The screening of the genes that involved in the determination of the neural area and the neural induction

神经区域决定及神经诱导相关基因的筛选

• 批准号: 14570034
• 负责人: MURAOKA Osamu
• 金额: $ 2.24万
• 依托单位: RIKEN (The Institute of Physical and Chemical Research)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570034/
• 关键词: zebrafish; neural area; kheper; mouse; ES cell; Expression cloning; Zebrafish; ES cell; Mouse; Expression cloning; ゼブラフイツシュ; 神経発生

We screened cDNA libraries using expression screen strategy to identify the factors that regulated the development of the neuroectoderm. We made the plasmid cDNA libraries from zebrafish embryos, mouse embryos, and early differentiated mouse embryonic stem (ES) cells. These were transformed into the E.coli and divided in groups which contained 200 clones each. We prepared the plasmid from each groups and made RNAs for the microinjection. These RNAs were injected into the zebrafish embryos at one to two cell stage. We checked the phenotype by microscopic view and the whole mount in situ hybridization with neural marker probes. As the neural marker, we used six3,eng3,krox20 and kheper. kheper is the new transcription factor which we cloned. kheper is expressed in the whole neuroectoderm and regulated the development of the neural tissue of zebrafish.We screened 23 x 10^4 clones and found many well known genes. In addition, we succeeded to identify new FGF family gene and new Bcl2 family gene and new wnt family gene. When these are overexpressed in zebrafish embryos, the neuroectoderm were enlarged. The expression of the new fgf was detected in the anterior region of the central nervous system. It is suggested that the new FGF regulates the development of the forebrain.We are going to do the epistatic analysis of these factors. We would like to know the relationship of these with kheper and how they regulate the development of neural tissue.

我们使用表达筛选策略筛选 cDNA 文库，以确定调节神经外胚层发育的因素。我们从斑马鱼胚胎、小鼠胚胎和早期分化的小鼠胚胎干 (ES) 细胞中构建了质粒 cDNA 文库。将它们转化到大肠杆菌中并分成每组包含 200 个克隆的组。我们制备了各组的质粒并制备了用于显微注射的RNA。这些 RNA 被注射到一到两个细胞阶段的斑马鱼胚胎中。我们通过显微镜观察和用神经标记探针进行整体原位杂交来检查表型。作为神经标记，我们使用 Six3、eng3、krox20 和 kheper。 kheper是我们克隆的新转录因子。 kheper在整个神经外胚层中表达，调控斑马鱼神经组织的发育。我们筛选了23 x 10^4克隆，发现了许多众所周知的基因。此外，我们还成功鉴定出新的FGF家族基因、新的Bcl2家族基因和新的wnt家族基因。当这些在斑马鱼胚胎中过度表达时，神经外胚层就会增大。在中枢神经系统的前部区域检测到新的 fgf 的表达。推测新的FGF调节前脑的发育。我们将对这些因素进行上位分析。我们想知道它们与 kheper 的关系以及它们如何调节神经组织的发育。

项目成果

Bae YK, Shimizu T, Yabe T, Kim CH, Hirata T, Nojima H, Muraoka O, Hirano T, Hibi M.: "A homeobox gene, pnx, is involved in the formation of posterior neurons in zebrafish."Development.. 130. 1853-1865 (2003)

Bae YK、Shimizu T、Yabe T、Kim CH、Hirata T、Nojima H、Muraoka O、Hirano T、Hibi M.：“同源盒基因 pnx 参与斑马鱼后部神经元的形成。”开发..

Bae YK, Shimizu T, Yabe T, Kim CH, Hirata T, Nojima H, Muraoka O, Hirano T, Hibi M.: "A homeobox gene, pnx, is involved in the formation of posterior neurons in zebrafish"Development. 130. 1853-1865 (2003)

Bae YK、Shimizu T、Yabe T、Kim CH、Hirata T、Nojima H、Muraoka O、Hirano T、Hibi M.：“同源盒基因 pnx 参与斑马鱼后部神经元的形成”开发。

Yabe T, Shimizu T, Muraoka O, Bae YK, Hirata T, Nojima H, Kawakami A, Hirano T, Hibi M.: "Ogon/Secreted Frizzled functions as a negative feedback regulator of Bmp signaling"Development. 130. 2705-2716 (2003)

Yabe T、Shimizu T、Muraoka O、Bae YK、Hirata T、Nojima H、Kawakami A、Hirano T、Hibi M.：“Ogon/Secreted Frizzled 充当 Bmp 信号传导的负反馈调节剂”开发。

Hisashi Hashimoto, Michael Rebagliati, Nadira Ahmad, Osamu Muraoka, Tadahide Kurokawa, Masahiko Hibi, Tohru Suzuki: "The Cerberus/Dan-family protein Charon is a negative regulator of Nodal signaling during left-right patterning in zebrafish"Development. 1

Hisashi Hashimoto、Michael Rebagliati、Nadira Ahmad、Osamu Muraoka、Tadahide Kurokawa、Masahiko Hibi、Tohru Suzuki：“Cerberus/Dan 家族蛋白 Charon 是斑马鱼左右模式期间 Nodal 信号的负调节因子”的开发。

Hideaki Nojima, Takashi Shimizu, Cheol-Hee Kim, Taijiro Yabe, Young-Ki Bae, Osamu Muraoka, Tsutomu Hirata, Ajay Chitnis, Toshio Hirano, Masahiko Hibi: "Genetic evidence for involvement of maternally derived Wnt canonical signaling in dorsal determination

Hideaki Nojima、Takashi Shimizu、Cheol-Hee Kim、Taijiro Yabe、Young-Ki Bae、Osamu Muraoka、Tsutomu Hirata、Ajay Chitnis、Toshio Hirano、Masahiko Hibi：“母源 Wnt 规范信号参与背侧决定的遗传证据