 35 2015年 [査読有り][通常論文]
     
    The sizes of nuclear bodies and other nuclear structures are normally no more than a few hundred nanometers. This size is below the resolution limit of light microscopy and thus requires electron microscopy for direct observation. Recent developments in super-resolution microscopy have extended the resolution of light microscopy to beyond 100 nm. Here, we describe a super-resolution technique, gated STED, for the analysis of the structure of nuclear bodies, with emphasis on the sample preparation and other technical tips that are important to obtain high-quality super-resolution images.
  • Hasegawa Y, Nakagawa S
    Methods in molecular biology (Clifton, N.J.) 1262 333 - 348 2015年 [査読有り][通常論文]
  • Laura Standaert, Carmen Adriaens, Enrico Radaelli, Alexandra Van Keymeulen, Cedric Blanpain, Tetsuro Hirose, Shinichi Nakagawa, Jean-Christophe Marine
    RNA 20 12 1844 - 1849 2014年12月 [査読有り][通常論文]
     
    The lncRNA Neat1 is an essential architectural component of paraspeckle nuclear bodies. Although cell-based studies identified Neat1-paraspeckles as key regulators of gene expression through retention of hyperdited mRNAs and/or transcription factors, it is unclear under which specific physiological conditions paraspeckles are formed in vivo and whether they have any biological relevance. Herein, we show that paraspeckles are assembled in luminal epithelial cells during mammary gland development. Importantly, genetic ablation of Neat1 results in aberrant mammary gland morphogenesis and lactation defects. We provide evidence that the lactation defect is caused by a decreased ability of Neat1-mutant cells to sustain high rates of proliferation during lobular-alveolar development. This study is the first to assign an important biological function to the lncRNA Neat1 and to link it to the presence of paraspeckles nuclear bodies in vivo.
  • Shinichi Nakagawa, Masayuki Shimada, Kaori Yanaka, Mari Mito, Takashi Arai, Eiki Takahashi, Youko Fujita, Toshihiko Fujimori, Laura Standaert, Jean-Christophe Marine, Tetsuro Hirose
    DEVELOPMENT 141 23 4618 - 4627 2014年12月 [査読有り][通常論文]
     
    Neat1 is a non-protein-coding RNA that serves as an architectural component of the nuclear bodies known as paraspeckles. Although cell-based studies indicate that Neat1 is a crucial regulator of gene expression, its physiological relevance remains unclear. Here, we find that Neat1 knockout (KO) mice stochastically fail to become pregnant despite normal ovulation. Unilateral transplantation of wildtype ovaries or the administration of progesterone partially rescued the phenotype, suggesting that corpus luteum dysfunction and concomitant low progesterone were the primary causes of the decreased fertility. In contrast to the faint expression observed in most of the adult tissues, Neat1 was highly expressed in the corpus luteum, and the formation of luteal tissue was severely impaired in nearly half of the Neat1 KO mice. These observations suggest that Neat1 is essential for the formation of the corpus luteum and for the subsequent establishment of pregnancy under a suboptimal condition that has not yet been identified.
  • Dimple Chakravarty, Andrea Sboner, Sujit S. Nair, Eugenia Giannopoulou, Ruohan Li, Sven Hennig, Juan Miguel Mosquera, Jonathan Pauwels, Kyung Park, Myriam Kossai, Theresa Y. MacDonald, Jacqueline Fontugne, Nicholas Erho, Ismael A. Vergara, Mercedeh Ghadessi, Elai Davicioni, Robert B. Jenkins, Nallasivam Palanisamy, Zhengming Chen, Shinichi Nakagawa, Tetsuro Hirose, Neil H. Bander, Himisha Beltran, Archa H. Fox, Olivier Elemento, Mark A. Rubin
    NATURE COMMUNICATIONS 5 5 5383  2014年11月 [査読有り][通常論文]
     
    The androgen receptor (AR) plays a central role in establishing an oncogenic cascade that drives prostate cancer progression. Some prostate cancers escape androgen dependence and are often associated with an aggressive phenotype. The oestrogen receptor alpha (ER alpha) is expressed in prostate cancers, independent of AR status. However, the role of ER alpha remains elusive. Using a combination of chromatin immunoprecipitation (ChIP) and RNA-sequencing data, we identified an ER alpha-specific non-coding transcriptome signature. Among putatively ER alpha-regulated intergenic long non-coding RNAs (lncRNAs), we identified nuclear enriched abundant transcript 1 (NEAT1) as the most significantly overexpressed lncRNA in prostate cancer. Analysis of two large clinical cohorts also revealed that NEAT1 expression is associated with prostate cancer progression. Prostate cancer cells expressing high levels of NEAT1 were recalcitrant to androgen or AR antagonists. Finally, we provide evidence that NEAT1 drives oncogenic growth by altering the epigenetic landscape of target gene promoters to favour transcription.
  • Shinichi Nakagawa
    Regulatory Non-Coding RNAs: Methods and Protocols 1206 107 - 122 2014年09月20日 [査読有り][通常論文]
     
    In situ hybridization is a powerful method of examining the spatial distribution of RNA molecules at the subcellular level and serves as a basic technique in the fields of cell and developmental biology. In this technique, target RNAs are fixed in cells using formaldehyde and then hybridized to complementary probes labeled with modified nucleotides that are subsequently detected by immunohistochemical methods. Here, the procedures that are commonly used for the detection of RNA in mammalian tissues and cells are described, focusing on technical tips that improve the sensitivity, productivity, and reproducibility.
  • Akira Ishizuka, Yuko Hasegawa, Kentaro Ishida, Kaori Yanaka, Shinichi Nakagawa
    GENES TO CELLS 19 9 704 - 721 2014年09月 [査読有り][通常論文]
     
    Gomafu/MIAT/Rncr2 is a long noncoding RNA that has been proposed to control retinal cell specification, stem cell differentiation and alternative splicing of schizophrenia-related genes. However, how Gomafu controls these biological processes at the molecular level has remained largely unknown. In this study, we identified the RNA-binding protein Celf3 as a novel Gomafu-associating protein. Knockdown of Celf3 led to the down-regulation of Gomafu, and cross-link RNA precipitation analysis confirmed specific binding between Celf3 and Gomafu. In the neuroblastoma cell line Neuro2A, Celf3 formed novel nuclear bodies (named CS bodies) that colocalized with SF1, another Gomafu-binding protein. Gomafu, however, was not enriched in the CS bodies; instead, it formed distinct nuclear bodies in separate regions in the nucleus. These observations suggest that Gomafu indirectly modulates the function of the splicing factors SF1 and Celf3 by sequestering these proteins into separate nuclear bodies.
  • Yukiko Murota, Hirotsugu Ishizu, Shinichi Nakagawa, Yuka W. Iwasaki, Shinsuke Shibata, Miharu K. Kamatani, Kuniaki Saito, Hideyuki Okano, Haruhiko Siomi, Mikiko C. Siomi
    CELL REPORTS 8 1 103 - 113 2014年07月 [査読有り][通常論文]
     
    PIWI-interacting RNAs (piRNAs) direct Piwi to repress transposons and maintain genome integrity in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear Yb bodies, the centers of piRNA biogenesis. Here, we show that piRNA intermediates arising from the piRNA cluster flamenco (flam) localize to perinuclear foci adjacent to Yb bodies, termed Flam bodies. RNAi-based screening of piRNA factors revealed that Flam body formation depends on Yb, the core component of Yb bodies, while Piwi and another Yb body component, Armitage, are dispensable for formation. Abolishing the RNA-binding activity of Yb disrupts both Flam bodies and Yb bodies. Yb directly binds flam, but not transcripts from neighboring protein-coding genes. Thus, Yb integrates piRNA intermediates and piRNA processing factors selectively into Flam bodies and Yb bodies, respectively. We suggest that Yb is a key upstream factor in the cytoplasmic phase of the piRNA pathway in ovarian somatic cells.
  • Nakagawa S, Kageyama Y
    Biochimica et biophysica acta 1839 3 215 - 222 2014年03月 [査読有り][通常論文]
  • Shinichi Nakagawa, Tatsuya Hirano
    NATURE STRUCTURAL & MOLECULAR BIOLOGY 21 3 207 - 208 2014年03月 [査読有り][通常論文]
     
    Long noncoding RNAs (IncRNAs) have divergent roles in the nuclei of higher eukaryotes, including chromatin modification and regulation of nuclear bodies. A new study adds a new IncRNA function to the current list: serving as a platform for trans-chromosomal associations. At least three gene loci located on different chromosomes are brought together around the transcription site of a IncRNA termed functional intergenic repeating RNA element (Firre).
  • G. Barry, J. A. Briggs, D. P. Vanichkina, E. M. Poth, N. J. Beveridge, V. S. Ratnu, S. P. Nayler, K. Nones, J. Hu, T. W. Bredy, S. Nakagawa, F. Rigo, R. J. Taft, M. J. Cairns, S. Blackshaw, E. J. Wolvetang, J. S. Mattick
    Molecular Psychiatry 19 4 486 - 494 2014年 [査読有り][通常論文]
     
    Schizophrenia (SZ) is a complex disease characterized by impaired neuronal functioning. Although defective alternative splicing has been linked to SZ, the molecular mechanisms responsible are unknown. Additionally, there is limited understanding of the early transcriptomic responses to neuronal activation. Here, we profile these transcriptomic responses and show that long non-coding RNAs (lncRNAs) are dynamically regulated by neuronal activation, including acute downregulation of the lncRNA Gomafu, previously implicated in brain and retinal development. Moreover, we demonstrate that Gomafu binds directly to the splicing factors QKI and SRSF1 (serine/arginine-rich splicing factor 1) and dysregulation of Gomafu leads to alternative splicing patterns that resemble those observed in SZ for the archetypal SZ-associated genes DISC1 and ERBB4. Finally, we show that Gomafu is downregulated in post-mortem cortical gray matter from the superior temporal gyrus in SZ. These results functionally link activity-regulated lncRNAs and alternative splicing in neuronal function and suggest that their dysregulation may contribute to neurological disorders. © 2014 Macmillan Publishers Limited.
  • Tetsuro Hirose, Giorgio Virnicchi, Akie Tanigawa, Takao Naganuma, Ruohan Li, Hiroshi Kimura, Takahide Yokoi, Shinichi Nakagawa, Marianne Benard, Archa H. Fox, Gerard Pierron
    MOLECULAR BIOLOGY OF THE CELL 25 1 169 - 183 2014年01月 [査読有り][通常論文]
     
