Researcher Profile and Settings

Affiliation

• Faculty of Fisheries Sciences　Marine Life Science　Aquaculture Genetics and Genomics

Job Title

• Assistant Professor

J-Global ID

• 201801002712190354

Research Interests

• 生殖細胞 不妊化技術 ゲノム編集 メダカ ドジョウ サケ類   

Research Areas

• Life sciences / Marine/Aquatic life sciences

Educational Organization

•  Bachelor's degree program　School of Fisheries Sciences
•  Master's degree program　Graduate School of Fisheries Sciences
•  Doctoral (PhD) degree program　Graduate School of Fisheries Sciences

Academic & Professional Experience

• 2016/03 - 2020/03 名古屋大学大学院理学研究科 生殖生物学グループ 助教
• 2015/04 - 2016/02 National Institute for Basic Biology

Research Activities

Published Papers

• In Vitro Storage of Functional Sperm at Room Temperature in Zebrafish and Medaka.

  Kazumasa Takemoto, Toshiya Nishimura, Toshihiro Kawasaki, Yukiko Imai, Karine Levy, Neta Hart, Ivan Olaya, Sean M Burgess, Yaniv M Elkouby, Minoru Tanaka, Noriyoshi Sakai

  Zebrafish 2023/11/20 

  The longevity of sperm in teleost such as zebrafish and medaka is short when isolated even in saline-balanced solution at a physiological temperature. In contrast, some internal fertilizers exhibit the long-term storage of sperm, >10 months, in the female reproductive tract. This evidence implies that sperm in teleost possesses the ability to survive for a long time under suitable conditions; however, these conditions are not well understood. In this study, we show that the sperm of zebrafish can survive and maintain fertility in L-15-based storage medium supplemented with bovine serum albumin, fetal bovine serum, glucose, and lactic acid for 28 days at room temperature. The fertilized embryos developed to normal fertile adults. This storage medium was effective in medaka sperm stored for 7 days at room temperature. These results suggest that sperm from external fertilizer zebrafish and medaka has the ability to survive for at least 4 and 1 week, respectively, in the body fluid-like medium at a physiological temperature. This sperm storage method allows researchers to ship sperm by low-cost methods and to investigate key factors for motility and fertile ability in those sperm.
• Generation of Self-Fertilizing Hermaphroditic Fish from Gonochoristic Fish, Medaka (<b><i>Oryzias latipes</i></b>)

  Toshiya Nishimura, Minoru Tanaka

  Sexual Development 16 (4) 283 - 288 1661-5425 2022/11/30 [Refereed][Not invited]
   

  <b><i>Introduction:</i></b> Hermaphroditism is a mode of reproduction involving an individual animal that possesses both a testis and an ovary either sequentially or simultaneously. The mechanism creating hermaphrodites remains unknown. Previously, we identified <i>foxl3</i> as the germline sex determination gene in a gonochoristic fish, medaka (<i>Oryzias latipes</i>). <i>foxl3</i> loss-of-function (<i>foxl3</i>^{<i>−/−</i>}) females produce functional sperm as well as eggs in the ovary. However, these two gametes are not self-fertilizing because of the histological separation of each gamete production. In this study, we attempted to generate self-fertilizing medaka from female medaka by modifying germline sex using <i>foxl3</i>^−/− mutants and by using exogenous androgen to induce partial sex reversal of somatic cells. <b><i>Methods:</i></b> <i>foxl3</i>^−/− XX females were treated with 11-ketotestosterone (11-KT), a potent teleost fish androgen, at the sexually mature stage for 30 days (90–120 dph). Then, the fish were kept under normal conditions until they were either being dissected or crossed with infertile males. <b><i>Results and Discussion:</i></b> We showed that the <i>foxl3</i>^−/− XX female medaka can be transformed into a self-fertilizing hermaphrodite by inducing the formation of a male-like structure with exogenous 11-KT. Self-fertilization occurs in either the ovarian cavity, the oviduct, or both where sperm is released from a tubule-like structure which is likely derived from germinal epithelium, suggesting that timely modification of 2 independent mechanisms, regulation of germline sex and partial sex reversal of somatic cells, are critical to change the reproduction mode. Our results will provide insights in developmental and evolutional occurrence of hermaphrodite vertebrates, facilitate an innovative technique to improve the efficient selection of fish with desirable traits, and contribute to the rescue of endangered species.
• Zygotic nanos3 Mutant Medaka (Oryzias latipes) Displays Gradual Loss of Germ Cells and Precocious Spermatogenesis During Gonadal Development

