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Study of the program underlying the mammalian complex brain formation

Research Project

Project/Area Number 18H04003
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section一般
Review Section Medium-sized Section 44:Biology at cellular to organismal levels, and related fields
Research InstitutionInstitute of Physical and Chemical Research

Principal Investigator

松崎 文雄  国立研究開発法人理化学研究所, 生命機能科学研究センター, チームリーダー (10173824)

Project Period (FY) 2018-04-01 – 2022-03-31
Project Status Granted (Fiscal Year 2021)
Budget Amount *help
¥43,940,000 (Direct Cost: ¥33,800,000、Indirect Cost: ¥10,140,000)
Fiscal Year 2021: ¥10,400,000 (Direct Cost: ¥8,000,000、Indirect Cost: ¥2,400,000)
Fiscal Year 2020: ¥11,180,000 (Direct Cost: ¥8,600,000、Indirect Cost: ¥2,580,000)
Fiscal Year 2019: ¥10,270,000 (Direct Cost: ¥7,900,000、Indirect Cost: ¥2,370,000)
Fiscal Year 2018: ¥12,090,000 (Direct Cost: ¥9,300,000、Indirect Cost: ¥2,790,000)
Keywords脳幹細胞 / 細胞系譜 / 複雑脳 / クローンサイズ / 神経幹細胞 / 神経発生 / フェレット / 多様性 / 脳回 / 高次能 / 幹細胞 / フェッレット / 大脳皮質 / 発生 / 動態 / 不均一性
Outline of Annual Research Achievements

昨年から本格的に始めた神経幹細胞の細胞系譜の解析の実験系の改良を引き続き行なった。基本としているのはBarclock法と呼ばれる、CRISPR-Cas9系によるgenome editingを用いる方法で、Cas9と deaminaseの融合タンパクであるBase-editor 4EXを用いて、target配列に変異を導入し、その蓄積から細胞系譜を再構成する方法である。技術の確立のため、マウスをモデルとしてテストすることになった。barcodeとbarclock modeleを合わせた長さが600bp程度になり、それを1base単位の正確さで読み取るために、PacBioという方法を採用している。この方法は高価なため、この研究費ではカバーできないため、新学術領域「先進ゲノム支援」に応募し、承諾を得て行っている。また、構築している細胞の経時的時間記録法が実態を反映しているかどうかを確認するため、HEK293という培養細胞系と、in utero electroporationで神経発生初期にマウス脳にbarclockシステム一式を導入し、そこから、primary cultureを作成し、live-imagingで個々の幹細胞の分裂パターンを追跡・記録した後、その幹細胞クローンの各構成細胞から横河SU10を用いて、細胞質を抽出し、RNAをcDNAとして増幅し、barclockの配列を調べた。そしてbarcloc上の変異導入から再構成した細胞系譜とlive-imagingにより得られた細胞系譜を比較し、一致するかどうか検証した。その結果、変異が導入されている細胞間では系譜関係が正しいことが確かめられた。ただし、barclockへの変異導入の頻度が予想より低いため、現在、変異導入頻度をあげるように実験系に改良を加えている。

Current Status of Research Progress
Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

barclock系を用いて、細胞系譜を正確に決定するには、幹細胞分裂1回あたり、1つ以上の変異が常に入ることが前提である。現在平均では1回を上回っているが、必ず1回は入るという条件をまだ満たしていず、システムの改良を行なっている。この点は重要なポイントであるため、改良を重ね安定してこの条件を満たすシステムを完成させるため、十分な時間をかけて行う価値があると考える。

Strategy for Future Research Activity

barclock systemの改良は進んでおり、今年度内に、細胞分裂1回あたり1頻度以上の変異導入が達成される見込みである。それによって、フェレットに実験系を移し、フェレットの細胞系譜を多数取得し、マウスと比較して、ランダムさがましていることを確認したのち、そのばらつきに潜むルールを見出すことにより、複雑脳の幹細胞系譜が脳構築に果たす役割の解析に進む予定である。

Report

(3 results)
  • 2020 Annual Research Report
  • 2019 Annual Research Report
  • 2018 Annual Research Report

Research Products

(21 results)

All 2020 2019 Other

All Int'l Joint Research (1 results) Journal Article (10 results) (of which Int'l Joint Research: 4 results,  Peer Reviewed: 10 results,  Open Access: 10 results) Presentation (10 results) (of which Int'l Joint Research: 4 results,  Invited: 4 results)