    Paraspeckles are subnuclear structures formed around nuclear paraspeckle assembly transcript 1 (NEAT1)/MEN epsilon/beta long noncoding RNA (IncRNA). Here we show that paraspeckles become dramatically enlarged after proteasome inhibition. This enlargement is mainly caused by NEAT1 transcriptional up-regulation rather than accumulation of undegraded paraspeckle proteins. Of interest, however, using immuno-electron microscopy, we find that key paraspeckle proteins become effectively depleted from the nucleoplasm by 50% when paraspeckle assembly is enhanced, suggesting a sequestration mechanism. We also perform microarrays from NEAT1-knockdown cells and find that NEAT1 represses transcription of several genes, including the RNA-specific adenosine deaminase B2 (ADARB2) gene. In contrast, the NEAT1-binding paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for ADARB2 transcription. This leads us to hypothesize that ADARB2 expression is controlled by NEAT1-dependent sequestration of SFPQ. Accordingly, we find that ADARB2 expression is strongly reduced upon enhanced SFPQ sequestration by proteasome inhibition, with concomitant reduction in SFPQ binding to the ADARB2 promoter. Finally, NEAT1(-/-) fibroblasts are more sensitive to proteasome inhibition, which triggers cell death, suggesting that paraspeckles/NEAT1 attenuates the cell death pathway. These data further confirm that paraspeckles are stress-responsive nuclear bodies and provide a model in which induced NEAT1 controls target gene transcription by protein sequestration into paraspeckles.
  • Nishimoto Y, Nakagawa S, Hirose T, Okano HJ, Takao M, Shibata S, Suyama S, Kuwako K, Imai T, Murayama S, Suzuki N, Okano H
    EMBO J 31 4020 - 4034 2013年07月 [査読有り][通常論文]
  • Yoshinori Nishimoto, Shinichi Nakagawa, Tetsuro Hirose, Hirotaka James Okano, Masaki Takao, Shinsuke Shibata, Satoshi Suyama, Ken-Ichiro Kuwako, Takao Imai, Shigeo Murayama, Norihiro Suzuki, Hideyuki Okano
    Molecular Brain 6 1 31  2013年 [査読有り][通常論文]
     
    Background: A long non-coding RNA (lncRNA), nuclear-enriched abundant transcript 1-2 (NEAT1-2), constitutes nuclear bodies known as "paraspeckles". Mutations of RNA binding proteins, including TAR DNA-binding protein-43 (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS), have been described in amyotrophic lateral sclerosis (ALS). ALS is a devastating motor neuron disease, which progresses rapidly to a total loss of upper and lower motor neurons, with consciousness sustained. The aim of this study was to clarify the interaction of paraspeckles with ALS-associated RNA-binding proteins, and to identify increased occurrence of paraspeckles in the nucleus of ALS spinal motor neurons. Results: In situ hybridization (ISH) and ultraviolet cross-linking and immunoprecipitation were carried out to investigate interactions of NEAT1-2 lncRNA with ALS-associated RNA-binding proteins, and to test if paraspeckles form in ALS spinal motor neurons. As the results, TDP-43 and FUS/TLS were enriched in paraspeckles and bound to NEAT1-2 lncRNA directly. The paraspeckles were localized apart from the Cajal bodies, which were also known to be related to RNA metabolism. Analyses of 633 human spinal motor neurons in six ALS cases showed NEAT1-2 lncRNA was upregulated during the early stage of ALS pathogenesis. In addition, localization of NEAT1-2 lncRNA was identified in detail by electron microscopic analysis combined with ISH for NEAT1-2 lncRNA. The observation indicating specific assembly of NEAT1-2 lncRNA around the interchromatin granule-associated zone in the nucleus of ALS spinal motor neurons verified characteristic paraspeckle formation. Conclusions: NEAT1-2 lncRNA may act as a scaffold of RNAs and RNA binding proteins in the nuclei of ALS motor neurons, thereby modulating the functions of ALS-associated RNA-binding proteins during the early phase of ALS. These findings provide the first evidence of a direct association between paraspeckle formation and a neurodegenerative disease, and may shed light on the development of novel therapeutic targets for the treatment of ALS. © 2013 Nishimoto et al. licensee BioMed Central Ltd.
  • Takao Naganuma, Shinichi Nakagawa, Akie Tanigawa, Yasnory F. Sasaki, Naoki Goshima, Tetsuro Hirose
    EMBO JOURNAL 31 20 4020 - 4034 2012年10月 [査読有り][通常論文]
     
    Paraspeckles are unique subnuclear structures built around a specific long noncoding RNA, NEAT1, which is comprised of two isoforms produced by alternative 3'-end processing (NEAT1_1 and NEAT1_2). To address the precise molecular processes that lead to paraspeckle formation, we identified 35 paraspeckle proteins (PSPs), mainly by colocalization screening with a fluorescent protein-tagged full-length cDNA library. Most of the newly identified PSPs possessed various putative RNA-binding domains. Subsequent RNAi analyses identified seven essential PSPs for paraspeckle formation. One of the essential PSPs, HNRNPK, appeared to affect the production of the essential NEAT1_2 isoform by negatively regulating the 3'-end polyadenylation of the NEAT1_1 isoform. An in vitro 3'-end processing assay revealed that HNRNPK arrested binding of the CPSF6-NUDT21 (CFIm) complex in the vicinity of the alternative polyadenylation site of NEAT1_1. In vitro binding assays showed that HNRNPK competed with CPSF6 for binding to NUDT21, which was the underlying mechanism to arrest CFIm binding by HNRNPK. This HNRNPK function led to the preferential accumulation of NEAT1_2 and initiated paraspeckle construction with multiple PSPs. The EMBO Journal (2012) 31, 4020-4034. doi:10.1038/emboj.2012.251; Published online 7 September 2012
  • Tetsuro Hirose, Shinichi Nakagawa
    Biomolecular Concepts 3 5 415 - 428 2012年10月01日 [査読有り][通常論文]
     
    The mammalian cell nucleus is a highly compartmentalized system in which multiple subnuclear structures, called nuclear bodies, exist in the nucleoplasmic spaces. Some of the nuclear bodies contain specific long non-coding RNAs (ncRNAs) as their components, and may serve as sites for long ncRNA functions that remain largely enigmatic. A paraspeckle is a nuclear body that is almost ubiquitously observed in mammalian cultured cells but is cell population-specific in adult mouse tissue. The paraspeckle structure is RNase-sensitive. Long ncRNAs, termed MENε/β ncRNAs (also referred to as NEAT1 ncRNAs), have been identified as the RNA components of the paraspeckles. Specific elimination has revealed that MENε/β ncRNAs are essential components for the formation of the intact paraspeckle structure. Paraspeckle formation requires the continual MENε/β ncRNA biogenesis process, including ongoing transcription, alternative 3′-end processing, and stabilization. Some paraspeckle-localized RNA-binding proteins (p54/nrb and PSF) direct paraspeckle formation through the selective stabilization of MENβ ncRNA. Both MENε/β ncRNA expression and their subsequent interactions with paraspeckle proteins can be regulated under environmental and developmental conditions, which are reflected in the size and number of the paraspeckles. However, how paraspeckles function remains largely unsolved. Paraspeckles appear to serve as the site of nuclear retention of specific mRNAs that are selectively transported to the cytoplasm upon certain signals. Alternatively, MENε/β ncRNAs may sequester paraspeckle proteins that function outside the paraspeckles. This review focuses on known aspects of paraspeckles and provides a model of their structure and function.
  • Shinichi Nakagawa, Tetsuro Hirose
    CELLULAR AND MOLECULAR LIFE SCIENCES 69 18 3027 - 3036 2012年09月 [査読有り][通常論文]
     
    The nucleus of higher eukaryotes, such as humans and mice, is compartmentalized into multiple nuclear bodies, an organization that allows for the regulation of complex gene expression pathways that are characteristic of these organisms. Paraspeckles are recently discovered, mammalian-specific nuclear bodies built on a long, non-protein-coding RNA, NEAT1 (nuclear-enriched abundant transcript 1), which assembles various protein components including RNA-binding proteins of the DBHS (Drosophila behavior and human splicing) family. Paraspeckles have been proposed to control several biological processes, such as stress responses and cellular differentiation, but their function at the whole animal level remains unclear. In this review, we summarize a series of studies on paraspeckles that have been carried out in the decade since their discovery and discuss their physiological function and molecular mechanism.
  • Shinichi Nakagawa, Joanna Y. Ip, Go Shioi, Vidisha Tripathi, Xinying Zong, Tetsuro Hirose, Kannanganattu V. Prasanth
    RNA-A PUBLICATION OF THE RNA SOCIETY 18 8 1487 - 1499 2012年08月 [査読有り][通常論文]
     
    Malat1 is an abundant long, noncoding RNA that localizes to nuclear bodies known as nuclear speckles, which contain a distinct set of pre-mRNA processing factors. Previous studies in cell culture have demonstrated that Malat1 interacts with pre-mRNA splicing factors, including the serine-and arginine-rich (SR) family of proteins, and regulates a variety of biological processes, including cancer cell migration, synapse formation, cell cycle progression, and responses to serum stimulation. To address the physiological function of Malat1 in a living organism, we generated Malat1-knockout (KO) mice using homologous recombination. Unexpectedly, the Malat1-KO mice were viable and fertile, showing no apparent phenotypes. Nuclear speckle markers were also correctly localized in cells that lacked Malat1. However, the cellular levels of another long, noncoding RNA-Neat1-which is an architectural component of nuclear bodies known as paraspeckles, were down-regulated in a particular set of tissues and cells lacking Malat1. We propose that Malat1 is not essential in living mice maintained under normal laboratory conditions and that its function becomes apparent only in specific cell types and under particular conditions.
  • Joanna Y. Ip, Shinichi Nakagawa
    DEVELOPMENT GROWTH & DIFFERENTIATION 54 1 44 - 54 2012年01月 [査読有り][通常論文]
     
