Building new foundation for biological sciences based on transposon-mediated gene trapping

2016 Fiscal Year Annual Research Report

• Project/Area Number: 15H02370
• Research Institution: National Institute of Genetics
• Principal Investigator: 川上 浩一 国立遺伝学研究所, 個体遺伝研究系, 教授 (70195048)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Keywords: ゲノム / 遺伝学 / 遺伝子 / 神経科学 / 発生・分化 / ゼブラフィッシュ / 遺伝子トラップ

Outline of Annual Research Achievements

（１）ゼブラフィッシュにおいて、Tol2トランスポゾンを用いた遺伝子トラップスクリーニングを実施し、細胞・組織・器官特異的に改変型酵母転写因子Gal4FFを発現するトランスジェニックフィッシュを新規に100系統作製した。 サザンブロッティング、インバースPCRによりこれらにおけるTol2挿入部位近傍のゲノムDNAの解析を行い、挿入部位を決定した。それら発現情報、遺伝子情報を とりこんだデータベースの整備を行った。
（２）Gal4FF発現トランスジェニックフィッシュを活用し、以下のことを明らかにした。トリコサンのアッセイシステムを確立（Tse博士らとの共同研究）。筋障害修復の過程で働くRax7陽性幹細胞を同定（Hughes博士ら）。細胞骨格を可視化するトランスジェニックフィッシュの開発（伊藤博士ら）。ニューレグリンの説普段を可視化（瀬原博士ら）。ヒレ再生におけるfgfシグナリングの役割の解明（川上厚志博士ら）。網膜における神経活動の波の発生メカニズムを解明（Du博士ら）。腸管神経細胞の移動メカニズムの解明（Heanue博士ら）。運動性繊毛の作り出す流れが嗅覚に重要であることを解明（Yaksi博士ら）。手綱核の視覚に依存する行動における役割を解明（Du博士ら）。GGGGCCリピートの蛋白質レベルでの毒性を証明（Schmid博士ら）。E3ユビキチンリガーぜASB2が心筋細胞の成熟に重要であることを解明（Stainier博士ら）。ヒレ再生過程における炎症の解消の重要性を解明（川上厚志博士ら）。
（３）これらのGal4FF発現トランスジェニックフィッシュは機能的神経回路の研究に有効に活用するために、ボツリヌス毒素遺伝子を用いてGal4FF発現神経細胞の機能を効率良く阻害することができるシステムの開発に成功した（Curr Biol 2016）。

Current Status of Research Progress

1: Research has progressed more than it was originally planned.

Reason

（１）ゼブラフィッシュにおいて、Tol2トランスポゾンを用いた遺伝子トラップスクリーニングを実施し、細胞・組織・器官特異的に改変型酵母転写因子Gal4FFを発現するトランスジェニックフィッシュを新規に100系統作製づることができた。
（２）平成28年度の研究において、Gal4FF発現トランスジェニックフィッシュを活用し、以下の共同研究を含む英文原著論文14報、総説１報、方法論を述べた著書３報を出版することができた。トリコサンのアッセイシステムを確立（Tse博士らと共著）。筋障害修復の過程で働くRax7陽性幹細胞を同定（Hughes博士ら）。細胞骨格を可視化するトランスジェニックフィッシュの開発（伊藤博士ら）。ニューレグリンの説普段を可視化（瀬原博士ら）。ヒレ再生におけるfgfシグナリングの役割の解明（川上厚志博士ら）。網膜における神経活動の波の発生メカニズムを解明（Du博士ら）。腸管神経細胞の移動メカニズムの解明（Heanue博士ら）。運動性繊毛の作り出す流れが嗅覚に重要であることを解明（Yaksi博士ら）。手綱核の視覚に依存する行動における役割を解明（Du博士ら）。GGGGCCリピートの蛋白質レベルでの毒性を証明（Schmid博士ら）。E3ユビキチンリガーぜASB2が心筋細胞の成熟に重要であることを解明（Stainier博士ら）。ヒレ再生過程における炎症の解消の重要性を解明（川上厚志博士ら）。
（３）ボツリヌス毒素遺伝子を用いて、非常に効率良くGal4FF発現神経細胞の機能を阻害するシステムの開発に成功し、論文を出版することができた（Curr Biol 2016）。