  Toshiya Nishimura, Minoru Tanaka

  Zoological Science 39 (3) 0289-0003 2022/03/22 [Refereed][Not invited]
  
• Chromosome Set Manipulation and Genome Manipulation in Aquaculture

  Takafumi Fujimoto, Toshiya Nishimura

  Nippon Shokuhin Kagaku Kogaku Kaishi 68 (7) 277 - 289 1341-027X 2021/07/15
• Dynamics of Spermatogenesis and Change in Testicular Morphology under ‘Mating’ and ‘Non-Mating’ Conditions in Medaka (Oryzias latipes)

  Ruka Sumita, Toshiya Nishimura, Minoru Tanaka

  Zoological Science 38 (5) 0289-0003 2021/07/13 [Refereed][Not invited]
  
• Observation of Larval by Immunohistochemistry and

  Toshiya Nishimura, Minoru Tanaka

  Methods in Molecular Biology 209 - 218 1064-3745 2021 [Not refereed][Invited]
  
• Critical roles of the ddx5 gene in zebrafish sex differentiation and oocyte maturation

  Ryota Sone, Kiyohito Taimatsu, Rie Ohga, Toshiya Nishimura, Minoru Tanaka, Atsuo Kawahara

  Scientific Reports 10 (1) 14157 - 14157 2020/12 

  DEAD-box helicase 5 (Ddx5) functions as an ATP-dependent RNA helicase and as a transcriptional coactivator for several transcription factors; however, the developmental function of the ddx5 gene in vertebrates is not fully understood. We found that the zebrafish ddx5 gene was expressed in developing gonads. Using the genome editing technology transcription activator-like effector nuclease, we established a ddx5-disrupted zebrafish and examined the morphological phenotypes of the mutant. We found that the majority of ddx5-deficient mutants developed as fertile males with normal testes and a small number of ddx5-deficient mutants developed as infertile females with small ovaries. Apoptotic cell death at 31 days post fertilization was increased in thick immature gonads (presumptive developing ovaries) of the ddx5-deficient mutant compared to those of heterozygous wild-type fish, while the number of apoptotic cells in thin immature gonads (presumptive developing testes) was comparable between the mutant and wild-type animals. Histological analysis revealed that ovaries of adult ddx5-deficient females had fewer vitellogenic oocytes and a larger number of stage I and II oocytes. The amount of cyclic adenosine monophosphate in the ddx5-deficient ovaries was high compared to that of wild-type ovaries, presumably leading to the mitotic arrest of oocyte maturation. Therefore, the ddx5 gene is dispensable for testis development, but it is essential for female sex differentiation and oocyte maturation in zebrafish.
• foxl3, a sexual switch in germ cells, initiates two independent molecular pathways for commitment to oogenesis in medaka

  Mariko Kikuchi, Toshiya Nishimura, Satoshi Ishishita, Yoichi Matsuda, Minoru Tanaka

  Proceedings of the National Academy of Sciences 117 (22) 12174 - 12181 0027-8424 2020/06/02 

  Germ cells have the ability to differentiate into eggs and sperm and must determine their sexual fate. In vertebrates, the mechanism of commitment to oogenesis following the sexual fate decision in germ cells remains unknown.Forkhead-box protein L3(foxl3) is a switch gene involved in the germline sexual fate decision in the teleost fish medaka (Oryzias latipes). Here, we show thatfoxl3organizes two independent pathways of oogenesis regulated byREC8 meiotic recombination protein a(rec8a), a cohesin component, andF-box protein(FBP) 47(fbxo47), a subunit of E3 ubiquitin ligase. In mutants of either gene, germ cells failed to undergo oogenesis but developed normally into sperm in testes. Disruption ofrec8aresulted in arrest at a meiotic pachytenelike stage specifically in females, revealing a sexual difference in meiotic progression. Analyses offbxo47mutants showed that this gene regulates transcription factors that facilitate folliculogenesis:LIM homeobox 8(lhx8b),factor in the germlineα (figla), andnewborn ovary homeobox(nobox). Interestingly, we found that thefbxo47pathway ensures that germ cells do not deviate from an oogenic pathway until they reach diplotene stage. The mutant phenotypes together with the timing of their expression imply that germline feminization is established during early meiotic prophase I.
• Seasonal changes in NRF2 antioxidant pathway regulates winter depression-like behavior

