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Mechanism of sex differentiation of germ cells

Research Project

Project/Area Number 17H06166
Research Category

Grant-in-Aid for Scientific Research (S)

Allocation TypeSingle-year Grants
Research Field Developmental biology
Research InstitutionNational Institute of Genetics

Principal Investigator

相賀 裕美子  国立遺伝学研究所, 遺伝形質研究系, 教授 (50221271)

Project Period (FY) 2017-05-31 – 2022-03-31
Project Status Granted (Fiscal Year 2021)
Budget Amount *help
¥203,060,000 (Direct Cost: ¥156,200,000、Indirect Cost: ¥46,860,000)
Fiscal Year 2021: ¥40,170,000 (Direct Cost: ¥30,900,000、Indirect Cost: ¥9,270,000)
Fiscal Year 2020: ¥38,740,000 (Direct Cost: ¥29,800,000、Indirect Cost: ¥8,940,000)
Fiscal Year 2019: ¥41,080,000 (Direct Cost: ¥31,600,000、Indirect Cost: ¥9,480,000)
Fiscal Year 2018: ¥51,740,000 (Direct Cost: ¥39,800,000、Indirect Cost: ¥11,940,000)
Fiscal Year 2017: ¥31,330,000 (Direct Cost: ¥24,100,000、Indirect Cost: ¥7,230,000)
Keywords生殖細胞 / 性分化 / RNA制御 / P-body / 蛋白分解 / RNA 結合因子 / mTORC系 / シングルセルRNA-seq / RNAの可視化 / RNA結合タンパク質 / 細胞周期 / NANOS2 / RNA編集 / 転写因子 / ES細胞 / キメラ解析 / RNA / 減数分裂
Outline of Annual Research Achievements

胎児期の生殖細胞において、NANOS2およびDND1が標的遺伝子を認識して発現抑制を行うことは明らかであるが、その詳細な分子機構はまだ不明であった。培養細胞にNANOS2の標的遺伝子であるDazl及びSohlh2の3'-UTRを結合させ、かつMS2の認識配列を有したmRFPレポーターを発現させて、実際にNANOS2およびDND1がどのように標的遺伝子を認識し、機能するかを解析した。その結果、NANOS2は自分のRNA結合ドメインを介して標的RNAを結合したDND1を認識して結合すること。また標的1RNAの認識にはDND1のみでは不十分であり、NANOS2もその認識段階に関与することがNANOS2-DND1融合タンパク質の変異体解析により明らかになった。
NANOS2の下流で雄性文化に直接関与する遺伝子の探索を行った、NANOS2のエンハンサーに遺伝子を結合して、雄で強制発現させることにより、雄性化を抑制する遺伝子候補が得られた。
GS細胞を用いてFlag-NANOS3を条件付きでKOできる細胞を確立した。そのRNA-seq解析結果は、in vivoの解析結果を反映することが明らかになった。この系を用いて、NANOS3のインタラクターおよび、標的遺伝子候補を同定した。
生殖細胞の雄化モデルとしてSMAD4-cKO/RA阻害剤処理マウスの生殖細胞を単離し、シングルセル解析を行った。E13.5における他の遺伝子型と比較して、雄化に向かう特徴的な細胞が同定できた。

Current Status of Research Progress
Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

NANOS2の下流で機能する細胞分裂制御因子としてmTORC1系の因子、TSC2およびNrpl2を同定した。これらの因子は培養細胞において、mTORC系を制御することが確認できた。ES細胞を用いて、それぞれの因子の条件付きKOを作成し、キメラ解析により、興味深い結果が得られている。しかし、やはりコンスタントに解析するには、マウスラインの確立が必要である。現在、TSC2およびNrpl2のcKOマウスラインを確立中である。また雄性化にかかわる因子に関しても候補が得れらてれおり、現在、DOXを用いた強制発現系マウスを作成中である。