    High-throughput analyses of mammalian transcriptomes have revealed that more than half of the transcripts produced by RNA polymerase II are non-protein-coding. One class of these non-coding transcripts is the long non-coding RNAs (lncRNAs), which are more than 200 nucleotides in length and are molecularly indistinguishable from other protein-coding mRNAs. Although the molecular functions of these lncRNAs have long remained unknown, emerging evidence implicates the functional involvement of lncRNAs in the regulation of gene expression through the modification of chromatin, maintenance of subnuclear structures, transport of specific mRNAs, and control of pre-mRNA splicing. Here, we discuss the functions of a distinct group of vertebrate-specific lncRNAs, NEAT1/MENe/beta/VINC, MALAT1/NEAT2, and Gomafu/RNCR2/MIAT, which accumulate abundantly within the nucleus as RNA components of specific nuclear bodies.
  • Kunimasa Ohta, Ayako Ito, Sei Kuriyama, Giuseppe Lupo, Mitsuko Kosaka, Shin-Ichi Ohnuma, Shinichi Nakagawa, Hideaki Tanaka
    Proceedings of the National Academy of Sciences of the United States of America 108 36 14962 - 14967 2011年09月06日 [査読有り][通常論文]
     
    The Wnt signaling pathway is essential for the development of diverse tissues during embryogenesis. Signal transduction is activated by the binding of Wnt proteins to the type I receptor lowdensity lipoprotein receptor-related protein 5/6 and the seven-pass transmembrane protein Frizzled (Fzd), which contains a Wntbinding site in the form of a cysteine-rich domain. Known extracellular antagonists of the Wnt signaling pathway can be subdivided into two broad classes depending on whether they bind primarily to Wnt or to low-density lipoprotein receptor-related protein 5/6. We show that the secreted protein Tsukushi (TSK) functions as a Wnt signaling inhibitor by binding directly to the cysteine-rich domain of Fzd4 with an affinity of 2.3 × 10 -10 M and competing with Wnt2b. In the developing chick eye, TSK is expressed in the ciliary/iris epithelium, whereasWnt2b is expressed in the adjacent anterior rimof the optic vesicle, where it controls the differentiation of peripheral eye structures, such as the ciliary body and iris. TSK overexpression effectively antagonizes Wnt2b signaling in chicken embryonic retinal cells both in vivo and in vitro and represses Wnt-dependent specification of peripheral eye fates. Conversely, targeted inactivation of the TSK gene in mice causes expansion of the ciliary body and upregulation of Wnt2b and Fzd4 expression in the developing peripheral eye. Thus, we uncover a crucial role for TSK as a Wnt signaling inhibitor that regulates peripheral eye formation.
  • Yuko Hasegawa, Shinichi Nakagawa
    RNA BIOLOGY 8 5 735 - 739 2011年09月 [査読有り][通常論文]
     
    Mammalian females repress gene expression from one of their two X chromosomes to compensate for the gene dosage difference between females and males, via a process called X chromosome inactivation (XCI). Since the first discovery of XCI 50 years ago, the knowledge of this phenomenon has greatly contributed to a better understanding of the molecular mechanism that controls the epigenetic regulation of gene expression. The key molecule that organizes the chromatin-level repression is an X-linked 17-kb non-coding RNA named Xist. The transcripts of Xist are localized along the entire length of the X chromosome and subsequently recruit a chromatin remodeling complex that introduces the repressive epigenetic modifications. In the present review, we will highlight the recent findings that have illustrated the close relationship between XCI and the structural component of the nucleus called the nuclear scaffold/matrix, with an emphasis on the function of the bonafide scaffold/matrix-binding protein hnRNP U/SAF-A.
  • Shinichi Nakagawa, Kannanganattu V. Prasanth
    TRENDS IN CELL BIOLOGY 21 6 321 - 327 2011年06月 [査読有り][通常論文]
     
    X-chromosome inactivation has long served as an experimental model system for understanding the epigenetic regulation of gene expression. Central to this phenomenon is the long, non-coding RNA Xist that is specifically expressed from the inactive X chromosome and spreads along the entire length of the chromosome in cis. Recently, two of the proteins originally identified as components of the nuclear scaffold/matrix (S/MAR-associated proteins) have been shown to control the principal features of X-chromosome inactivation; specifically, context-dependent competency and the chromosome-wide association of Xist RNA. These findings implicate the involvement of nuclear S/MAR-associated proteins in the organization of epigenetic machinery. Here, we describe a model for the functional role of S/MAR-associated proteins in the regulation of key epigenetic processes.
  • Hitomi Tsuiji, Rei Yoshimoto, Yuko Hasegawa, Masaaki Furuno, Minoru Yoshida, Shinichi Nakagawa
    GENES TO CELLS 16 5 479 - 490 2011年05月 [査読有り][通常論文]
     
    Gomafu (also referred to as RNCR2/MIAT) was originally identified as a noncoding RNA expressed in a particular set of neurons. Unlike protein-coding mRNAs, the Gomafu RNA escapes nuclear export and stably accumulates in the nucleus, making a unique nuclear compartment. Although recent studies have revealed the functional relevance of Gomafu in a series of physiological processes, the underlying molecular mechanism remains largely uncharacterized. In this report, we identified a chicken homologue of Gomafu using a comparative genomic approach to search for functionally important and conserved sequence motifs among evolutionarily distant species. Unexpectedly, we found that all Gomafu RNA examined shared a distinctive feature: tandem repeats of UACUAAC, a sequence that has been identified as a conserved intron branch point in the yeast Saccharomyces cerevisiae. The tandem UACUAAC Gomafu RNA repeats bind to the SF1 splicing factor with a higher affinity than the divergent branch point sequence in mammals, which affects the kinetics of the splicing reaction in vitro. We propose that the Gomafu RNA regulates splicing efficiency by changing the local concentration of splicing factors within the nucleus.
  • Shinichi Nakagawa, Takao Naganuma, Go Shioi, Tetsuro Hirose
    JOURNAL OF CELL BIOLOGY 193 1 31 - 39 2011年04月 [査読有り][通常論文]
     
    Nuclei of higher organisms are well structured and have multiple, distinct nuclear compartments or nuclear bodies. Paraspeckles are recently identified mammal-specific nuclear bodies ubiquitously found in most cells cultured in vitro. To investigate the physiological role of paraspeckles, we examined the in vivo expression patterns of two long noncoding RNAs, NEAT1_1 and NEAT1_2, which are essential for the architectural integrity of nuclear bodies. Unexpectedly, these genes were only strongly expressed in a particular subpopulation of cells in adult mouse tissues, and prominent paraspeckle formation was observed only in the cells highly expressing NEAT1_2. To further investigate the cellular functions of paraspeckles, we created an animal model lacking NEAT1 by gene targeting. These knockout mice were viable and fertile under laboratory growth conditions, showing no apparent phenotypes except for the disappearance of paraspeckles. We propose that paraspeckles are nonessential, subpopulation-specific nuclear bodies formed secondary to particular environmental triggers.
  • Peter D. Westenskow, Jon B. McKean, Fumi Kubo, Shinichi Nakagawa, Sabine Fuhrmann
    INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE 51 10 5328 - 5335 2010年10月 [査読有り][通常論文]
     
    PURPOSE. Development of the retinal pigment epithelium (RPE) is controlled by intrinsic and extrinsic regulators including orthodenticle homeobox 2 (Otx2) and the Wnt/beta-catenin pathway, respectively. Otx2 and beta-catenin are necessary for the expression of the RPE key regulator microphthalmia-associated transcription factor (Mitf); however, neither factor is sufficient to promote Mitf expression in vivo. The study was conducted to determine whether Otx2 and beta-catenin act in a combinatorial manner and tested whether co-expression in the presumptive chick retina induces ectopic Mitf expression. METHODS. The sufficiency of Wnt/beta-catenin activation and/or Otx2 expression to induce RPE-specific gene expression was examined in chick optic vesicle explant cultures or in the presumptive neural retina using in ovo-electroporation. Luciferase assays were used to examine the transactivation potentials of Otx2 and beta-catenin on the Mitf-D enhancer and autoregulation of the Mitf-D and Otx2T0 enhancers. RESULTS. In optic vesicles explant cultures, RPE-specific gene expression was activated by lithium chloride, a Wnt/beta-catenin agonist. However, in vivo, Mitf was induced only in the presumptive retina if both beta-catenin and Otx2 are co-expressed. Furthermore, both Mitf and Otx2 can autoregulate their own enhancers in vitro. CONCLUSIONS. The present study provides evidence that beta-catenin and Otx2 are sufficient, at least in part, to convert retinal progenitor cells into presumptive RPE cells expressing Mitf. Otx2 may act as a competence factor that allows RPE specification in concert with additional RPE-promoting factors such as beta-catenin. (Invest Ophthalmol Vis Sci. 2010;51:5328-5335) DOI:10.1167/iovs.09-5015
  • Yuko Hasegawa, Neil Brockdorff, Shinji Kawano, Kimiko Tsutui, Ken Tsutui, Shinichi Nakagawa
    DEVELOPMENTAL CELL 19 3 469 - 476 2010年09月 [査読有り][通常論文]
     
    In XX female mammals, one of the two X chromosomes is epigenetically inactivated to equalize gene expression with XY males. The formation of the inactive X chromosome (Xi) is regulated by an X-linked long noncoding RNA Xist, which accumulates on the entire length of the chromosome in cis and induces heterochromatin formation. However, the mechanism by which Xist RNA "paints" the Xi has long remained elusive. Here, we show that a matrix protein hnRNP U/SP120/SAF-A is required for the accumulation of Xist RNA on the Xi. Xist RNA and hnRNP U interact and upon depletion of hnRNP U, Xist RNA is detached from the Xi and diffusely localized into the nucleoplasm. In addition, ES cells lacking hnRNP U expression fail to form the Xi. We propose that the association with matrix proteins is an essential step in the epigenetic regulation of gene expression by Xist RNA.
  • Fumi Kubo, Shinichi Nakagawa
    DEVELOPMENTAL DYNAMICS 239 9 2492 - 2500 2010年09月 [査読有り][通常論文]
     