  • [Int'l Joint Research] British Columbia大学(米国)

    • Related Report
      2019 Annual Research Report
  • [Journal Article] Amyloidogenic processing of amyloid β protein precursor (APP) is enhanced in the brains of alcadein α-deficient mice2020

    • Author(s)
      Gotoh N, Saito Y, Hata S, Saito H, Ojima D, Murayama C, Shigeta M, Abe T, Konno D, Matsuzaki F, Suzuki T, Yamamoto T
    • Journal Title

      The Journal of Biological Chemistry

      Volume: 295 Pages: 9650-9662

    • DOI

      10.1074/jbc.ra119.012386

    • NAID

      120007116485

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Comparative Analysis of Brain Stiffness Among Amniotes Using Glyoxal Fixation and Atomic Force Microscopy2020

    • Author(s)
      Iwashita Misato、Nomura Tadashi、Suetsugu Taeko、Matsuzaki Fumio、Kojima Satoshi、Kosodo Yoichi
    • Journal Title

      Frontiers in Cell and Developmental Biology

      Volume: 8 Pages: 1-15

    • DOI

      10.3389/fcell.2020.574619

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Mechanical forces drive ordered patterning of hair cells in the mammalian inner ear2020

    • Author(s)
      Cohen Roie、Amir-Zilberstein Liat、Hersch Micha、Woland Shiran、Loza Olga、Taiber Shahar、Matsuzaki Fumio、Bergmann Sven、Avraham Karen B.、Sprinzak David
    • Journal Title

      Nature Communications

      Volume: 11 Pages: 1-12

    • DOI

      10.1038/s41467-020-18894-8

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Blood and lymphatic systems are segregated by the FLCN tumor suppressor2020

    • Author(s)
      Tai-Nagara I, Hasumi Y, Kusumoto D, Hasumi H, Okabe K, Ando T, Matsuzaki F, Itoh F, Saya H, Hirashima M, Suzuki Y, Furuya M, Baba M, Kubota Y
    • Journal Title

      Nature Communications volume

      Volume: 11 Pages: 1-12

    • DOI

      10.1038/s41467-020-20156-6

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Notch1 and Notch2 collaboratively maintain radial glial cells in mouse neurogenesis2020

    • Author(s)
      Mase Shun、Shitamukai Atsunori、Wu Quan、Morimoto Mitsuru、Gridley Thomas、Matsuzaki Fumio
    • Journal Title

      Neuroscience Research

      Volume: Available online 11 December Pages: 1-11

    • DOI

      10.1016/j.neures.2020.11.007

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Endfoot regeneration restricts radial glial state and prevents translocation into the outer subventricular zone in early mammalian brain development2020

    • Author(s)
      Fujita Ikumi、Shitamukai Atsunori、Kusumoto Fumiya、Mase Shun、Suetsugu Taeko、Omori Ayaka、Kato Kagayaki、Abe Takaya、Shioi Go、Konno Daijiro、Matsuzaki Fumio
    • Journal Title

      Nature Cell Biology

      Volume: 22 Pages: 2637-2637

    • DOI

      10.1038/s41556-019-0436-9

    • Related Report
      2019 Annual Research Report 2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Dmrt factors determine the positional information of cerebral cortical progenitors via differential suppression of homeobox genes.2019

    • Author(s)
      Konno D, Kishida C, Maehara K, Ohkawa Y, Kiyonari H, Okada S, Matsuzaki F
    • Journal Title

      Development.

      Volume: 146 Pages: 1-13

    • DOI

      10.1242/dev.174243

    • Related Report
      2019 Annual Research Report 2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Reconstruction of Par-dependent polarity in apolar cells reveals a dynamic process of cortical polarization2019

    • Author(s)
      Kono K, Yoshiura S, Fujita I, Okada Y, Shitamukai A, Shibata T, Matsuzaki F
    • Journal Title

      ELIFE

      Volume: 8 Pages: 1-31

    • DOI

      10.7554/elife.45559

    • NAID

      120006653006

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Isl1-expressing non-venous cell lineage contributes to cardiac lymphatic vessel development.2019

