脊椎動物中枢神経系における前後軸・背腹軸の成立機構

• Project/Area Number: 10220203
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Saitama University (2002-2003); The University of Tokyo (2001); National Institute of Genetics (1999-2000); Nagoya University (1998)
• Principal Investigator: 弥益 恭 (2002-2003) 埼玉大学, 理学部, 助教授 (60230439); 武田 洋幸 (1998-2001) 東京大学, 大学院・理学系研究科, 教授 (80179647)
• Co-Investigator(Kenkyū-buntansha): 武田 洋幸 東京大学, 大学院・理学系研究科, 教授 (80179647) ; 川上 厚志 東京大学, 大学院・理学系研究科, 助手 (00221896)
• Project Period (FY): 1998 – 2003
• Project Status: Completed (Fiscal Year 2003)
• Budget Amount: ¥48,700,000 (Direct Cost: ¥48,700,000); Fiscal Year 2003: ¥7,000,000 (Direct Cost: ¥7,000,000); Fiscal Year 2002: ¥7,000,000 (Direct Cost: ¥7,000,000); Fiscal Year 2001: ¥10,000,000 (Direct Cost: ¥10,000,000); Fiscal Year 2000: ¥10,000,000 (Direct Cost: ¥10,000,000); Fiscal Year 1999: ¥7,200,000 (Direct Cost: ¥7,200,000); Fiscal Year 1998: ¥7,500,000 (Direct Cost: ¥7,500,000)
• Keywords: 脳形成 / 個体発生 / 体軸 / 脊椎動物 / 中枢神経系 / ゼブラフィッシュ / 転写調節 / ミュータント / 前後軸 / 背腹軸 / 脳の領域化 / パターニング / 転写制御 / プロモーター解析 / ポジショナルクローニング / 神経誘導 / FGF / 突然変異体 / 終脳 / ソニックヘッジホッグ / BMP / フロアープレート細胞 / オーガナイザー / Wnt

Research Abstract

本研究者は、脊椎動物の中枢神経系が体軸に沿って領域化される機構を明らかにするため、ゼブラフィッシュを用いて以下の研究に取り組んだ。
1.中脳後脳境界(MHB)と後脳前端領域の形成におけるgbx2ホメオボックス遺伝子の機能:MHB領域の決定に関わることが予想されるgbx2の機能を検討するため、まずゼブラフィッシュ胚でgbx2の強制発現あるいは機能阻害実験を行い、gbx2が前脳・中脳形成に抑制的であること、また峡部の形成と維持に密接に関わることを示した。ひき続き、MHBの位置情報を与える機構を解明するため、gbx2遺伝子周辺DNAの転写調節活性をEGFP遺伝子を用いたレポーター解析により検討し、後脳前端と間脳での発現には上流-7kbに存在する0.7kb領城で十分であることを見出した。
2.fgf8遺伝子の転写制御機構:脊椎動物脳形成において、前脳及びMHBの形成に関わる分泌性因子Fgf8の領域特異的な発現の制御機構を知るため、ゼブラフィッシュfgf8遣伝子のプロモーター解析を進めた結果、上流4.5kb領域内には頭部表皮、下流+5.1kbには心臓、+9.7kbには終脳後方と後方神経管、+14.8kbには耳胞、+15.1kbにMHBでの発現を制御するゲノム領城を同定した。このように、fgf8の多様な発現と機能が、ゲノム中に存在する多重エンハンサーにより可能となっているという分子機構が見えてきている。
3.脳の前後軸に沿った領域化に関わる突然変異体のスクリーニング:脳の領域化に関わる遺伝子群を明らかにするため、ゼブラフィッシュを用いたmutagenesisの準備を進めて来ており、パイロットスクリーンによりmutagenesisの条件を確立した。一方、様々な脳領域特異的にGFPを発現するゼブラフィッシュ系統を作製しており、今後、これらのGFP発現Tg fishを利用したmutagenesisを進めていく予定である。