    Basic helix-loop-helix (bHLH) transcription factors play important roles in cell type specification and differentiation during the development of the nervous system. In this study, we identified a chicken homolog of Atonal 8/ath6 (Cath6) and examined its role in the developing retina. Unlike other Atonal-family proneural genes that induce neuronal differentiation, Cath6 was expressed in stem cell-like progenitor cells in the marginal region of the retina, and its overexpression inhibited neuronal differentiation. A Cath6 fused with a VP16 transactivation domain recapitulated the inhibitory effect of Cath6 on neuronal differentiation, indicating that Cath6 functions as a transcription activator. These results demonstrate that Cath6 constitutes a unique member of the Atonal-family of genes in that it acts as a negative regulator of neuronal differentiation. Developmental Dynamics 239:2492-2500, 2010. (c) 2010 Wiley-Liss, Inc.
  • Akiko Suga, Masanori Taira, Shinichi Nakagawa
    DEVELOPMENTAL BIOLOGY 330 2 318 - 328 2009年06月 [査読有り][通常論文]
     
    In the nervous system, transcription factor expression in progenitor and/or nascent neurons regulates cell type specification. Although the functions of these transcription factors at early stages are well established, whether or not they are required during late developmental periods remains an open question. To address this issue, we conditionally manipulated gene expression using a recently developed transposon-mediated gene transfer system combined with in ovo electroporation. In chicken retinas, horizontal cells are classified into three subtypes according to their characteristic neuronal morphology. Two LIM family transcription factors, Lim1 and Isl1, begin to be expressed in a distinct subset of nascent retinal neurons, which results in complementary expression of these genes in mature retinas in type I and type II/III horizontal cells, respectively. Overexpression of Isl1 in post-migratory horizontal cells represses endogenous Lim1 expression and increases the number of neurons with a dendritic morphology characteristic of type II horizontal cells, which normally express Isl1. Inhibition of Lim1 function by expression of a dominant negative form Lim1 perturbs axonal morphogenesis of type I horizontal cells. Therefore, we propose that LIM family transcription factors are required for subtype-specific morphogenesis of horizontal cells at later stages of retinal development. (C) 2009 Elsevier Inc. All rights reserved.
  • Fumi Kubo, Shinichi Nakagawa
    DEVELOPMENT 136 11 1823 - 1833 2009年06月 [査読有り][通常論文]
     
    In the vertebrate retina, stem cell-like progenitor cells are maintained in a distinct region called the ciliary marginal zone (CMZ). Canonical Wnt signaling regulates the maintenance of the progenitor cells in the CMZ. However, its downstream molecular mechanisms have remained largely unclear. Here, we show that chick Hairy1, an established Notch signaling effector, mediates the Wnt-dependent maintenance of CMZ progenitor cells in chicken. Interestingly, unlike other developmental contexts in which Hes gene expression is regulated by Notch signaling, Hairy1 expression in the CMZ is regulated by Wnt signaling. Hairy1 is necessary and sufficient for the expression of a set of molecular markers characteristic of the CMZ, and Wnt2b fails to induce CMZ markers when Hairy1 activity is inhibited. Furthermore, microarray analysis identifies multiple Wnt-responsive transcription factors that activate Hairy1 expression. We thus propose that Hairy1 functions as a node downstream of Wnt signaling to maintain progenitor cells in the chick CMZ.
  • Fumi Kubo, Shinichi Nakagawa
    DEVELOPMENT GROWTH & DIFFERENTIATION 50 4 245 - 251 2008年05月 [査読有り][通常論文]
     
    In the vertebrate retina, stem cells with prolonged proliferative capacities reside in the most peripheral region, the ciliary marginal zone (CMZ), and they persist even after the functional eye has formed. These stem cells contribute to the formation of the retinal structures during the postnatal period in vivo, or can expand as neurospheres in vitro. Despite the wealth of anatomical descriptions of the characteristics of CMZ cells, molecular mechanisms for their specification or maintenance have long been uncharacterized. Recent studies provide evidence that certain secreted signaling molecules act as key regulators at multiple steps during these processes. In this review, we discuss the molecular basis for the regulation of retinal stem cells and their related cell types, especially focusing on the role of Wnt signaling.
  • Yoshiko Takahashi, Tadayoshi Watanabe, Shinichi Nakagawa, Koichi Kawakami, Yuki Sato
    AVIAN EMBRYOLOGY, 2ND EDITION 87 271 - + 2008年 [査読有り][通常論文]
  • Kunimasa Ohta, Ayako Ito, Sei Kuriyama, Rika Nakayama, Naoko Oshima, Mitsuko Kosaka, Shinichi Ohnuma, Shinichi Nakagawa, Hideaki Tanaka
    NEUROSCIENCE RESEARCH 61 S42 - S42 2008年 [査読有り][通常論文]
  • Dai Chida, Shinichi Nakagawa, So Nagai, Hiroshi Sagara, Harumi Katsumata, Toshihiro Imaki, Harumi Suzuki, Fumiko Mitani, Tadashi Ogishima, Chikara Shimizu, Hayato Kotaki, Shigeru Kakuta, Katsuko Sudo, Takao Koike, Mitsurnasa Kubo, Yoichiro Iwakura
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 104 46 18205 - 18210 2007年11月 [査読有り][通常論文]
     
    ACTH (i.e., corticotropin) is the principal regulator of the hypothalamus-pituitary-adrenal axis and stimulates steroidogenesis in the adrenal gland via the specific cell-surface melanocortin 2 receptor (MC2R). Here, we generated mice with an inactivation mutation of the MC2R gene to elucidate the roles of MC2R in adrenal development, steroidogenesis, and carbohydrate metabolism. These mice, the last of the knockout (KO) mice to be generated for melanocortin family receptors, provide the opportunity to compare the phenotype of proopiomelanocortin KO mice with that of MCIR-MC5R KO mice. We found that the MC2R KO mutation led to neonatal lethality in three-quarters of the mice, possibly as a result of hypoglycemia. Those surviving to adulthood exhibited macroscopically detectable adrenal glands with markedly atrophied zona fasciculata, whereas the zona glomerulosa and the medulla remained fairly intact. Mutations of MC2R have been reported to be responsible for 25% of familial glucocorticoid deficiency (FGD) cases. Adult MC2R KO mice resembled FGD patients in several aspects, such as undetectable levels of corticosterone despite high levels of ACTH, unresponsiveness to ACTH, and hypoglycemia after prolonged (36 h) fasting. However, MC2R KO mice differ from patients with MC2R-null mutations in several aspects, such as low aldosterone, levels and unaltered body length. These results indicate that MC2R is required for postnatal adrenal development and adrenal steroidogenesis and that MC2R KO mice provide a useful animal model by which to study FGD.
  • Masamitsu Sone, Tetsutaro Hayashi, Hiroshi Tarui, Kiyokazu Agata, Masatoshi Takeichi, Shinichi Nakagawa
    JOURNAL OF CELL SCIENCE 120 15 2498 - 2506 2007年08月 [査読有り][通常論文]
     
    Recent transcriptome analyses have revealed that a large body of noncoding regions of mammalian genomes are actually transcribed into RNAs. Our understanding of the molecular features of these noncoding RNAs is far from complete. We have identified a novel mRNA-like noncoding gene, named Gomafu, which is expressed in a distinct set of neurons in the mouse nervous system. Interestingly, spliced mature Gomafu RNA is localized to the nucleus despite its mRNA-like characteristics, which usually act as potent export signals to the cytoplasm. Within the nucleus, Gomafu RNA is detected as numerous spots that do not colocalize with known nuclear domain markers. Gomafu RNA is extremely insoluble and remains intact after nuclear matrix preparation. Furthermore, heterokaryon assays revealed that Gomafu RNA does not shuttle between the nucleus and cytoplasm, but is retained in the nucleus after its transcription. We propose that Gomafu RNA represents a novel family of mRNA-like noncoding RNA that constitutes a cell-type-specific component of the nuclear matrix.
  • Kunimasa Ohta, Ayako Ito, Sei Kuriyama, Shinichi Ohnuma, Mitsuko Kosaka, Shinichi Nakagawa, H. Tanaka
    DEVELOPMENTAL BIOLOGY 306 1 391 - 391 2007年06月 [査読有り][通常論文]
  • Tadayoshi Watanabe, Daisuke Saito, Koji Tanabe, Rinako Suetsugu, Yukiko Nakaya, Shinichi Nakagawa, Yoshiko Takahashi
    DEVELOPMENTAL BIOLOGY 305 2 625 - 636 2007年05月 [査読有り][通常論文]
     
    The in ovo electroporation technique in chicken embryos has enabled investigators to uncover the functions of numerous developmental genes. In this technique, the ubiquitous promoter, CAGGS (CMV base), has often been used for overexpression experiments. However, if a given gene plays a role in multiple steps of development and if overexpression of this gene causes fatal consequences at the time of electroporation, its roles in later steps of development would be overlooked. Thus, a technique with which expression of an electroporated DNA can be controlled in a stage-specific manner needs to be formulated. Here we show for the first time that the tetracycline-controlled expression method, "tet-on" and "tet-off', works efficiently to regulate gene expression in electroporated chicken embryos. We demonstrate that the onset or termination of expression of an electroporated DNA can be precisely controlled by timing the administration of tetracycline into an egg. Furthermore, with this technique we have revealed previously unknown roles of RhoA, cMeso-1 and Pax2 in early somitogenesis. In particular, cMeso-1 appears to be involved in cell condensation of a newly forming somite by regulating Pax2 and NCAM expression. Thus, the novel molecular technique in chickens proposed in this study provides a useful tool to investigate stage-specific roles of developmental genes. (c) 2007 Elsevier Inc. All rights reserved.
  • Yuki Sato, Toshiharu Kasai, Shinichi Nakagawa, Koji Tanabe, Tadayoshi Watanabe, Koichi Kawakami, Yoshiko Takahashi
    DEVELOPMENTAL BIOLOGY 305 2 616 - 624 2007年05月 [査読有り][通常論文]
     
