Studies on embryonic axis determinants in Xenopus laevis oocytes

非洲爪蟾卵母细胞胚胎轴决定因素的研究

• 批准号: 07680806
• 负责人: KINOSHITA Tsutomu
• 金额: $ 1.41万
• 依托单位: Kwansei Gakuin University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07680806/
• 关键词: Xenopus laevis; cytoplasmic factors; determinants; gene cloning; maternal mRNA; PCR; axis formation; localization of mRNA; 多発性外骨腫症; 胚軸形成; ディファレンシャルディスプレイ

In amphibian development, determination of spemann's organizer is a key step for morphogenesis of the embryo. Since molecular mechanism of the organizer formation probably depends on cytoplasmic factors called determinants, molecular cloning of the determinants is a prerequisite step for clarifying mechanism of the embryogenesis. In the present study, we cloned various cDNAs as candidates for determinants, basing on their temporary and spatially regulated expression.As the first approach, we researched cDNA clones which are transcribed during the period of oogenesis and thereafter translated rapidly. RT-PCR cloning with mix primers for transcription factors showed a cDNA clone, Xext, which has a high homology with human EXT1 gene. As the second approach, we compared molecular species of mRNAs among 4 blastomeres of 8-cell stage embryos. Screening of ovary cDNA library with blastomere-specific cDNA probe selected a candidate clone which encodes a protein of 330 amino acid residues with WD40 repeat within the molecule. As the third approach, we compared mRNAs between normal and hyperdorsalized embryos. Differential display showed a cDNA clone, Xcea, which has a high homology with mouse CEA gene. Functional analyzes of these clones will be performed to evaluate their roles as axis determinants.

在两栖动物发育过程中，Spemann‘s组织器的确定是胚胎形态发生的关键步骤。由于组织者形成的分子机制可能依赖于被称为决定因素的细胞质因素，因此决定因素的分子克隆是阐明胚胎发生机制的先决条件。在本研究中，我们基于其临时性和空间调节的表达，克隆了各种作为决定因素的候选基因。作为第一种方法，我们研究了在卵子发生期间转录并随后快速翻译的cDNA克隆。用转录因子混合引物进行RT-PCR克隆，得到一个与人EXT1基因同源性较高的cDNA克隆Xext。作为第二种方法，我们比较了4个8-细胞期胚胎的卵裂球中mRNAs的分子种类。卵裂球特异的cDNA探针筛选卵巢cDNA文库筛选出一个候选克隆，编码330个氨基酸残基，分子内有WD40重复序列。作为第三种方法，我们比较了正常胚胎和超背向胚胎的mRNAs。差异显示克隆XceA与小鼠CEA基因高度同源。将对这些克隆进行功能分析，以评估它们作为轴决定因素的作用。