  Tomoya Nakayama, Kousuke Okimura, Jiachen Shen, Ying-Jey Guh, T. Katherine Tamai, Akiko Shimada, Souta Minou, Yuki Okushi, Tsuyoshi Shimmura, Yuko Furukawa, Naoya Kadofusa, Ayato Sato, Toshiya Nishimura, Minoru Tanaka, Kei Nakayama, Nobuyuki Shiina, Naoyuki Yamamoto, Andrew S. Loudon, Taeko Nishiwaki-Ohkawa, Ai Shinomiya, Toshitaka Nabeshima, Yusuke Nakane, Takashi Yoshimura

  Proceedings of the National Academy of Sciences 117 (17) 9594 - 9603 0027-8424 2020/04/28 

  Seasonal changes in the environment lead to depression-like behaviors in humans and animals. The underlying mechanisms, however, are unknown. We observed decreased sociability and increased anxiety-like behavior in medaka fish exposed to winter-like conditions. Whole brain metabolomic analysis revealed seasonal changes in 68 metabolites, including neurotransmitters and antioxidants associated with depression. Transcriptome analysis identified 3,306 differentially expressed transcripts, including inflammatory markers, melanopsins, and circadian clock genes. Further analyses revealed seasonal changes in multiple signaling pathways implicated in depression, including the nuclear factor erythroid-derived 2-like 2 (NRF2) antioxidant pathway. A broad-spectrum chemical screen revealed that celastrol (a traditional Chinese medicine) uniquely reversed winter behavior. NRF2 is a celastrol target expressed in the habenula (HB), known to play a critical role in the pathophysiology of depression. Another NRF2 chemical activator phenocopied these effects, and an NRF2 mutant showed decreased sociability. Our study provides important insights into winter depression and offers potential therapeutic targets involving NRF2.
• Starvation causes female-to-male sex reversal through lipid metabolism in the teleost fish, medaka (Olyzias latipes)

  Yuta Sakae, Akira Oikawa, Yuki Sugiura, Masatoshi Mita, Shuhei Nakamura, Toshiya Nishimura, Makoto Suematsu, Minoru Tanaka

  Biology Open 9 (4) bio050054 - bio050054 2020/04/15 

  The teleost fish, medaka (Oryzias latipes), employs the XX/XY genetic sex determination system. We show here that the phenotypic sex of medaka is affected by changes in lipid metabolism. Medaka larvae subjected to 5 days of starvation underwent female-to-male sex reversal. Metabolomic and RT-qPCR analyses indicated that pantothenate metabolism was suppressed by starvation. Consistently, inhibiting the pantothenate metabolic pathway caused sex reversal. The final metabolite in this pathway is coenzyme A, an essential factor for lipogenesis. Inhibiting fatty acid synthesis, the first step of lipogenesis, also caused sex reversal. The expression of dmrt1, a critical gene for male development, was suppressed by starvation, and a dmrt1 (Δ13) mutant did not show sex reversal under starvation. Collectively, these results indicate that fatty acid synthesis is involved in female-to-male sex reversal through ectopic expression of male gene dmrt1 under starvation.
• Seasonal regulation of the lncRNA LDAIR modulates self-protective behaviours during the breeding season

  Tomoya Nakayama, Tsuyoshi Shimmura, Ai Shinomiya, Kousuke Okimura, Yusuke Takehana, Yuko Furukawa, Takayuki Shimo, Takumi Senga, Mana Nakatsukasa, Toshiya Nishimura, Minoru Tanaka, Kataaki Okubo, Yasuhiro Kamei, Kiyoshi Naruse, Takashi Yoshimura