    The in ovo electroporation in chicken embryos has widely been used as a powerful tool to study roles of genes during embryogenesis. However, the conventional electroporation technique fails to retain the expression of transgenes for more than several days because transgenes are not integrated into the genome. To overcome this shortcoming, we have developed a transposon-mediated gene transfer, a novel technique in chicken manipulations. It was previously reported that the transposon To12, originally found in medaka fish, facilitates an integration of a transgene into the genome when co-acting with To12 transposase. In this study, we co-electroporated a plasmid containing a CAGGS-EGFP cassette cloned in the To12 construct along with a transposase-encoding plasmid into early presomitic mesoderm or optic vesicles of chicken embryos. This resulted in persistent expression of EGFP at least until embryonic day 8 (E8) and E12 in somite-derived tissues and developing retina, respectively. The integration of the transgene was confirmed by genomic Southern blotting using chicken cultured cells. We further combined this transposon-mediated gene transfer with the tetracycline-dependent conditional expression system that we also developed recently. With this combined method, expression of a stably integrated transgene could be experimentally induced upon tetracycline administration at relatively late stages such as E6, where a variety of organogenesis are underway. Thus, the techniques proposed in this study provide a novel approach to study the mechanisms of late organogenesis, for which chickens are most suitable model animals. (c) 2007 Elsevier Inc. All rights reserved.
  • Koji Tanabe, Yoshiko Takahashi, Yuki Sato, Koichi Kawakami, Masatoshi Takeichi, Shinichi Nakagawa
    DEVELOPMENT 133 20 4085 - 4096 2006年10月 [査読有り][通常論文]
     
    Dendrite morphology of neurons provides a structural basis for their physiological characteristics, and is precisely regulated in a cell type-dependent manner. Using a unique transposon-mediated gene transfer system that enables conditional and cell-type specific expression of exogenous genes, we investigated the role of cadherin on dendritic morphogenesis of horizontal cells in the developing chicken retina. We first visualized single horizontal cells by overexpressing membrane-targeted EGFP, and confirmed that there were three subtypes of horizontal cells, the dendritic terminals of which projected to distinct synaptic sites in the outer plexiform layer. Expression of a dominant-negative cadherin decreased the dendritic field size, and perturbed the termination of dendritic processes onto the photoreceptor cells. The cadherin blockade also impaired the accumulation of GluR4, a postsynaptic marker, at the cone pedicles. We thus provide in vivo evidence that cadherin is required for dendrite morphogenesis of horizontal cells and subsequent synapse formation with photoreceptor cells in the vertebrate retina.
  • H Aoki, A Hara, S Nakagawa, T Motohashi, M Hirano, Y Takahashi, T Kunisada
    EXPERIMENTAL EYE RESEARCH 82 2 265 - 274 2006年02月 [査読有り][通常論文]
     
    A culture system to generate eye-like structures consisting of lens, neural retina, and retinal pigmented epithelium (RPE) cells from undifferentiated embryonic stem cells has been established. Precursors of RPE cells that differentiated in the cultures were responsive to Wnt2b signaling and identified retrospectively to form secondary colonies consisting of only RPE-like cells in eye-like structures. These transplanted eye-like structures were capable of populating the developing chick eye as neuronal retina and RPE cells. The outgrowth of a single cell layer of mature RPE cells from the grafted eye-like structures confirmed the existence of precursors for RPE cells. These results suggest that the eye-like structures resulted from the normal developmental pathway responsible for generating eyes in vivo. If a functional effect of these cells can be established, such eye-like structures may be potentially used to establish therapy models for various eye diseases. (c) 2005 Elsevier Ltd. All rights reserved.
  • F Kubo, M Takeichi, S Nakagawa
    DEVELOPMENT 132 12 2759 - 2770 2005年06月 [査読有り][通常論文]
     
    During the development of the central nervous system, cell proliferation and differentiation are precisely regulated. In the vertebrate eye, progenitor cells located in the marginal-most region of the neural retina continue to proliferate for a much longer period compared to the ones in the central retina, thus showing stem-cell-like properties. Wnt2b is expressed in the anterior rim of the optic vesicles, and has been shown to control differentiation of the progenitor cells in the marginal retina. In this paper, we show that stable overexpression of Wnt2b in retinal explants inhibited cellular differentiation and induced continuous growth of the tissue. Notably, Wnt2b maintained the undifferentiated progenitor cells in the explants even under the conditions where Notch signaling was blocked. Wnt2b downregulated the expression of multiple proneural bHLH genes as well as Notch. In addition, expression of Cath5 under the control of an exogenous promoter suppressed the negative effect of Wnt2b on neuronal differentiation. Importantly, Wnt2b inhibited neuronal differentiation independently of cell cycle progression. We propose that Wnt2b maintains the naive state of marginal progenitor cells by attenuating the expression of both proneural and neurogenic genes, thus preventing those cells from launching out into the differentiation cascade regulated by proneural genes and Notch.
  • K Tanabe, M Takeichi, S Nakagawa
    DEVELOPMENTAL DYNAMICS 229 4 899 - 906 2004年04月 [査読有り][通常論文]
     
    Classic cadherins mediate calcium-dependent cell-cell adhesion in a variety of animals, but there are marked differences in their domain structures between chordate and nonchordate animals. The extracellular domain of chordate-type classic cadherins (type I and type 11 classic cadherins) consists of five tandem repeats of conserved sequences called EC domains, whereas that of nonchordate-type classic cadherins (designated as type III classic cadherin) contains a variable number of EC domains, followed by a characteristic domain complex made of laminin-A globular domains and EGF-like repeats. In the present study, we identified a novel vertebrate type III cadherin showing high sequence similarity to Drosophila N-cadherin, and named this molecule chicken Hz-cadherin (cHz-cadherin), because of the distinct expression in horizontal cells of the neural retina. cHz-cadherin functioned as an adhesion molecule when introduced into cultured cells. Database search revealed one cHz-cadherin homologue in zebrafish and two in puffer fish, but none in other vertebrate species examined. These observations indicate that type III classic cadherins have been conserved in vertebrate species, being expressed by limited cells types, but lost in particular phylogenic groups of the vertebrates. Developmental Dynamics 229.899-906, 2004. (C) 2004 Wiley-Liss, Inc.
  • S Nakagawa, S Takada, R Takada, M Takeichi
    DEVELOPMENTAL BIOLOGY 260 2 414 - 425 2003年08月 [査読有り][通常論文]
     
    To study the molecular mechanism that controls the laminar organization of the retina, we utilized reaggregation cultures of dissociated retina] cells prepared from chicken embryos. These cells cannot generate laminated structures by themselves and, instead, form rosettes within the reaggregates. However, the dissociated cells can organize into a correctly laminated structure when cultured in the presence of a putative laminar inducing factor coming from particular tissue or cells, but its molecular identity of this factor has long remained elusive. In this study, we found that the anterior rim of the retina sends a signal to rearrange the rosette-forming cells into a neuroepithelial structure characteristic of the undifferentiated retinal layer. This activity of the anterior rim was mimicked by Wnt-2b expressed in this tissue, and was neutralized by a soluble form of Frizzled, which works as a Wnt antagonist. Furthermore, the neuroepithelial structure induced by Wnt-2b subsequently developed into correctly laminated retinal layers. These observations suggest that the anterior rim functions as a layer-organizing center in the retina, by producing Wnt-2b. (C) 2003 Elsevier Inc. All rights reserved.
  • F Kubo, M Takeichi, S Nakagawa
    DEVELOPMENT 130 3 587 - 598 2003年02月 [査読有り][通常論文]
     
    The ciliary marginal zone of the vertebrate retina contains undifferentiated progenitor cells that continue to proliferate and add new neurons and glia peripherally during the embryonic stages - even after the formation of a functional retina. To understand the molecular mechanism that controls the prolonged progenitor cell proliferation in the ciliary marginal zone, we employed a candidate molecule approach, focusing on Wnt2b (formerly know as Wnt13), which is expressed in the marginal most tip of the retina. Frizzled 4 and 5, seven-pass transmembrane Writ receptors, were expressed in the peripheral and central part of the retina, respectively. LEF1, a downstream Writ signaling component, was expressed at high levels in the ciliary marginal zone with expression gradually decreasing towards the central retina. The LEF1-expressing region, which is where Writ signaling is supposedly activated, expressed a set of molecular markers that are characteristic of the progenitor cells in the ciliary marginal zone. Overexpression of Wnt2b by use of in ovo electroporation in the central retina inhibited neuronal differentiation and induced the progenitor cell markers. Blocking of the Wnt downstream signaling pathway by a dominant-negative LEF1 inhibited proliferation of the cells in the marginal area, which resulted in their premature neuronal differentiation. The progenitor cells in the ciliary marginal zone differentiated into all the neuronal and glial cell types when cultured in vitro, and they proliferated for a longer period than did centrally located progenitor cells that underwent a limited number of cell divisions. In addition, the proliferation of these progenitor cells was promoted in the presence of Wnt2b. These results suggest that Wnt2b functions to maintain undifferentiated progenitor cells in the ciliary marginal zone, and thus serves as a putative stem cell factor in the retina.
  • Takeichi M, Nakagawa S
    Current protocols in cell biology Chapter 9 Unit 9.3  2001年05月 [査読有り][通常論文]
     
    Differential treatment of cells with trypsin can be used to distinguish Ca(2+)-dependent adhesion (CDS) from Ca(2+)-independent adhesion (CIDS). Cadherins appear to be a unique family of molecules whose structure and function as adhesion molecules are protected from trypsin in the presence of Ca(2+). This unit provides protocols for preparation and analysis of cells for cadherin-dependent adhesion in short-term and long-term aggregation assays. The functions of different cadherins can be assessed in mixed aggregate assays. Fluorescence antibody-based assays are used to identify specific cadherins and their associated catenins, and transformation of cells with specific constructs can be used to assay adhesion in cells with loss of cadherin activity.
  • M Takeichi, S Nakagawa, S Aono, T Usui, T Uemura
    PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON SERIES B-BIOLOGICAL SCIENCES 355 1399 885 - 890 2000年07月 [査読有り][通常論文]
     