Research Products

• [Publications] H, Kikuta, M.Kanai, Y.Ito, K.Yamasu: "gbx2 homeobox gene is required for the maintenance of the isthmic region in the zebrafish embryonic brain."Developmental Dynamics. 228. 433-450 (2003)
• [Publications] K.Horii, G.Suzuki, T.Suyemitsu, K.Yamasu: "Characterization of the upstream region that regulates the transcription of the gene for the precursor to EGF-related peptides, exogastrula-inducing peptides, of the sea urchin Anthocidaris crassispina."Comparative Biochemistry and Physiology Part B Biochemistry and Molecular.Biology.. 136. 15-26 (2003)
• [Publications] Y.Hirate, H.Okamoto, K.Yamasu: "Structure of the zebrafish fasciclin I-related extracellular matrix protein (βig-h3) and its characteristic expression during embryogenesis."Gene Expression Patterns. 3. 331-336 (2003)
• [Publications] A.F.Giusti, F.J.O'Neill, K.Yamasu, K.R.Foltz, L.A.Jaffe: "Function of a sea urchin egg src family kinase in initiating Ca^<2+> release at fertilization."Developmental Biology. 256. 367-378 (2003)
• [Publications] Y.Hirate: "Structure of the zebrafish fasciclin 1-related extracellular matrix protein(βig-h3) and its characteristic expression during embryogenesis"Gene Expression Patterns. (in press).
• [Publications] A.F.Giusti: "Function of a sea urchin egg src family kinase in initiating Ca^<2+> release at fertilization"Developmental Biology. (in press).
• [Publications] N.Tonou-Fujimori: "Expression of the FGF receptor 2 gene(fgfr2) during embryogenesis in the zebrafish Danio rerio"Gene Expression Patterns. 2・3-4.
• [Publications] Y.Haruguchi: "Genomic organization of the gene that encodes the precursor to EGF-related peptides, exogastrula-inducing peptides, of the sea urchin Anthocidaris crassispina"Biochimica et Biophysica Acta. 1574・3. 311-320 (2002)
• [Publications] K.Tomita: "Role of syndecan in the elongation of postoral arms in sea urchin larvae"Development Growth & Differentiation. 44・1. 45-53 (2002)
• [Publications] S.Koshida: "Inhibition of BMP activity by the FGF signal promotes posterior neural development in zebrafish"Developmental Biology. 244・1. 9-20 (2002)
• [Publications] A.Nomura-Kitabayashi: "Hypomorphic Mesp allele distinguishes establishment of rotrocaudal polarity and segment border formation in somitogenesis"Development. 129・10. 2473-2481 (2002)
• [Publications] N.Nikaido: "Tbx24, a novel T-box gene, is mutated in the zebrafish somite segmentation mutant fused somites"Nature Genetics. 31・2. 195-199 (2002)
• [Publications] A.Picker: "A novel positive transcriptional feed back loop in midbrain-hindbrain boundary development is revealed through analysis of the zebrafish pax2.1 promoter in transgenic lines"Development. 129・13. 3227-3239 (2002)
• [Publications] H.Yokoi: "sox9 in a teleost fish, medaka(Orizias latipes) : evidence for diversified function of Sox9 in gonad differentiation"Molecular Reproduction and Development. 63・1. 5-16 (2002)
• [Publications] H.Fujii: "Anteroposterior patterning in Xenopus embryos : egg fragment assay system reveals a synergy of dorsalizing and posteriorizing embryonic domains"Developmental Biology. 252・1. 15-30 (2002)
• [Publications] Takuya Sakaguchi: "Expression of Zebrafish big-b, an anti-proliferative cofactor, during early eubryogenesis"Mechanisms of Development. 104. 113-115 (2001)
• [Publications] Takuya Sakaguchi: "A novel sox gene, 226D7, acts downstream of Nodal signaling to specify endoderm precursors in zebrafish"Mechanism of Development. 107. 25-38 (2001)
• [Publications] Minori Shinya: "Fgf signalling through MAPK cascade is required for development of the subpallial telencephalon in zebrafish embryos"Development. 128. 4153-4154 (2001)
• [Publications] Atsushi Sawada: "Fgf/MAPK signalling is a crucial positional cue in somite boundary formation"Development. 128. 4873-4880 (2001)
• [Publications] Sumito Koshida: "Inhibition of BMP activity by the FGF signal promotes posterior neural development in zebrafish"Developmental Biology. (in press). (2001)
• [Publications] Yumiko Saga: "The making of the somite : molecular events in vertebrate segmentation"Nature Reviews Genetics. 2. 835-845 (2001)
• [Publications] 武田 洋幸: "動物のからだづくり -形態発生の分子メカニズム-"朝倉書店. 138 (2001)
• [Publications] Atsushi Sawada: "Zebrafish Mesp family genes, mesp-a and mesp-b are segmentally expressed in the presomitic mesoderm, and Mesp-b confers the anterior identity to the developing somites."Development. 128. 1691-1702 (2000)
• [Publications] Hisato Kondo: "Zebrafish mutations in Gli mediated hedgehog signaling lead to lens transdifferentiation from the adenohypophysis anlage."Mechanisms of Development. 96. 165-174 (2000)