  Nature Ecology & Evolution 3 (5) 845 - 852 2019/05 

  To cope with seasonal environmental changes, animals adapt their physiology and behaviour in response to photoperiod. However, the molecular mechanisms underlying these adaptive changes are not completely understood. Here, using genome-wide expression analysis, we show that an uncharacterized long noncoding RNA (lncRNA), LDAIR, is strongly regulated by photoperiod in Japanese medaka fish (Oryzias latipes). Numerous transcripts and signalling pathways are activated during the transition from short- to long-day conditions; however, LDAIR is one of the first genes to be induced and its expression shows a robust daily rhythm under long-day conditions. Transcriptome analysis of LDAIR knockout fish reveals that the LDAIR locus regulates a gene neighbourhood, including corticotropin releasing hormone receptor 2, which is involved in the stress response. Behavioural analysis of LDAIR knockout fish demonstrates that LDAIR affects self-protective behaviours under long-day conditions. Therefore, we propose that photoperiodic regulation of corticotropin releasing hormone receptor 2 by LDAIR modulates adaptive behaviours to seasonal environmental changes.
• Novel components of germline sex determination acting downstream of foxl3 in medaka

  Mariko Kikuchi, Toshiya Nishimura, Daisuke Saito, Shuji Shigenobu, Ritsuko Takada, José Arturo Gutierrez-Triana, Juan Luis Mateo Cerdán, Shinji Takada, Joachim Wittbrodt, Mikita Suyama, Minoru Tanaka

  Developmental Biology 445 (1) 80 - 89 0012-1606 2019/01 

  Germline sex determination is an essential process for the production of sexually dimorphic gametes. In medaka, Forkhead box L3 (foxl3) was previously identified as a germ cell-intrinsic regulator of sex determination that suppresses the initiation of spermatogenesis in female germ cells. To reveal the molecular mechanism of germline sex determination by foxl3, we conducted the following four analyses: Comparison of transcriptomes between wild-type and foxl3-mutant germ cells; epistatic analysis; identification of the FOXL3-binding motif; and ChIP-qPCR assay using a FOXL3-monoclonal antibody. We identified two candidate genes acting downstream of foxl3: Rec8a and fbxo47. It has been known that Rec8 regulates sister chromatid cohesion and Fbxo47 acts as a ubiquitin E3 ligase. These functions have not been, however, associated with sexual differentiation in germ cells. Our results uncover novel components acting downstream of foxl3, providing insights into the mechanism of germline sex determination.
• Germ cells in the teleost fish medaka have an inherent feminizing effect

  Toshiya Nishimura, Kazuki Yamada, Chika Fujimori, Mariko Kikuchi, Toshihiro Kawasaki, Kellee R. Siegfried, Noriyoshi Sakai, Minoru Tanaka

  PLOS Genetics 14 (3) e1007259 - e1007259 2018/03/29
• Dynamic plasticity in phototransduction regulates seasonal changes in color perception

  Tsuyoshi Shimmura, Tomoya Nakayama, Ai Shinomiya, Shoji Fukamachi, Masaki Yasugi, Eiji Watanabe, Takayuki Shimo, Takumi Senga, Toshiya Nishimura, Minoru Tanaka, Yasuhiro Kamei, Kiyoshi Naruse, Takashi Yoshimura

  NATURE COMMUNICATIONS 8 2041-1723 2017/09 

  To cope with seasonal changes in the environment, organisms adapt their physiology and behavior. Although color perception varies among seasons, the underlying molecular basis and its physiological significance remain unclear. Here we show that dynamic plasticity in phototransduction regulates seasonal changes in color perception in medaka fish. Medaka are active and exhibit clear phototaxis in conditions simulating summer, but remain at the bottom of the tank and fail to exhibit phototaxis in conditions simulating winter. Mate preference tests using virtual fish created with computer graphics demonstrate that medaka are more attracted to orange-red-colored model fish in summer than in winter. Transcriptome analysis of the eye reveals dynamic seasonal changes in the expression of genes encoding photopigments and their downstream pathways. Behavioral analysis of photopigment-null fish shows significant differences from wild type, suggesting that plasticity in color perception is crucial for the emergence of seasonally regulated behaviors.
• foxl3 is a germ cell-intrinsic factor involved in sperm-egg fate decision in medaka