    • Author(s)
      Maruyama K, Miyagawa-Tomita S, Mizukami K, Matsuzaki F, Kurihara H.
    • Journal Title

      Developmental Biology

      Volume: 452 Pages: 134-143

    • DOI

      10.1016/j.ydbio.2019.05.002

    • Related Report
      2019 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Lzts1 controls both neuronal delamination and outer radial glial-like cell generation during mammalian cerebral development2019

    • Author(s)
      Kawaue T.、Shitamukai A.、Nagasaka A.、Tsunekawa Y.、Shinoda T.、Saito K.、Terada R.、Bilgic M.、Miyata T.、Matsuzaki F.、Kawaguchi A.
    • Journal Title

      Nature Communications volume

      Volume: 10 Pages: 2780-2780

    • DOI

      10.1038/s41467-019-10730-y

    • NAID

      120006649601

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Presentation] Regeneration ability of the epithelial structure in neural stem cells during mammalian brain development.2020

    • Author(s)
      Ikumi Fujita, Atsunori Shitamukai, Fumiya Kusumoto, Shun Mase, Taeko Suetsugu, Ayaka Omori, Kagayaki Kato, Takaya Abe, Go Shioi, Daijiro Konno, Fumio Matsuzaki
    • Organizer
      第72回日本細胞生物学会大会
    • Related Report
      2020 Annual Research Report
  • [Presentation] Regeneration of the epithelial structure in neural stem cells during mammalian brain development.2020

    • Author(s)
      Ikumi Fujita, Atsunori Shitamukai, Fumiya Kusumoto, Shun Mase, Taeko Suetsugu, Ayaka Omori, Kagayaki Kato, Takaya Abe, Go Shioi, Daijiro Konno, Fumio Matsuzaki
    • Organizer
      Cell Bio Virtual 2020 Meeting (The American Society for Cell Biology)
    • Related Report
      2020 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Regeneration ability of the epithelial structure in neural stem cells during mammalian brain development.2020

    • Author(s)
      Ikumi Fujita, Atsunori Shitamukai, Fumiya Kusumoto, Shun Mase, Taeko Suetsugu, Ayaka Omori, Kagayaki Kato, Takaya Abe, Go Shioi, Daijiro Konno, Fumio Matsuzaki
    • Organizer
      第43回日本分子生物学会年会
    • Related Report
      2020 Annual Research Report
  • [Presentation] The radial fiber-mediated FGF signal ensures the feedback signaling system from neurons to radial glial cells in the developing cerebral cortex.2020

    • Author(s)
      Atsunori Shitamukai,Fumio Matsuzaki
    • Organizer
      第43回日本分子生物学会年会
    • Related Report
      2020 Annual Research Report
  • [Presentation] Structural plasticity of neural stem cells in mammalian brain development2019

    • Author(s)
      Fumio Matsuzaki
    • Organizer
      Baikal Neuroscience Meeting 2019
    • Related Report
      2019 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Structural plasticity of neural stem cells in mammalian brain development.2019

    • Author(s)
      Fumio Matsuzaki
    • Organizer
      The 10th IBRO World Congress of Neuroscience IBRO 2019
    • Related Report
      2019 Annual Research Report 2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] How has the brain expanded and acquired complexity in the mammalian evolution?2019

    • Author(s)
      Fumio Matsuzaki
    • Organizer
      第42回分子生物学会
    • Related Report
      2019 Annual Research Report
  • [Presentation] Structural plasticity of neural stem cells in mammalian brain development.2019

    • Author(s)
      Fumio Matsuzaki
    • Organizer
      Baikal Neuroscience Meeting 2019
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] How has the brain expanded and acquired complexity in the mammalian2019

    • Author(s)
      Fumio Matsuzaki,Yuji Tsunekawa, Taeko Suetsugu, Ikumi Fujita, Quan Wu, Atsunori Shitamukai, Ayaka Omori, Osamu Nishimura, Shigehiro Kuraku
    • Organizer
      第42回分子生物学会
    • Related Report
      2018 Annual Research Report
  • [Presentation] 非分裂細胞に対するノックイン技術HITIとその展望2019

    • Author(s)
      恒川雄二
    • Organizer
      ウイルスベクター開発研究センターキックオフシンポジウム
    • Related Report
      2018 Annual Research Report
    • Invited

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Published: 2018-04-23   Modified: 2022-12-28  

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