    During morphogenesis, cell cell association patterns are dynamically altered. We are interested in how ce adhesion molecule scan regulate the patterning of cellular assemblies. Cadherins, a group of cell-cell adhesion receptors, are crucial for the organized assembly of many cell types, but they also regulate dynamic aspects of cell association. For example, during neural crest emigration from the neural tube, the cadherin subtypes expressed by crest cells ar switched from one subtype to another. Artificial perturbation of this switch results in blocking of their escape from the neural tube. Intracellular modulations of cadherin activity also seem to play a role in regulation of cell adhesion. We identified p120(ctn) as a regulator of cadherin function in carcinoma cells. With such regulators, cells may make a choice as to whether they should maintain stable cell contacts or disrupt their association. Finally, we found another type of cadherin-mediated cell patterning: Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity in Drosophila imaginal discs. Thus, the cadherin superfamily receptors control the patterning of cell assemblies through a variety of mechanisms.
  • C Redies, M Ast, S Nakagawa, M Takeichi, M Martinez-de-la-Torre, L Puelles
    JOURNAL OF COMPARATIVE NEUROLOGY 421 4 481 - 514 2000年06月 [査読有り][通常論文]
     
    The expression of four cadherins (cadherin-6B, cadherin-7, R-cadherin, and N-cadherin) was mapped in the diencephalon of chicken embryos at 11 days and 15 days of incubation and was compared with Nissl stains and radial glial topology. Results showed that each cadherin is expressed in a restricted manner by a different set of embryonic divisions, brain nuclei, and their subregions. An analysis of the segmental organization based on the prosomeric model indicated that, in the mature diencephalon, each prosomere persists and forms a coherent domain of gray matter extending across the entire transverse dimension of the neural tube, from the ventricular surface to the pial surface. Moreover, the results suggest the presence of a novel set of secondary subdivisions for the dorsal thalamus (dorsal, intermediate, and ventral tiers and anteroventral subregion). They also confirm the presence of secondary subdivisions in the pretectum (commissural, juxtacommissural, and precommissural). At most of the borders between the prosomeres and their secondary subdivisions, changes in radial glial fiber density were observed. The diencephalic brain nuclei that derive from each of the subdivisions were determined. In addition, a number of previously less well-characterized gray matter regions of the diencephalon were defined in more detail based on the mapping of cadherin expression. The results demonstrate in detail how the divisions of the early embryonic diencephalon persist and transform into mature gray matter architecture during brain morphogenesis, and they support the hypothesis that cadherins play a role in this process by providing a framework of potentially adhesive specificities. J. Comp. Neurol. 421:481-514, 2000. (C) 2000 Wiley-Liss, Inc.
  • Y Honjo, S Nakagawa, M Takeichi
    GENES TO CELLS 5 4 309 - 318 2000年04月 [査読有り][通常論文]
     
    Background: Synaptic junctions have cadherin-catenin complexes, but their functions are poorly understood. Using retinal neurones, we investigated the role of this adhesion machinery in synaptic organization. Results: In cultures of chicken retinal cells, cadherin-6B (cad6B) and cadherin-7 (cad7) are expressed by distinct neurones, each being distributed in a punctate pattern along their neurites as well as in the soma. Double-immunostaining for cad6B and PSD-95/SAP90 or other PSD-95 family members, known to localize in the postsynaptic density, showed that their distributions overlapped each other. To assess the role for cad6B, we incubated retinal cells with antibodies that could specifically block cad6B-mediated adhesion. In the antibody-treated neurones, the localization pattern of PSD-95 family proteins was altered, that is, their staining signals tended to be reduced or disarranged. We then examined whether cadherins interacted molecularly with PSD-95: Cadherin immunoprecipitates from brain lysates did not contain PSD-95; nevertheless, this protein was co-precipitated with alpha N- and beta-catenins. When PSD-95 proteins were ectopically expressed in epithelial cells, some of these molecules were concentrated in cell-cell junctions, co-localizing with E-cadherin, and this junctional localization of PSD-95 was abolished by blocking of E-cadherin activity. Conclusion: These results suggest that cadherins play a role in the subcellular organization of postsynaptic density components through some, perhaps indirect, molecular interactions.
  • S Nakagawa, C Brennan, KG Johnson, D Shewan, WA Harris, CE Holt
    NEURON 25 3 599 - 610 2000年03月 [査読有り][通常論文]
     
    In Xenopus tadpoles, all retinal ganglion cells (RGCs) send axons contralaterally across the optic chiasm. At metamorphosis, a subpopulation of EphB-expressing RGCs in the ventrotemporal retina begin to project ipsilaterally. However, when these metamorphic RGCs are grafted into embryos, they project contralaterally, suggesting that the embryonic chiasm lacks signals that guide axons ipsilaterally. Ephrin-B is expressed discretely at the chiasm of metamorphic but not premetamorphic Xenopus. When expressed prematurely in the embryonic chiasm, ephrin-B causes precocious ipsilateral projections from the EphB-expressing RGCs. Ephrin-B is also found in the chiasm of mammals, which have ipsilateral projections, but not in the chiasm of fish and birds, which do not. These results suggest that ephrin-B/EphB interactions play a key role in the sorting of axons at the vertebrate chiasm.
  • S Aono, S Nakagawa, AB Reynolds, M Takeichi
    JOURNAL OF CELL BIOLOGY 145 3 551 - 562 1999年05月 [査読有り][通常論文]
     
    p120(ctn) binds to the cytoplasmic domain of cadherins but its role is poorly understood. Cole 205 cells grow as dispersed cells despite their normal expression of E-cadherin and catenins. However, in these cells we can induce typical E-cadherin-dependent aggregation by treatment with staurosporine or trypsin. These treatments concomitantly induce an electrophoretic mobility shift of p120(ctn) to a faster position. To investigate whether p120(ctn) plays a role in this cadherin reactivation process, we transfected Cole 205 cells with a series of p120(ctn) deletion constructs. Notably, expression of NH2-terminally deleted p120(ctn) induced aggregation. Similar effects were observed when these constructs were introduced into HT-29 cells. When a mutant N-cadherin lacking the p120(ctn)-binding site was introduced into Cole 205 cells, this molecule also induced cell aggregation, indicating that cadherins can function normally if they do not bind to p120(ctn). These findings suggest that in Cole 205 cells, a signaling mechanism exists to modify a biochemical state of p120(ctn) and the modified p120(ctn) blocks the cadherin system. The NH2 terminus-deleted p120(ctn) appears to compete with the endogenous p120(ctn) to abolish the adhesion-blocking action.
  • JCP Wohrn, S Nakagawa, M Ast, M Takeichi, C Redies
    NEUROSCIENCE 90 3 985 - 1000 1999年 [査読有り][通常論文]
     
    The expression of four cadherins (N-cadherin, R-cadherin, cadherin-6B and cadherin-7) was mapped in the developing tectal system of the chicken embryo from four to 19 days of incubation. Each of the cadherins is expressed in a restricted fashion in specific tectal layers, with partial overlap between the cadherins. In some layers, subpopulations of neurons differentially express the cadherins, e.g., in the stratum griseum centrale. Double labeling demonstrates that many of the projection neurons in this layer co-express at least two cadherins. Fibers of the efferent (tectofugal) pathways originating in these neurons also differentially express the cadherins, most prominently at around 11 days of incubation. While the different subpopulations of cadherin-expressing projection neurons are dispersed and mixed within the tectum, their neurites sort out and fasciculate according to which cadherin they express, as they collect in the major output of the tectum, the brachium colliculi superioris. From here, cadherin-expressing fascicles follow separate paths to their respective target areas, some of which also express the respective cadherins, in a matching fashion. We propose that the preferentially homophilic binding of cadherins provides a potential adhesive basis for the sorting and selective fasciculation of specific subpopulations of neurites, similar to the well-established sorting and aggregation of cells expressing cadherins. The combinatorial expression of cadherins by the tectal projection neurons may contribute to the complexity and specificity of functional connections in this system. (C) 1999 IBRO. Published by Elsevier Science Ltd.
  • S Nakagawa, M Takeichi
    DEVELOPMENT 125 15 2963 - 2971 1998年08月 [査読有り][通常論文]
     
    During the emergence of neural crest cells from the neural tube, the expression of cadherins dynamically changes. In the chicken embryo, the early neural tube expresses two cadherins, N-cadherin and cadherin-6B (cad6B), in the dorsal-most region where neural crest cells are generated. The expression of these two cadherins is, however, downregulated in the neural crest cells migrating from the neural tube; they instead begin expressing cadherin-7 (cad7). As an attempt to investigate the role of these changes in cadherin expression, we overexpressed various cadherin constructs, including N-cadherin, cad7, and a dominant negative N-cadherin (cN390 Delta), in neural crest-generating cells. This was achieved by injecting adenoviral expression vectors encoding these molecules into the lumen of the closing neural tube of chicken embryos at stage 14, In neural tubes injected with the viruses, efficient infection was observed at the neural crest-forming area, resulting in the ectopic cadherin expression also in migrating neural crest cells. Notably, the distribution of neural crest cells with the ectopic cadherins changed depending on which constructs were expressed. Many crest cells failed to escape from the neural tube when N-cadherin or cad7 overexpressed. Moreover, none of the cells with these ectopic cadherins migrated along the dorsolateral (melanocyte) pathway. When these samples were stained for Mitf, an early melanocyte marker, positive cells were found accumulated within the neural tube, suggesting that the failure of their migration was not due to differentiation defects. In contrast to these phenomena, cells expressing non-functional cadherins exhibited a normal migration pattern. Thus, the overexpression of a neuroepithelial cadherin (N-cadherin) and a crest cadherin (cad7) resulted in the same blocking effect on neural crest segregation from neuroepithelial cells, especially for melanocyte precursors. These findings suggest that the regulation of cadherin expression or its activity at the neural crest-forming area plays a critical role in neural crest emigration from the neural tube.
  • JCP Wohrn, L Puelles, S Nakagawa, M Takeichi, C Redies
    JOURNAL OF COMPARATIVE NEUROLOGY 396 1 20 - 38 1998年06月 [査読有り][通常論文]
     