タンパクノックダウン系を確立するためにAID-tagをNANOS2, CNOT1, DDX6, DCP2遺伝子座にノックインしたマウスの確立に成功した。

Strategy for Future Research Activity

胎児期の生殖細胞の分化制御にかかわるRNA制御因子の機能解析をおこなっているが、遺伝子をノックアウトしてもタンパク質が維持されており、解析が不可能である場合が多い。特にNANOS2, CNOT1, DCP2など非常に重要なタンパク質は安定であることが明らかになった。今後オーキシンデグロン系を用いたタンパク分解系が重要になると考えられる。まずその系を確立するために必要なマウスの系を確立する。
一方、強制発現系はDOX系を利用する、NANOS2のエンハンサーで機能する、rtTA発現マウスを確立するとともに、発現誘導する遺伝子はROSAあるいはTigre遺伝子座に導入する。

Assessment Rating
Result (Rating)

A: Progress in the research is steadily towards the initial goal. Expected research results are expected.

Report

(8 results)
  • 2020 Annual Research Report   Abstract(Research Progress Assessment) ( PDF )   Research Progress Assessment (Result) ( PDF )
  • 2019 Annual Research Report
  • 2018 Annual Research Report
  • 2017 Abstract ( PDF )   Comments on the Screening Results ( PDF )   Annual Research Report

Research Products

(38 results)

All 2021 2020 2019 2018 2017 Other

All Journal Article (11 results) (of which Int'l Joint Research: 1 results,  Peer Reviewed: 11 results,  Open Access: 9 results,  Acknowledgement Compliant: 1 results) Presentation (26 results) (of which Int'l Joint Research: 13 results,  Invited: 9 results) Remarks (1 results)

  • [Journal Article] Genetic and structural analysis of the in vivo functional redundancy between murine NANOS2 and NANOS32021

    • Author(s)
      Danelle Wright, Makoto Kiso, Yumiko Saga
    • Journal Title

      Development .

      Volume: 148

    • DOI

      10.1242/dev.191916

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Mouse dead end1 acts with Nanos2 and Nanos3 to regulate testicular teratoma incidence2020

    • Author(s)
      Imai A, Hagiwara Y, Niimi Y, Tokumoto T, Saga Y, Suzuki A.
    • Journal Title

      PLOS ONE

      Volume: 15

    • DOI

      10.1371/journal.pone.0232047

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice2020

    • Author(s)
      Yesbolatova Aisha、Saito Yuichiro、Kitamoto Naomi、Makino-Itou Hatsune、Ajima Rieko、Nakano Risako、Nakaoka Hirofumi、Fukui Kosuke、Gamo Kanae、Tominari Yusuke、Takeuchi Haruki、Saga Yumiko、Hayashi Ken-ichiro、Kanemaki Masato T.
    • Journal Title

      Nat Commun .

      Volume: 11 Pages: 1-13

    • DOI

      10.1038/s41467-020-19532-z

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] ELAVL2‐directed RNA regulatory network drives the formation of quiescent primordial follicles2019

    • Author(s)
      Kato Yuzuru、Iwamori Tokuko、Ninomiya Youichirou、Kohda Takashi、Miyashita Jyunko、Sato Mikiko、Saga Yumiko
    • Journal Title

      EMBO Reports

      Volume: 20

    • DOI

      10.15252/embr.201948251

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Essential role of mouse Dead end1 in the maintenance of spermatogonia.2019

    • Author(s)
      Niimi Y, Imai A, Nishimura H, Yui K, Kikuchi A, Koike H, Saga Y, Suzuki A
    • Journal Title

      Developmental Biology

      Volume: 445 Pages: 103-112

    • DOI

      10.1016/j.ydbio.2018.11.003

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] ES-mediated chimera analysis revealed requirement of DDX6 for NANOS2 localization and function in mouse germ cells2019

    • Author(s)
      Shimada R, Kiso M, Saga Y
    • Journal Title

      Scientific Report

      Volume: 9 Pages: 515-515

    • DOI

      10.1038/s41598-018-36502-0

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Requirement of the 3′-UTR-dependent suppression of DAZL in oocytes for pre-implantation mouse development2018

    • Author(s)
      Kurumi Fukuda, Aki Masuda, Takuma Naka, Atsushi Suzuki, Yuzuru Kato, Yumiko Saga
    • Journal Title

      PLos Genetics

      Volume: 14 (6): e1007436

    • DOI

      10.1371/journal.pgen.1007436

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] NANOS2 acts as an intrinsic regulator of gonocytes-to-spermatogonia transition in the murine testes.2018

    • Author(s)
      Pui HP, Saga Y.
    • Journal Title

      Mech Dev.