• [Publications] Takuya Sakaguchi: "Expression of zebrafish BTG-b, anti-proliferative cofactor, during early embryogenesis"Mechanisms of Development. (in press). (2001)
• [Publications] 澤田篤志: "体節の形成と分化"蛋白質核酸酵素. 45. 2766-2773 (2000)
• [Publications] 新屋みのり: "神経誘導と神経組織の体軸に沿ったパターニング"蛋白質核酸酵素. 45. 2738-2744 (2000)
• [Publications] 川上厚志: "ゼブラフィッシュのジェネティックスとジェノミックス"蛋白質核酸酵素. 45. 2853-2863 (2000)
• [Publications] Atsushi Sawada: "Zebrafish Mesp family genes, mesp-a and mesp-b are segmentally expressed in the presomitic mesoderm, and Mesp-b confers the anterior identity to the developing somites."Development. 128. 1691-1702 (2000)
• [Publications] Hisato Kondo: "Zebrafish mutations in Gli mediated hedgehog signaling lead to lens transdifferentiation from the adenohypophysis anlage."Mechanisms of Development. 96. 165-174 (2000)
• [Publications] Takuya Sakaguchi: "Expression of zebrafish BTG-b, anti-proliferative cofactor, during early embryogenesis"Mechanisms of Development. (in press). (2001)
• [Publications] Akihiko Koga: "The medaka fish transpozable element Tol2 : Low of interspecific sequence divergence points to recent invation of the fish genome"Genetics. 155. 273-281 (2000)
• [Publications] Akihiko Koga: "Detection of de novo insertion of the medaka fish transposable element Tol2"Genetics. 155. 1243-1247 (2000)
• [Publications] Masaru Matsuda: "Construction of a BAC library derived from the inbred Hd-rR strain of the teleost fish, Orizias latipes"Genes & Genetic Sysmtems. (in press). (2001)
• [Publications] Atsushi Sawada: "Zebrafish Mesp family genes, mesp-a and mesp-b are segmentally expressed in the presomitic soderm, and Mesp-b confers the anterior identity to the developing somites"Development. (印刷中). (2000)
• [Publications] Kimiko Amanuma: "Transgenic zebrafish for the detection of mutations caused by compounds in aquatic environments"Nature Biotechnology. 18. 62-65 (2000)
• [Publications] Minori Shinya: "Mosaic analysis with oep mutant reveales a repressiveinteraction between floor-plate and non-floor plate mutant cells in the zebrafish neural tube"Development Growth & Differentiation. 41. 135-142 (1999)
• [Publications] Toshiro Mizuno: "Removal of vegetal yolk causes dorsal deficiencies and impairs dorsal inducing ability of the yolk cell in zebrafish"Mechanisms of Development. 81. 51-63 (1999)
• [Publications] Adam Rodaway: "Induction of the mesendoderm in the zebrafish germ rign by yolk cell-drived TGF-b family signals and dicrimination of mesoderm and endoderm by FGF"Development. 126. 3067-3078 (1999)
• [Publications] Masataka Nikaido: "In vitro analysis using variants of zebrafish BMPR-IA: range of action and involvement of BMP in ectoderm patterning"Development. 126. 181 (1999)
• [Publications] Toshiro Mizuno: "Moleculer Methods in Developmental Biology-Xenopus and Zebrafish Ed. M. Guile, Humana Press Inc."Cell and tissue transplantation in zebrafish embryos. 15-28 (1999)
• [Publications] Sumito Koshida: "Initial anteroposterior pattern of zebrafish central nervous system is determined by differential competence of the epiblast." Development. 125. 1957-1966 (1998)
• [Publications] Makoto Kobayashi: "Overexpression of the forebrain-specific homeobox gene six3 induces rostral forebrain en-largement in zebrafish." Development. 125. 2973-2982 (1998)
• [Publications] Yojiro Yamanaka: "A novel homeobox gene,dharma,can induce the organizer in a non-cell autonomous manner." Genes & Development. 12. 2345-2353 (1998)
• [Publications] Ryosuke Makita: "Zebrafish wntll:pattern and regulation of the expression by the yolk cell and No tail activity." Mechanisms of Development. 71. 165-176 (1998)
• [Publications] Etsuro Yamaha: "Dorsal specification in the blastoderm at the blastula stage in goldfish,Carassius auratus." Development,Growth & Differentiation. 40. 267-275 (1998)
• [Publications] Masataka Nikaido: "In vivo analysis using variants of zebrafish BMPR-IA:range of action and involvement of BMP in ectoderm patterning." Development. 126. 181-190 (1999)
• [Publications] Minori Shinya: "Mosaic analysis with oep mutant reveals a repressive interaction between floor-plate and non-floor plate mutant cells in the zebrafish neural tube." Development,Growth & Dfferentiation. (印刷中). (1999)
• [Publications] Toshiro Mizuno: "Removal of vegetal yolk causes dorsal deficiencies and impairs dorsal inducing ability of the yolk cell in zebrafish." Mechanisms of Development. (印刷中). (1999)
• [Publications] 武田 洋幸: "ゼブラフィッシュの現状" 医学のあゆみ. 185巻6号. 395-398 (1998)
• [Publications] Toshiro Mizuno: "Molecular Methods in Developmental Biology-Xenopus and Zebrafish" Matt Guille(University of Portsmouth)編(印刷中), (1999)