    The expression of two calcium-dependent adhesion molecules of the cadherin superfamily (cadherin-6B and cadherin-7) was mapped in the embryonic neural retina and retinofugal pathways of the chicken embryo and compared with the expression of R-cadherin, N-cadherin, and B-cadherin, studied previously. Whereas B-cadherin is only found in Muller glia, the other four cadherins are each expressed by specific subpopulations of retinal neurons. For example, different (but partly overlapping) populations of bipolar cells express R-cadherin, cadherin-6B, and cadherin-7. Cadherin-6B and cadherin-7 are also expressed by subsets of amacrine cells. In the inner plexiform layer, cadherin-6B and cadherin-7 immunoreactivities are restricted to specific sublaminae associated with synapsin-I-positive nerve terminals. In addition, cadherin-6B and cadherin-7 are expressed by a subset of ganglion cells that project to several retinorecipient nuclei forming part of the accessory optic system (e.g., nucleus of the basal optic root and external pretectal nucleus). Together with their connecting fiber tracts, these nuclei also express cadherin-6B and cadherin-7 in their neurons and neuropile. The expression patterns of the two cadherins overlap but show distinct differences. Some other visual nuclei express cadherin-7 but not cadherin-6B. The expression patterns differ from those previously described for N- and R-cadherin. Together, these results demonstrate that cadherins could provide a system of adhesive cues that specify developing retinal circuits and other functional connections and subsystems in the embryonic chicken visual system. (C) 1998 Wiley-Liss, Inc.
  • K Arndt, S Nakagawa, M Takeichi, C Redies
    MOLECULAR AND CELLULAR NEUROSCIENCE 10 5-6 211 - 228 1998年04月 [査読有り][通常論文]
     
    In the developing chicken cerebellar cortex, three cadherins (Cad6B, Cad7, and R-cadherin) are expressed in distinct parasagittal segments that are separated from each other by ribbons of migrating interneurons and granule cells which express R-cadherin and Cad7, respectively. The segment/ribbon pattern is respected by the expression of other types of molecules, such as engrailed-2 and SC1/BEN/DM-GRASP. The cadherin-defined segments contain young Purkinje cells which are connected to underlying nuclear zones expressing the same cadherin, thereby forming parasagittal cortico-nuclear zones of topographically organized connections. In addition, R-cadherin-positive mossy fiber terminals display a periodic pattern in the internal granular layer. In this layer, Cad7 and R-cadherin are associated with synaptic complexes. These results suggest that cadherins play a pivotal role in the formation of functional cerebellar architecture by providing a three-dimensional scaffold of adhesive information.
  • K Arndt, S Nakagawa, M Takeichi, C Redies
    MOLECULAR AND CELLULAR NEUROSCIENCE 10 5-6 211 - 228 1998年04月 [査読有り][通常論文]
     
    In the developing chicken cerebellar cortex, three cadherins (Cad6B, Cad7, and R-cadherin) are expressed in distinct parasagittal segments that are separated from each other by ribbons of migrating interneurons and granule cells which express R-cadherin and Cad7, respectively. The segment/ribbon pattern is respected by the expression of other types of molecules, such as engrailed-2 and SC1/BEN/DM-GRASP. The cadherin-defined segments contain young Purkinje cells which are connected to underlying nuclear zones expressing the same cadherin, thereby forming parasagittal cortico-nuclear zones of topographically organized connections. In addition, R-cadherin-positive mossy fiber terminals display a periodic pattern in the internal granular layer. In this layer, Cad7 and R-cadherin are associated with synaptic complexes. These results suggest that cadherins play a pivotal role in the formation of functional cerebellar architecture by providing a three-dimensional scaffold of adhesive information.
  • S Nakagawa, M Takeichi
    DEVELOPMENT GROWTH & DIFFERENTIATION 39 4 451 - 455 1997年08月 [査読有り][通常論文]
     
    The developing heart primordium strongly expresses N-cadherin. In order to investigate the role of this adhesion molecule in heart morphogenesis, chicken embryos were cultured al stages 5-12, and injected with anti-N-cadherin antibodies that can specifically block the activity of this cadherin. In the injected embryos, the epimyocardial layers, which develop bilaterally from the splanchnic mesoderm, did not fuse to form a single cardiac tube. Moreover, each of the unfused layers became fragmented into epithelioid clusters. At the cellular level, large intercellular gaps were observed in the antibody-treated myocardial layers. These disorganized myocardial layers beat to some extent, suggesting that their differentiation was not blocked; however, their contraction was not coordinated. Morphogenesis of other tissues, not only N-cadherin-negative but also N-cadherin-positive tissues, such as the neural tube and notochord, proceeded normally even in the presence of anti-N-cadherin antibodies. These results suggest that N-cadherin is indispensable for heart formation, but not for morphogenesis of the other tissues, at the developmental stages examined. For the latter processes, expression of other cadherin subtypes presumably compensated for the loss of N-cadherin activity.
  • N Matsuyoshi, K Toda, Y Horiguchi, T Tanaka, S Nakagawa, M Takeichi, S Imamura
    Proceedings of the Association of American Physicians 109 4 362 - 71 1997年07月 
    Vascular endothelial cell-cell adhesion is crucial for the regulation of vascular functions and is associated with many circulatory disorders. We isolated a rat monoclonal antibody (VECD1) recognizing the mouse vascular endothelial cell adhesion molecule and found that it inhibited vascular endothelial cell-cell association. We sequenced a full-length cDNA of the antigen that was identical to mouse cadherin-5. L-cells transfected with its cDNA acquired cell-cell adhesiveness, and these transfectants reacted with VECD1 at cell-cell contact areas. We studied the role of mouse cadherin-5 in vascular functions. The addition of VECD1 antibody to a cultured vascular endothelial cell line (F-2) caused the detachment of each cell. Although normal F-2 cells formed tubular structures on Matrigel, VECD1 disturbed the tubulogenesis. VECD1 also increased the permeability through the F-2 cell layer. To clarify the in vivo function of mouse cadherin-5, we intraperitoneally injected the hybridomas producing VECD1 into adult mice. Severe venous stasis and subcutaneous hemorrhage were induced within several days after the injection, resulting in the early death of the animals. These findings are evidence of an essential role of cadherin-5 in the regulation of vascular endothelial cell-cell adhesion in vivo.
  • Shinichi Nakagawa, Hiroaki Matsunami, Masatoshi Takeichi
    Current Topics in Developmental Biology 36 C 197 - 210 1997年01月01日 [査読有り][通常論文]
     
    This chapter illustrates how to assay the role of adhesion mechanisms in selective adhesion of embryonic brain cells and fibroblastic cell lines. The methods described in the chapter include how to dissociate cells while leaving a particular set of adhesion mechanisms intact, how to let them reaggregate, and how to evaluate selective cell adhesion. Cell-cell adhesion mechanisms are complex, and comprise multiple systems, Animals cells are known to exhibit selective adhesiveness. When cells derived from different tissues are mixed, they sort out. This property of cells is thought to play a key role in the organization of complex tissue architecture. Studies suggest that the selective adhesiveness of cells also contributes to regionalization in the embryonic brain. To understand the molecular basis of patterning in the developing brain, therefore, it is critical to accumulate much more information on the adhesion properties of the cells that belong to each compartment. There are many ways to measure and quantify cell-cell adhesiveness. A classic method is to dissociate cell masses into single cells, and observe their reaggregation process. This chapter presents an updated version of classic methods, illustrates how to dissociate cells, how to allow them to reaggregate, and how to assay their selective adhesiveness. © 1998 Academic Press, Inc.
  • S NAKAGAWA, M TAKEICHI
    DEVELOPMENT 121 5 1321 - 1332 1995年05月 [査読有り][通常論文]
     
    We identified two cadherins, c-cad6B and c-cad7, expressed by neural crest cells at their premigratory and migratory stages, respectively, in chicken embryos, cDNA transfection experiments showed that both were homophilic adhesion endowing cells with molecules, specific adhesiveness. During development, c-cad6B appeared in the neural fold, localizing at the future neural crest area. This expression was maintained during neural tube closure, but disappeared after neural crest cells had left the neural tube, suggesting its role in neural fold fusion and/or in the formation and maintenance of the presumptive neural crest domain in the neural plate/tube. Crest cells emerging from the neural tube lost c-cad6B, and a subpopulation of them began to express c-cad7. This subpopulation-specific expression of c-cad7 persisted during their migration. The migrating c-cad7-positive cells clustered together, and eventually populated restricted regions including the dorsal and ventral roots but very little ganglia. The latter was populated with N-cadherin-positive crest cells, Migrating neural crest cells expressed alpha- and beta-catenin at cell-cell contacts, indicating that their cadherins are functioning. These results suggest that the migrating crest cells are grouped into subpopulations expressing different cadherins. The cadherin-mediated specific interaction between crest cells likely plays a role in intercellular signaling between homotypic cells as well as in sorting of heterotypic cells.