      Volume: 149 Pages: 27-40

    • DOI

      10.1016/j.mod.2018.01.001

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Gonocytes-to-spermatogonia transition initiates prior to birth in murine testes and it requires FGF signaling.2017

    • Author(s)
      Pui HP, Saga Y.
    • Journal Title

      Mech Dev

      Volume: 印刷中 Pages: 125-139

    • DOI

      10.1016/j.mod.2017.03.002

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Roles of MIWI, MILI and PLD6 in small RNA regulation in mouse growing oocytes.2017

    • Author(s)
      Kabayama Y, Toh H, Katanaya A, Sakurai T, Chuma S, Kuramochi-Miyagawa S, Saga Y, Nakano T, Sasaki
    • Journal Title

      Nucleic acids research

      Volume: 印刷中 Pages: 5387-5398

    • DOI

      10.1093/nar/gkx027

    • NAID

      120006355769

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] NEDD4 controls spermatogonial stem cell homeostasis and stress response by regulating messenger ribonucleoprotein complexes.2017

    • Author(s)
      Zhou, Z., Kawabe, H., Suzuki, A., Shinmyozu, K., and Saga, Y.
    • Journal Title

      Nature Commun.

      Volume: 8 Pages: 15662-15662

    • DOI

      10.1038/ncomms15662

    • Related Report
      2017 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Presentation] Genetics without making mouse lines: P-body requirement for the function of NANOS2 in male germ cell development2020

    • Author(s)
      Yumiko Saga
    • Organizer
      The 6 th International Symposium toward the Future of Advanced Researches in Shizuoka University
    • Related Report
      2019 Annual Research Report
    • Invited
  • [Presentation] Germ cell specific conditional knockout without making mouse lines2019

    • Author(s)
      Yumiko Saga
    • Organizer
      The 15th Transgenic technology meeting
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] scRNA-Seqを利用したマウス生殖細胞オス化機構の研究2019

    • Author(s)
      島田龍輝、相賀裕美子
    • Organizer
      日本発生生物学会
    • Related Report
      2019 Annual Research Report
  • [Presentation] マウス初期発生におけるDDX6の役割2019

    • Author(s)
      Jessica Kim, Yumiko Saga
    • Organizer
      日本発生生物学会
    • Related Report
      2019 Annual Research Report
  • [Presentation] Toward understanding the molecular mechanism of sexual fate decision in murine germ cells2018

    • Author(s)
      相賀 裕美子
    • Organizer
      日本発生生物学会年会
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] Analysis of granulosa cell progenitor differentiation during primordial follicle formation in mice.2018

    • Author(s)
      福田 胡桃, 加藤 譲, 相賀 裕美子
    • Organizer
      日本発生生物学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] To explore the feminizing genes mediated by SMAD4 in germ cell2018

    • Author(s)
      島田 龍輝, 相賀 裕美子
    • Organizer
      日本発生生物学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Structural association between mouse NANOS and DND1 RNA binding proteins2018

    • Author(s)
      ダネル ライト, 相賀 裕美子
    • Organizer
      日本発生生物学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Requirement of DDX6 medicated P-body formation for the function of NANOS2 In male germ cell differentiation2018

    • Author(s)
      Yumiko Saga
    • Organizer
      COMBIO2018
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Analysis of the gene expression changes during male germ cell differentiation with scRNA-Seq2018

    • Author(s)
      島田 龍輝, 相賀 裕美子
    • Organizer
      CSH-Germ Cells Meeting
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research
  • [Presentation] DND1 protein is stabilized via interaction with its partner protein NANOS in mouse germ cells.2018