  T. Nishimura, T. Sato, Y. Yamamoto, I. Watakabe, Y. Ohkawa, M. Suyama, S. Kobayashi, M. Tanaka

  Science 349 (6245) 328 - 331 0036-8075 2015/07/17 

  Sex determination is an essential step in the commitment of a germ cell to a sperm or egg. However, the intrinsic factors that determine the sexual fate of vertebrate germ cells are unknown. Here, we show that foxl3, which is expressed in germ cells but not somatic cells in the gonad, is involved in sperm-egg fate decision in medaka fish. Adult XX medaka with disrupted foxl3 developed functional sperm in the expanded germinal epithelium of a histologically functional ovary. In chimeric medaka, mutant germ cells initiated spermatogenesis in female wild-type gonad. These results indicate that a germ cell-intrinsic cue for the sperm-egg fate decision is present in medaka and that spermatogenesis can proceed in a female gonadal environment.
• Analysis of a novel gene, Sdgc, reveals sex chromosome-dependent differences of medaka germ cells prior to gonad formation

  T. Nishimura, A. Herpin, T. Kimura, I. Hara, T. Kawasaki, S. Nakamura, Y. Yamamoto, T. L. Saito, J. Yoshimura, S. Morishita, T. Tsukahara, S. Kobayashi, K. Naruse, S. Shigenobu, N. Sakai, M. Schartl, M. Tanaka

  Development 141 (17) 3363 - 3369 0950-1991 2014/09/01
• Hyperproliferation of mitotically active germ cells due to defective anti-Mullerian hormone signaling mediates sex reversal in medaka

  S. Nakamura, I. Watakabe, T. Nishimura, J.-Y. Picard, A. Toyoda, Y. Taniguchi, N. di Clemente, M. Tanaka

  Development 139 (13) 2283 - 2287 0950-1991 2012/07/01
• Analysis of Medaka sox9 Orthologue Reveals a Conserved Role in Germ Cell Maintenance

  Shuhei Nakamura, Ikuko Watakabe, Toshiya Nishimura, Atsushi Toyoda, Yoshihito Taniguchi, Minoru Tanaka

  PLoS ONE 7 (1) e29982 - e29982 2012/01/12
• Sexual dimorphism of gonadal structure and gene expression in germ cell-deficient loach, a teleost fish

  T. Fujimoto, T. Nishimura, R. Goto-Kazeto, Y. Kawakami, E. Yamaha, K. Arai

  Proceedings of the National Academy of Sciences National Academy of Sciences 107 (40) 17211 - 17216 0027-8424 2010/10/05 

  Germ cell deficient fish usually develop as phenotypic males. Thus, the presence of germ cells is generally considered to be essential for female gonadal differentiation or the maintenance of ovarian structure. However, little is known of the role of germ cells in the determination of the sexual fate of gonadal somatic cells. We have established an inducible germ cell deficiency system in the loach (Misgurnus anguillicaudatus, Cypriniformes: Cobitidae), a small fresh-water fish, using knock-down of the dead end gene with a morpholino antisense oligonucleotide. Interestingly, loach lacking germ cells could develop as either phenotypic males or females, as characterized morphologically by the presence or absence of bony plates in the pectoral fins, respectively. The phenotypic males and females had testicular and ovarian structures, respectively, but lacked germ cells. Gene expression patterns in these male and female germ cell deficient gonads were essentially the same as those in gonads of normal fish. Our observations indicate that sexually dimorphic gonads can develop in germ cell deficient loach. In contrast to the situation in other model fish species, the gonadal somatic cells in phenotypic females autonomously differentiated into ovarian tissues and also played a role in the maintenance of gonadal structure. On the basis of our observations, we propose two possible models to explain the role of germ cells in sex determination in fish.
• Identification of Germline Stem Cells in the Ovary of the Teleost Medaka

  S. Nakamura, K. Kobayashi, T. Nishimura, S.-i. Higashijima, M. Tanaka

  Science 328 (5985) 1561 - 1563 0036-8075 2010/06/18

Books etc

• Sexual plasticity and gametogenesis in fishes

  Senthilkumaran, Balasubramanian (ContributorFunction of Germ Cells in Sex Differentiation)
  Nova Science Publishers 2013 (ISBN: 9781626188488) xii, 456 p.