Strategy for Future Research Activity

平成28年度の研究を継続して発展させる。

Research Products

• [Int'l Joint Research] ICM/Universite Paris/INSERM(フランス)
  • Country Name: FRANCE
  • Counterpart Institution: ICM/Universite Paris/INSERM
• [Int'l Joint Research] Hong Kong Baptist University/Chinese Academy of Sciences(中国)
  • Country Name: CHINA
  • Counterpart Institution: Hong Kong Baptist University/Chinese Academy of Sciences
• [Int'l Joint Research] King’s College London/Oxford University/The Francis Crick Institute(英国)
  • Country Name: UNITED KINGDOM
  • Counterpart Institution: King’s College London/Oxford University/The Francis Crick Institute
• [Int'l Joint Research] NTNU(ノルウェー)
  • Country Name: NORWAY
  • Counterpart Institution: NTNU
• [Int'l Joint Research] Ludwig-Maximilians University/DZNE/Max Planck Institute(ドイツ)
  • Country Name: GERMANY
  • Counterpart Institution: Ludwig-Maximilians University/DZNE/Max Planck Institute
• [Int'l Joint Research]
  • # of Other Countries: 1
• [Journal Article] A novel zebrafish ret heterozygous model of Hirschsprung disease identifies a functional role for mapk10 as a modifier of enteric nervous system phenotype severity (2017)
  • Author(s): Heanue, T.A., Boesmans, W., Bell, D.M., Kawakami, K., Vanden Berghe, P., and Pachnis, V.
  • Journal Title: PLoS Genetics Volume: 12 Pages: e1006439
  • DOI: 10.1371/journal.pgen.1006439
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Motile-cilia-mediated flow improves sensitivity and temporal resolution of olfactory computations (2017)
  • Author(s): Reiten, I., Emre Uslu, F., Fore, S., Pelgrims, R., Ringers, C., Diaz Verdugo, C., Hoffman, M., Lal, P., Kawakami, K., Pekkan, K., Yaksi, E., and Jurisch-Yaksi, N.
  • Journal Title: Current Biology Volume: 27 Pages: 166-174
  • DOI: 10.1016/j.cub.2016.11.036
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Left habenula mediates light-preference behavior in zebrafish via an asymmetrical visual pathway (2017)
  • Author(s): Bai-bing, Z., Yao, Y.-y., Zhang, H.-f., Kawakami, K., and Du, J.-l.
  • Journal Title: Neuron Volume: 93 Pages: 914-928
  • DOI: 10.1016/j.neuron.2017.01.011
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Glycine-alanine dipeptide repeat protein contributes to toxicity in a zebrafish model of C9orf72 associated neurodegeneration (2017)
  • Author(s): Ohki, Y., Wenninger-Weinzierl, A., Hruscha, A., Asakawa, K., Kawakami, K., Haass, C., Edbauer, D., and Schmid, B.
  • Journal Title: Molecular Neurodegeneration Volume: 12 Pages: 6
  • DOI: 10.1186/s13024-016-0146-8
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Proteolysis regulates cardiomyocyte maturation and tissue integration (2017)
  • Author(s): Fukuda, R., Gunawan, F., Beisaw, A., Jimenez-Amilburu, V., Maischein, H.-M., Kostin, S., Kawakami, K., and Stainier, D.Y.R.
  • Journal Title: Nature Communications Volume: 8 Pages: 14495
  • DOI: 10.1038/ncomms14495
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Transient inflammatory response mediated by interleukin-1β is required for proper regeneration in zebrafish fin fold (2017)
  • Author(s): Hasegawa, T., Hall, C.J., Crosier, P.H., Abe, G., Kawakami, K., Kudo, A., and Kawakami, A.
  • Journal Title: eLife Volume: 6 Pages: e22716
  • DOI: 10.7554/eLife.22716
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Triclosan (TCS) exposure impairs lipid metabolism in zebrafish embryos (2016)
  • Author(s): Ho, J.C.H., Hsiao, C.D., Kawakami, K., and William, K.F.
  • Journal Title: Aquatic Toxicology Volume: 173 Pages: 29-35
  • DOI: 10.1016/j.aquatox.2016.01.001
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Cellular dynamics of regeneration reveals role of two distinct Pax7 stem cell populations in larval zebrafish muscle repair (2016)
  • Author(s): Pipalia, T.G., Koth, J., Roy, S.D., Hammond, C.L., Kawakami, K., and Hughes, S.M.
  • Journal Title: Disease Models and Mechanisms Volume: 9 Pages: 671-684
  • DOI: 10.1242/dmm.022251
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Gal4 driver transgenic zebrafish: powerful tools to study developmental biology, organogenesis, and neuroscience (2016)
  • Author(s): Kawakami, K., Asakawa, K., Hibi, M., Itoh, M., Muto, A., and Wada, H.
  • Journal Title: Advances in Genetics Volume: 95 Pages: 65-87
  • DOI: 10.1016/bs.adgen.2016.04.002
  • Peer Reviewed
• [Journal Article] Zebrafish lines expressing UAS-driven red probes for monitoring cytoskeletal dynamics (2016)
