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

为了了解海胆胚胎细胞命运决定的分子机制，我们寻找了编码微粒决定因子和微粒释放的肠主诱导配体并被锂离子激活的关键基因。我们差异筛选了在微粒或后代细胞中表达受限且在中粒后代细胞中依赖锂离子的基因。通过差异显示，我们获得了大约120个似乎是微粒和/或后代特异性的DNA片段。然后，我们进行Southern blot分析以检查特异性。我们从微粒和中粒及其后代中制备了poly (A^+) rna，并将其作为探针。结果，我们获得了10个DNA片段，它们对微粒探针显示出更强的信号。通过减法PCR，我们还检测到1个微粒特异性DNA片段，2个微粒后代特异性DNA片段和1个锂离子依赖性DNA片段。目前，我们正在利用原位杂交技术检测它们在胚胎中的表达，并尝试了表达克隆技术分离出关键基因。我们从卵巢、受精卵和有16个细胞的胚胎中制备了poly (A^+) RNA。通过将rna注射到16个细胞胚胎的一个中粒中，我们检测了将注射细胞转化为微粒表型的活性，并诱导邻近细胞形成主肠突。然而，我们没有检测到任何rna的活性。

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.