Conference Activities & Talks

• メダカ半数性生殖細胞はクローン配偶子を産生するか?  [Not invited]

  竹内萌, 山羽悦郎, 藤本貴史, 西村俊哉

  令和５年度 日本水産学会春季大会  2023/03
• CRISPR-Cas13d システムを用いた汎用性の高い不妊化技術の確立  [Not invited]

  西村俊哉, 飯野菜帆, 藤本貴史, 高橋英佑, 山羽悦郎

  令和５年度 日本水産学会春季大会  2023/03
• クローン系統ドジョウにおける生殖細胞可視化系統の樹立  [Not invited]

  中村風歌, 藤本貴史, 西村俊哉

  令和５年度 日本水産学会春季大会  2023/03

MISC

• The Mechanism of Germline Sex Determination in Vertebrates

  T. Nishimura, M. Tanaka  Biology of Reproduction  95-  (1)  30  -30  2016/07/28
• Identification of the gene for germline sex determination in medaka

  西村俊哉, 田中実  臨床免疫・アレルギー科  65-  (2)  164  -168  2016/02  [Not refereed][Invited]
  
• A Structurally and Functionally Common Unit in Testes and Ovaries of Medaka (Oryzias latipes), a Teleost Fish

  Toshiya Nishimura, Shuhei Nakamura, Minoru Tanaka  Sexual Development  10-  (3)  159  -165  2016
• Gonadal Development in Fish

  Toshiya Nishimura, Minoru Tanaka  Sexual Development  8-  (5)  252  -261  2014
• Germline stem cells and sex differentiation in medaka (Oryzias latipes)

  NAKAMURA Shuhei, KOBAYASHI Kayo, NISHIMURA Toshiya, TANAKA Minoru  生化學  83-  (7)  627  -632  2011/07/25
• Ovarian Germline Stem Cells in the Teleost Fish, Medaka (Oryzias latipes)

  Shuhei Nakamura, Kayo Kobayashi, Toshiya Nishimura, Minoru Tanaka  International Journal of Biological Sciences  7-  (4)  403  -409  2011
• 配偶子幹細胞:世代をつなぐキープレーヤーの正体と可能性 メダカ卵巣の配偶子幹細胞

  中村修平, 小林佳代, 西村俊哉, 田中実  細胞工学  29-  (7)  664-669  -669  2010/06/22  [Not refereed][Not invited]
  
• メダカ(Oryzias latipes)卵巣生殖幹細胞の同定

  中村修平, 小林佳代, 西村俊哉, 西村俊哉, 東島眞一, 田中実, 田中実  日本動物学会大会予稿集  81st-  2010

Research Grants & Projects

• 鰭(ヒレ)から魚を創る

  科学技術振興機構：戦略的な研究開発の推進 創発的研究支援事業

  Date (from‐to) : 2022 -2028 

  Author : 西村 俊哉

   

  本研究ではヒレから魚を再生させる技術開発を目指します。このようなことが実現すれば、魚類において「精子と卵=唯一の個体再生可能な細胞」という概念が破壊されるため、水産育種をはじめ、遺伝資源の保存や絶滅危惧種の再生に新たな技術変革をもたらします。また、ヒレから魚を創るプロセスを解き明かすことで、分化細胞から多能性幹細胞へのリプログラミングや生殖細胞が形成する仕組みの理解に繋がります。
• The molecular basis for germ cell development in teleost fish

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2021/04 -2024/03 

  Author : 西村 俊哉

   