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  • 文部科学省:科学研究費補助金(挑戦的萌芽研究)
    研究期間 : 2012年 -2012年 
    代表者 : 中川 真一
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2011年 -2012年 
    代表者 : 中川 真一
  • 文部科学省:科学研究費補助金(基盤研究(S))
    研究期間 : 2009年 -2012年 
    代表者 : 吉田 稔, 中川 真一
     
    抗癌剤候補化合物として発見されたFR901464を改変して作成した安定誘導体Spliceostatin A (SSA)はスプライシング因子複合体SF3bに結合してスプライシング反応を阻害し、その結果pre-mRNAからタンパク質が合成され、イントロンの配列の翻訳を引き起こす。スプライシング阻害とイントロン配列翻訳のメカニズムを明らかにするため、in vitroスプライシングに対する効果を詳細に調べたところ、U1、SF1、U2AFなどが結合するE complexの形成には影響がなく、SF1とU2が入れ替わってA complexが形成されるステップで阻害が起こっていることがわかった。また、スプライシング阻害の程度は、イントロン内のBranch point配列に依存していることも明らかになった。さらにイントロン内にMS2配列を導入したプローブを用いたイメージング法によっても同様の結果が得られた。次にSSAの標的分子であるSF3bによって安定性が制御されていることが示唆されていた非コードRNAのXistの局在を制御する分子を同定するために、RNA結合ドメインを持つタンパク質に対するカスタムsiRNAライブラリーを作製し、特定の遺伝子をノックダウンした細胞におけるXistの局在を調べた。その結果、hnRNP Uというタンパク質がXistの染色体上への局在に必要なこと、hnRNP Uの機...
  • 文部科学省:科学研究費補助金(新学術領域研究(研究領域提案型))
    研究期間 : 2009年 -2012年 
    代表者 : 影山 裕二, 中川 真一
     
    21年度までの解析により、ショウジョウバエMRE32は胚発生期において中枢神経系特異的に発現するnon-coding RNAであることが明らかになっている。また、MRE32遺伝子の欠失変異体は発生に遅れが生じることから、MRE32が神経系において重要な機能を担っていることが推察されている。ショウジョウバエMRE32遺伝子の発現組織・細胞を同定するため、in situハイブリダイゼーションによる解析を行ったところ、従来明らかになっていた胚発生期の中枢神経系に加え、成虫視葉の二次狭窄を構成する神経細胞にもMRE32の発現が見られた。また、それ以外の多くの中枢神経細胞にも発現が見られたが、これらについては細胞の同定のためにさらなる解析が必要であると考えられる。胚発生期の発現細胞については、前胸腺特異的な分子マーカとの二重染色により、少なくとも食道神経節細胞の一部で発現していることが推察された。また、MRE32の転写領域を確定するため、RT-PCRによるマッピングを行ったところ、逆向きのトランスポゾン配列(roo element、約10kb)を含む約30kbがMRE32遺伝子被転写領域であると考えられた。この領域では、RNA-seq法により複数のタグが検出されており、転写活性は低いものの、何らかの転写産物が合成されているという従来の知見とよく一致する。なお、雌特異的と考えられたMRE...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2007年 -2009年 
    代表者 : 中川 真一
     
    網膜の最もマージン側の領域には長期間にわたって多分化能と分裂能を保つ網膜幹細胞が存在しており、それらは分泌性のシグナル分子であるWnt2bによって維持されている。本研究においては、Wntの下流で働いている転写因子に注目し、それらが作るネットワーク構造を解明することを目指した。その結果、Wntによって活性化されるβ-catenin/LEF1複合体がc-mycを含む複数の転写因子群を活性化していること、それらが協調して転写抑制因子であるc-hairy1を活性化していること、c-hairy1はWntの幹細胞維持活性を細胞に伝えるためのノードとして働いている事などを明らかにした。
  • 文部科学省:科学研究費補助金(特定領域研究)
    研究期間 : 2005年 -2006年 
    代表者 : 中川 真一
     
    我々は、中枢神経系の層構造の形成時に細胞タイプ特異的な振る舞いを制御する遺伝子を同定するために、それぞれの細胞タイプで特異的に発現している遺伝子を単一細胞サブトラクション法を用いて同定して来た。その過程で、通常のmRNAと異なり転写産物が核マトリクスに多数のスポットとして観察される、全長9kbのユニークなmRNA型ノンコーディングRNAを同定し、その特徴的な核内局在からこの遺伝子をGomafu(胡麻斑)と名付けた。本年度はGomafuの性質についてより詳細な解析を行なった。まず、GomafuをHelaやNeuro2Aなどの培養細胞に強制発現したところ、それらの細胞でも生体内と同様に核内に局在する事が分かった。また、これらの細胞を、Gomafuを発現していない親株と融合させてヘテロカリオンを作製したところ、Gomafu RNAはトランスフェクタント由来の核でのみ観察された事から、Gomafu RNAは転写後細胞質に輸送される事なく核内に保持されている事が分かった。また、これまでに知られているXist、Air、Evflなどの核内に局在するmRNA型ノンコーディングRNAと局在を比較したところ、GomafUはユニークな場所に局在する事が分かった。また、興味深い事に、Gomafu RNAは熱処理や翻訳阻害などのストレスによって消失する事が分かった。熱処理によっても新規タンパク質の合成...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2004年 -2006年 
    代表者 : 中川 真一
     
    器官形成の過程では細胞の増殖・分化は正確に制御されており、生み出された細胞が個々の形質に応じて適切な位置に配置されることで機能的な組織が形づくられる。我々はこれまでの研究において、分泌性のシグナル分子であるWnt2bが網膜の再集合培養の系で正しい層形成を誘導する事が出来る事、さらに実際の生体内では網膜幹細胞の維持に関わっている事などを明らかにして来た。本研究においては、これらの成果をさらに発展させ、どのようなシグナルが細胞内に伝わる事で幹細胞としての性質が維持されるかについて詳細な解析を行なった。神経細胞の分化はプロニューラル遺伝子と呼ばれる一群の転写因子によって促進され、その活性はNotch受容体からのシグナルで抑制されていることが知られている。そこで網膜幹細胞においてWntのシグナルとこれらの細胞分化抑制機構がどのような関係にあるかを調べたところ、興味深い事に、WntにはNotchの活性に非依存的にプロニューラル遺伝子の活性を抑制できることが明らかとなった。さらにWntの下流で働いている遺伝子を明らかにするためにWntのシグナルが活性化している細胞とそうでない細胞の間でサグトラクションスクリーニングを行ない、複数の遺伝子を得る事が出来た。これらのWnt応答性遺伝子の発現を実際の生体内において調べたところ、そのいくつかは網膜の幹細胞で特異的に発現しており、幹細胞においてWn...
  • 文部科学省:科学研究費補助金(特定領域研究)
    研究期間 : 2005年 -2005年 
    代表者 : 中川 真一
     
    中枢神経系の層形成の過程において、それぞれの細胞がどのようにして細胞タイプに応じた特定の層へ移動してゆくのかを調べるために、これまでに網膜をモデルシステムとして用いて、発生期に細胞タイプ特異的な発現を示す遺伝子を複数同定して来た。本年度はそれらの候補遺伝子を網膜全体に強制発現させ、それぞれの細胞の振る舞いに変化が起きるかを調べたが、顕著な異常を観察する事は出来なかった。一方、網膜への遺伝子導入技術を改良している過程で、時期特異的、かつ細胞タイプ特異的に遺伝子を発現する系を立ち上げる事に成功した。この技術を用いて網膜の水平細胞をEGFPでラベルしたところ、樹状突起の形態および軸索の有無から明確に区別出来る3種類の水平細胞が存在する事を見出した。さらに、これらの樹状突起の形態形成を制御する分子メカニズムを明らかにするために、神経系における主要な細胞間接着分子の一つであるカドヘリンの機能阻害実験を試みた。その結果、カドヘリンの機能を阻害すると、樹状突起の伸展が大きく阻害される事が分かった。しかしながら、短いながらもそれぞれの樹状突起の末端は適切な視細胞の末端に投射していた。ただし、そこでのシナプスマーカーの集積は阻害されていた。これらの事から、カドヘリンは樹状突起の伸展、および最終的なシナプス形成の過程を制御しているが、局所的な標的認識過程には関与していない事が明らかとなった。
  • 文部科学省:科学研究費補助金(特定領域研究)
    研究期間 : 2003年 -2004年 
    代表者 : 中川 真一
     
    中枢神経系の様々な領域で、特定の機能を持ったニューロンおよびグリア細胞が決められた位置に一列に並んだような、規則的な層構造が見られる。この層構造が形成される過程でそれぞれの細胞が細胞タイプに応じた目的地をどのように認識しそこへ移動してゆくのかを調べるために、我々は網膜をモデルシステムとして研究を進めてきた。網膜の神経節細胞と錐体光受容細胞は発生の初期、ほぼ同じ時期におなじ神経上皮のアピカル側で最終分裂を行うが、その後生み出された細胞は正反対の方向へ移動してゆく。すなわち神経節細胞は基底膜側に移動してゆくのに対し、錐体光受容細胞はアピカル側に戻ってきてそこで細胞層を形成する。我々はこのような振る舞いの違いはそれぞれの細胞タイプで特異的に発現している遺伝子によって制御されていると考え、以下のような実験を行った。まず、これらの細胞が移動をしているような時期のマウスの網膜を単一の細胞に解離し、それぞれの細胞からPCRによるバイアスを抑えるような条件のもとでcDNAを合成した。次にサザンプロット上でマーカー遺伝子の発現を調べることで、それぞれのcDNAがどの細胞タイプに由来するものかを遡及的に同定した。このようにして選んだ神経節細胞由来のcDNAと錐体光受容細胞由来のcDNAを用いてサブトラクションライブラリーを作製し、それぞれの細胞由来のプローブでディファレンシャルスクリーニングを行...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2001年 -2003年 
    代表者 : 中川 真一
     
    我々は、脊椎動物の中枢神経系に見られる層構造の形成が、どのような分子メカニズムによって制御されているか調べるために、特に網膜に注目して研究を行っている。未分化な網膜の細胞を単一細胞に乖離してから培養条件下で再集合させると、それらは組織塊を形成し、網膜に見られるすべての種類の神経細胞およびグリア細胞を作り出すことができる。しかしながらそのような組織には正常網膜に見られるような層構造は見られず、ロゼットと呼ばれる異常な構造体が形成される。我々は、この異常なロゼット形成を阻害するような領域が発生期の眼胞組織に存在するかどうかを調べるために、網膜の様々な領域から組織片を作製し、乖離した網膜前駆細胞と共培養を行った。その結果、レンズに隣接する網膜のもっともマージン側の領域にロゼット形成を阻害する活性があることが分かった。さらに、この領域で発現しているシグナル分子Wnt2bにも同様のロゼット形成阻害活性があることを見い出し、このタンパク質の存在下では単一細胞に乖離した網膜前駆細胞からも正しい層構造を持った網膜が再生することが分かった。次に、実際に発生過程におけるWnt2bの役割を調べるために、網膜全体でこの分子を発現させたところ、神経細胞への分化が抑制されることが分かった。また、ドミナントネガティブ分子を発現することによってWntの下流のシグナルを阻害すると、網膜のマージン側の領域で意志...

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