Blastomere-Specific Genes of the 16-cell Sea Urchin Embryo

16 细胞海胆胚胎的卵裂球特异性基因

• 批准号: 07836006
• 负责人: YAMAGUCHI Masaaki
• 金额: $ 1.47万
• 依托单位: Kanazawa 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-07836006/
• 关键词: sea urchin; development; differentiation; micromere; determinant; induction; lithium ion; Liイオン; 植物極化; 大割球; 中割球; エルトリエータ

To understand molecular mechanisms of cell fate determination of the sea urchin embryo, we searched for key genes that encode the micromere-determinant and the archenteron-inducing ligand released from the micromere, and that are activated by lithium ion.We differentially screened genes whose expression is restricted in the micromeres or the descendants, and is dependent of lithium ion in the mesomere-descendent cells. By the differential display, we obtained approximately 120 DNA fragments that seemed to be the micromere and/or the descendant-specific. Then, we performed the Southern blot analysis to check the specificity. We prepared poly (A^+) RNAs from the micromere and the mesomere as well as their descendants, and used the cDNAs as probes. As the result, we obtained 10 DNA fragments that showed stronger signals to the micromere-probe. By the subtraction PCR,we also detected 1 micromere-specific, 2 micromere descendant-specific, and 1 lithium ion-dependent DNA fraqments. Now, we are under way to examine their expression in the embryo by in situ hybridizationWe also tried the expression cloning to isolate the key genes. We prepared poly (A^+) RNA from the ovary, the fertilized egg, as well as the 16-cell embryo. By injecting the RNAs into one of the mesomeres of the 16-cell embryo, we examined the activity that turns the injected cell into the micromere-phenotype, and that induces the adjacent cells to form the archenteron. We however, detected no activity with any RNAs.

为了解海胆胚胎细胞命运决定的分子机制，我们寻找了编码小孢子决定子和小孢子释放的原肠诱导配体的关键基因，并对锂离子激活的关键基因进行了差异筛选，筛选出了在小孢子或后代中表达受限的基因，以及在中间体后代细胞中表达依赖锂离子的基因。通过差异显示，我们获得了大约120个DNA片段，似乎是微节和/或后代特异性的。然后，我们进行Southern印迹分析以检查特异性。我们制备了来自微节、中节及其后代的poly（A^+）RNA，并将cDNA用作探针。结果，我们获得了10个DNA片段，显示出较强的信号，以微米探针。通过差减PCR，我们还检测到1个微节特异性DNA片段，2个微节后代特异性DNA片段和1个锂离子依赖性DNA片段。目前，我们正在通过原位杂交的方法检测它们在胚胎中的表达，并尝试表达克隆的方法分离关键基因。我们从卵巢、受精卵和16细胞胚胎中制备了多聚A^+ RNA。通过将RNA注射到16细胞胚胎的一个中粒中，我们检查了将注射的细胞转化为微粒表型的活性，并诱导相邻细胞形成原肠。然而，我们没有检测到任何RNA的活性。