  本研究の目的は、魚類における生殖細胞の分子基盤を明らかにし、魚類で汎用的に利用可能な不妊化技術を確立することである。そこで２０２１年度は、CRISPR-Cas13dシステムを用いた遺伝子ノックダウンの効果の確認と生殖腺へ到達する前の生殖細胞（PGC)の遺伝子発現プロファイルを得た。 CRISPR-Cas13dシステムをにおけるgRNAの設計方法、ターゲットmRNAへの結合部位のミスマッチの許容について調べた。哺乳類の研究を元に開発されたgRNA設計ツールであるCas13 design(https://cas13design.nygenome.org/)を用いることで、メダカにおいてノックダウン効果のあるgRNA（23nt)を設計できることが明らかとなった。生殖顆粒因子であるnanos3及びdnd1をターゲットするgRNAを３つずつ設計したところ、nanos3では３つ、dnd1では２つのgRNAに、ほぼ１００％の効率で生殖細胞を欠損できるノックダウン効果があることが分かった。またgRNAに、stem loop側から17~20番目の塩基に２塩基のミスマッチを入れると著しくノックダウン効果が落ちることから、23ntのgRNAを用いた場合、この領域にシード配列があると考えられる。さらに、上記と同様のgRNAの設計方法で、産業重要種であるサケ科魚類においても、dnd1をターゲットするgRNAによって生殖細胞を欠損させることに成功した。 生殖細胞形成に必要な分子基盤を明らかにするために、生殖腺へ到達する前の生殖細胞（PGC)をセルソーターで分取し、そこから得られた微量RNAを用いて、RNAseqによる遺伝子発現プロファイルを得た。現在、リアルタイムPCR及びin situ hybridizationによる遺伝子発現の調査を進めている。
• Mechanism of diploid gamete formation and its application for genetic breeding

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2021/04 -2024/03 

  Author : 藤本 貴史, 西村 俊哉, 田中 啓介, 黒田 真道

   

  ①ゲノム倍加を生じる系統間ゲノムの違い、②ゲノム倍加の分子機構の解明、③雑種由来の二倍性配偶子の育種応用に向けた実証研究の3項目の概要を下記に示す。 ①ドジョウB系統の倍加半数体の遺伝子型が完全同型接合であることを確認し、倍加半数体の高分子DNAをロングリードシーケンサーによるゲノム解析に供した。得られたリードデータを用いて、ゲノム構造解析の基盤整備を行った。また、B系統特異的なFISHプローブを開発するとともに、人為的に作出した二系統間雑種でGISHを行った結果、A系統をプローブDNAに用いた場合はA系統由来と推定される染色体の全域が染色されたのに対し、B系統をプローブDNAに用いた場合はB系統と推定される染色体のセントロメア領域が強く染色され、系統間におけるゲノム構造の変異が示唆された。 ②クローンドジョウの卵巣をトリプシンやコラゲナーゼ等の消化酵素を用いて細胞を開始した後、メッシュフィルターを通して大型卵母細胞を除去することにより、小型の細胞のみを回収した。これらの細胞を固定後、Vasa抗体を用いて免疫蛍光染色に供した結果、比較的大型の核を有する卵原細胞が確認された。また、FACSによる分析では、Vasa陽性の細胞がDAPI蛍光強度で3種類に分類できることが明らかとなった。一方、生殖細胞の可視化にむけた遺伝子組換えクローンドジョウでは、piwiあるいはvasaのプロモーター領域とEGFPからなるコンストラクトを作製し、これらを顕微注入した遺伝子組換え候補個体の作出を行った。 ③サケ科ではイワナ属の種間雑種では受精能を有する半数性の配偶子形成が確認された。他のサケ科雑種に関しては成熟に至らず、現在も継続して飼育している。ドジョウ雑種ではカラドジョウ雌と各系統のドジョウ雄の雑種雄は不妊性を示すことが確認された。メダカ属雑種では、雑種個体を誘起し成熟に向けて飼育を継続している。
• Studies on sexual dimorphism of chromosome and nucleolus dynamics in germ cells

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))

  Date (from‐to) : 2018/10 -2022/03 

  Author : Nishimura Toshiya

   