  • Author(s): Mizoguchi, T., Kawakami, K., and Itoh, M.
  • Journal Title: Genesis Volume: 54 Pages: 483-489
  • DOI: 10.1002/dvg.22955
  • Peer Reviewed / Open Access
• [Journal Article] Visualization of Neuregulin 1 ectodomain shedding reveals its local processing in vitro and in vivo (2016)
  • Author(s): Kamezaki, A., Sato, F., Aoki, K., Asakawa, K., Kawakami, K., Matsuzaki, F., and Sehara-Fujisawa, A.
  • Journal Title: Scientific reports Volume: 6 Pages: 28873
  • DOI: 10.1038/srep28873
  • Peer Reviewed / Open Access
• [Journal Article] Fgf signalling controls diverse aspects of fin regeneration (2016)
  • Author(s): Shibata, E., Yokota, Y., Horita, N., Kudo, A., Abe, G., Kawakami, K., and Kawakami, A.
  • Journal Title: Development Volume: 143 Pages: 2920-2929
  • DOI: 10.1242/dev.140699
  • Peer Reviewed / Open Access
• [Journal Article] Optimization of a neurotoxin to investigate the contribution of excitatory interneurons to speed modulation in vivo (2016)
  • Author(s): Sternberg, J.R., Severi, K.E., Fidelin, K., Gomez, J., Ihara, H., Alcheikh, Y., Hubbard, J. M., Kawakami, K., Suster, M., and Wyatt, C.
  • Journal Title: Current Biology Volume: 17 Pages: 2319-2328
  • DOI: 10.1016/j.cub.2016.06.037
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Stereotyped initiation of retinal waves by bipolar cells via presynaptic NMDA autoreceptors (2016)
  • Author(s): Zhang, R.-w., Li, X.-q., Kawakami, K., and Du, J.-l.
  • Journal Title: Nature Communications Volume: 7 Pages: 12650
  • DOI: 10.1038/ncomms12650
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Calcium dysregulation contributes to neurodegeneration in FTLD patient iPSC-derived neurons. (2016)
  • Author(s): Imamura, K., Sahara, N., Kanaan, N. M., Tsukita, K., Kondo, T., Kutoku, Y., Ohsawa, Y., Sunada, Y., Kawakami, K., Hotta, A., Yawata, S., Watanabe, D., Hasegawa, M., Trojanowski, J. Q., Lee, V. M.-Y., Suhara, T., Higuchi, M., and Inoue, H.
  • Journal Title: Scientific reports Volume: 6 Pages: 34904
  • DOI: 10.1038/srep34904
  • Peer Reviewed / Open Access
• [Journal Article] Fluorescence-activated cell sorting and gene expression profiling of GFP-positive cells from transgenic zebrafish lines (2016)
  • Author(s): Tanabe, H., Seki, M., Itoh, M., Deepak, A., Lal, P., Horiuchi, T., Suzuki, Y., and Kawakami, K.
  • Journal Title: Methods in Molecular Biology Volume: 1451 Pages: 93-106
  • DOI: 10.1007/978-1-4939-3771-4_7
• [Journal Article] Calcium imaging of neuronal activity in free-swimming larval zebrafish (2016)
  • Author(s): Muto, A., and Kawakami, K.
  • Journal Title: Methods in Molecular Biology Volume: 1451 Pages: 333-341
  • DOI: 10.1007/978-1-4939-3771-4_23
• [Journal Article] Tol2-mediated transgenesis, gene trapping, enhancer trapping, and Gal4-UAS system (2016)
  • Author(s): Kawakami, K., Asakawa, K., Muto, A., and Wada, H.
  • Journal Title: Methods in Cell Biology Volume: 135 Pages: 19-37
  • DOI: 10.1016/bs.mcb.2016.01.011
• [Presentation] The zebrafish amygdala and hippocampus (2017)
  • Author(s): Koichi Kawakami
  • Organizer: 7th Strategic Conference of Zebrafish Investigators
  • Int'l Joint Research / Invited
• [Presentation] Activation of the hypothalamic feeding centre upon visual prey detection (2016)
  • Author(s): Akira Muto, Koichi Kawakami
  • Organizer: The 39th Annual Meeting of the Japan Neuroscience Society
  • Int'l Joint Research / Invited
• [Presentation] The study of hippocampal functions in zebrafish (2016)
  • Author(s): Hideyuki Tanabe, Pradeep Lal, Akira Muto, Koichi Kawakami
  • Organizer: 第22回小型魚類研究会
• [Presentation] ゼブラフィッシュの扁桃体、海馬に相当する機能的神経回路の遺伝学的解析 (2016)
  • Author(s): 川上浩一
  • Organizer: 日本進化学会 第18回東京大会
  • Invited
• [Presentation] The zebrafish amygdala and hippocampus (2016)
  • Author(s): Koichi Kawakami
  • Organizer: The 9th Zebrafish Disease Models Conference
  • Int'l Joint Research / Invited
• [Presentation] The zebrafish amygdala and hippocampus (2016)
  • Author(s): Koichi Kawakami
  • Organizer: 7th Asia Oceania Zebrafish Meeting
  • Int'l Joint Research / Invited
• [Presentation] The zebrafish amygdala and hippocampus (2016)
  • Author(s): Koichi Kawakami
  • Organizer: The 4th Imaging Structure and Function in the Zebrafish Brain Conference
  • Int'l Joint Research / Invited
• [Book] Zebrafish: Methods and Protocols (2016)
  • Author(s): Kawakami, K., Patton, E.E., Orger, M.
  • Total Pages: 366
  • Publisher: Springer
  • ISBN: 978-1-4939-3771-4