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子。我们从斑马鱼胚胎、小鼠胚胎和早期分化的小鼠胚胎干（ES）细胞中制备了质粒cDNA文库。将这些转化到大肠杆菌中，并分成每组含有200个克隆的组。我们从每组中制备质粒并制备用于显微注射的RNA。将这些RNA注射到处于一到两个细胞阶段的斑马鱼胚胎中。通过显微镜观察和神经标记探针的整体原位杂交检测细胞表型。作为神经标记，我们使用six3、eng 3、krox 20和kheper。kheper是我们克隆的新的转录因子。kheper基因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 × 10^4个克隆，发现了许多已知的基因。此外，我们还成功地鉴定了新的FGF家族基因、新的Bcl 2家族基因和新的wnt家族基因。当这些在斑马鱼胚胎中过表达时，神经外胚层扩大。在中枢神经系统的前区检测到新的fgf的表达。这表明新的FGF对前脑的发育有调节作用，我们将对这些因素进行上位性分析。我们想知道这些与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。

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。

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。

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