    • Author(s)
      ダネル ライト, 相賀 裕美子
    • Organizer
      CSH-Germ Cells Meeting
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research
  • [Presentation] MOLECULAR DISSECTION OF NANOS-MEDIATED RNA-REGULATION2018

    • Author(s)
      相賀 裕美子
    • Organizer
      CSH-Germ Cells Meeting
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] THE EXPRESSION NETWORK BETWEEN RNA-BINDING PROTEINS ELAV2 AND DDX6 IS REQUIRED FOR PRIMORDIAL FOLLICLE FORMATION2018

    • Author(s)
      加藤 譲, 相賀 裕美子
    • Organizer
      CSH-Germ Cells Meeting
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research
  • [Presentation] マウス生殖細胞の性分化を制御するシグナル系2018

    • Author(s)
      相賀 裕美子
    • Organizer
      日本分子生物学会年会
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] Essential role of the expression network between two RNA-binding proteins in the formation of sustainable primordial follicles2018

    • Author(s)
      加藤 譲, 相賀 裕美子
    • Organizer
      日本分子生物学会年会
    • Related Report
      2018 Annual Research Report
  • [Presentation] マウス生殖細胞分化に必須なRNA結合タンパク質の機能を司る構造基盤の解析2017

    • Author(s)
      ダネル ライト, 相賀 裕美子
    • Organizer
      日本分子生物学会
    • Related Report
      2017 Annual Research Report
  • [Presentation] 生殖細胞特異的NANOS2-DND1経路の体細胞における再構成2017

    • Author(s)
      平野 孝昌, 相賀 裕美子
    • Organizer
      日本分子生物学会
    • Related Report
      2017 Annual Research Report
  • [Presentation] Post-transcriptional suppression of Dazl via its 3’UTR plays an important role in pre-implantation embryonic development.2017

    • Author(s)
      福田 胡桃, 相賀 裕美子
    • Organizer
      日本発生生物学会
    • Related Report
      2017 Annual Research Report
  • [Presentation] Requirement of DDX6-mediated P-body formation in male germ cell development2017

    • Author(s)
      島田 龍輝, 相賀 裕美子
    • Organizer
      日本発生生物学会
    • Related Report
      2017 Annual Research Report
  • [Presentation] Post-transcriptional suppression of Dazl via its 3’UTR plays an important role in pre-implantation embryonic development.2017

    • Author(s)
      福田 胡桃, 相賀 裕美子
    • Organizer
      Gordon Research Conference
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Requirement of DDX6-mediated P-body formation in male germ cell development2017

    • Author(s)
      島田 龍輝, 相賀 裕美子
    • Organizer
      Gordon Research Conference
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Primordial follicle formation: insights from a study of an RNA-binding protein2017

    • Author(s)
      加藤 譲, 相賀 裕美子
    • Organizer
      Gordon Research Conference
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Role of Nanos Proteins in Male Germ Cell Development2017

    • Author(s)
      相賀 裕美子
    • Organizer
      Gordon Research Conference
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Role of DDX6-mediated P-body formation in NANOS2 function2017

    • Author(s)
      島田 龍輝, 相賀 裕美子
    • Organizer
      The International Research Symposium on Regulation of Germ cell Development in vivo and in vitro
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Primordial follicles: insights from a study of RNA-binding proteins for its development and activation2017

    • Author(s)
      加藤 譲, 相賀 裕美子
    • Organizer
      The International Research Symposium on Regulation of Germ cell Development in vivo and in vitro
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Egg or sperm: how germ cell sex is determined.2017

    • Author(s)
      相賀 裕美子
    • Organizer
      The International Research Symposium on Regulation of Germ Cell Development in vivo and in vitro
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research / Invited
  • [Remarks]

    • URL

      https://www.nig.ac.jp/nig/ja/2017/06/research-highlights_ja/20170607.html

    • Related Report
      2017 Annual Research Report

URL: 

Published: 2017-06-01   Modified: 2022-12-28  

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