  The goal of the project was to reveal how chromosome and nucleolus dynamics differ between male and female germ cells and the mechanism behind the sexual dimorphism. To achieve the goal, live imaging of medaka germ cells was established in collaboration with Dr. Huynh in the College de France, who has pioneered live imaging technique using Drosophila germ cells. By culturing testes in a microfluidic device during live imaging with a spinning disc confocal laser microscopy, we successfully monitored medaka germ cells for more than 24 hours at a high resolution. We also generated transgenic medaka in which synaptonemal complex, a protein structure forms between homologous chromosomes during meiotic prophase, was able to be visualized by EGFP, which allows monitoring the dynamics of chromosome by live imaging.
• Identification of genes involved in germline sex determination by global screening

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)

  Date (from‐to) : 2018/06 -2020/03 

  Author : Nishimura Toshiya

   

  Germ cells are the only cells that can generate the next generation and produce either sperm and eggs. In this study, a genetic screening system was constructed to elucidate the mechanism of sperm-egg fate decision in germ cells using medaka. First, comparative gene expression analyses were performed between male and female germ cells to narrow down the number of candidate genes. Second, an efficient generation of DNA templates for gRNAs in CRISPR/Cas9 system was established. Third, transgenic lines in which spermatogenesis and oogenesis could be visualized by fluorescence were established. Upon gene knock-out, the phenotype could be easily distinguished by observing alive medaka under a fluorescent microscopy.
• Sex-reversal of germ cells in response to environments

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

  Date (from‐to) : 2016/04 -2018/03 

  Author : Nishimura Toshiya

   

  The applicant identified foxl3 as a sperm-egg switch gene in medaka. FOXL3 is expressed in female germ cells, and foxl3 loss-of-function (foxl3-/-) females initiate spermatogenesis in ovaries. However, foxl3-/- females also produce eggs by unknown mechanism. In this study, the applicant examined the downstream targets of foxl3 to understand how egg fate decision occurs, and asked whether foxl3 and figla, which is required for follicle formation, could be determinants of eggs. Genes related to chromosomal dynamics were found in targets of foxl3. Among then, a novel ubiquitin ligase likely regulated female-specific mitosis in germ cells. The applicant also generated transgenic medaka in which foxl3/figla genes could be induced in germ cells of testes using Cre/loxP system. In the future, production of eggs in testes will be examined using the transgenic medaka.
• 生殖細胞による体細胞のメス化メカニズムの解明

  日本学術振興会：科学研究費助成事業 研究活動スタート支援

  Date (from‐to) : 2015/08 -2016/03 

  Author : 西村 俊哉

   

  生殖細胞は、精子と卵の元となる細胞であるが、ただ配偶子を作るだけでなく、生殖腺の性分化、特にメス化に重要であることがメダカを用いた研究で明らかとなった。しかし、生殖細胞からどのような因子が分泌され、生殖腺をメス化するのか、そのメカニズムは全く分かっていない。本研究の目的は生殖細胞の分化段階に着目し、生殖細胞が特定の分化段階で止まる変異体を作出することで、生殖腺をメス化するために重要な生殖細胞の分化段階を特定することである。２７年度では、以下の変異体を作出することで生殖細胞をある特定の分化段階で停止させ、その変異体のXX個体の生殖腺が卵巣へ分化するのか調べた。 figla変異体：figla遺伝子は減数分裂へ進行した卵母細胞で発現することが知られている。figla変異体の生殖細胞は減数分裂のzygotene後期で停止し、濾胞を形成しなかった。ところがfigla変異体のXX個体は、第二次性徴がメスで、さらに生殖腺も卵巣であった。 moto変異体：moto変異体の生殖細胞は幹細胞型 (TypeI) からシスト型 (TypeII) への移行が抑制された。興味深いことにTypeIのみの生殖細胞を持つXX変異体は、第二次性徴がメスとなり、生殖腺も卵巣であることが明らかとなった。 dazl変異体：dazl変異体の生殖細胞は形態的な観察から始原生殖細胞様であることが明らかとなった。今後は同変異体を成魚まで育て、第二次性徴、生殖腺を調べる予定である。 以上のことから少なくともType I生殖細胞が存在すればXX個体において卵巣は形成されるという新たな知見が得られた。これは生殖細胞が体細胞をメス化させる因子の同定に貢献できると成果と考えられる。

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