Studies on embryonic axis determinants in Xenopus laevis oocytes

在两栖动物发育过程中，精子组织器的确定是胚胎形态发生的关键步骤。由于组织体形成的分子机制可能取决于被称为决定因子的细胞质因子，因此决定因子的分子克隆是阐明胚胎发生机制的先决条件。在目前的研究中，我们克隆了各种cdna作为决定因素的候选者，基于它们的临时和空间调控表达。作为第一种方法，我们研究了在卵发生期间转录并随后快速翻译的cDNA克隆。转录因子混合引物RT-PCR克隆得到与人EXT1基因高度同源的cDNA克隆Xext。作为第二种方法，我们比较了8细胞期胚胎的4个卵裂球中mrna的分子种类。利用卵裂球特异性cDNA探针筛选子房cDNA文库，筛选到一个编码330个氨基酸残基、WD40重复序列的候选克隆。作为第三种方法，我们比较了正常和过度活化胚胎之间的mrna。差异显示cDNA克隆Xcea与小鼠CEA基因具有高度同源性。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。

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.