Analysis of determinants for epidermis and muscle differentiation in ascidian egg cytoplasm.

碱性磷酸酶(AP)活性在海鞘幼虫的内胚层细胞表达。此前有报道称，AP活性的表达对转录抑制剂放线菌素D的处理具有抵抗力。AP对放线菌素D处理的抗性表明，AP活性的出现不依赖于合子转录，它的出现是由存在于海囊卵中的母体AP mRNA的翻译激活所介导的。为了直接检测母体AP转录本是否存在于卵的细胞质中，分离纯化了内胚层特异的碱性磷酸酶，并测定了其N-端氨基酸序列。根据该序列，我们克隆了该酶的全长3017个碱基的cDNA(HrES-AP)，其中含有完整的开放阅读框架和多聚腺苷酸。HrES-AP与非特异性小鼠AP有52.6%的同源性，并且在尾芽胚胎的内胚层前体细胞中特异表达。Northern杂交和原位杂交的结果表明，在未受精卵的细胞质中未检测到AP转录本，而在内胚层前体细胞的神经胚期首次检测到AP转录本。这些结果表明，至少在盐碱鱼中，胚胎AP的活性完全依赖于合子AP基因的激活，这可能是由卵子中存在的细胞质决定因素所决定的。

Alkaline phosphatase (AP) activity is expressed in endodermal cells of ascidian larvae. It was reported previously that the expression of AP activity is resistant to treatment with actinomycin D,a transcription inhibitor. The resistance of AP expression to actinomycin D treatment suggests that appearance of AP activity does not depend on zygotic transcription and that its appearance is mediated by the translational activation of maternal AP mRNA present in ascidian eggs. To directly examine whether or not maternal AP transcripts are present in the cytoplasm of eggs, the endoderm-specific alkaline phosphatase was purified and its N-terminal amino acid sequence was determined in Halocynthia roretzi. Based on this sequence, we isolated a cDNA of this enzyme (HrES-AP) which is 3017-bp long and contains the complete open reading frame and polyadenvlic acid. HrES-AP exhibited a 52.6% homology to a non-specific mouse AP and is specificaly expressed in endoderm precursor cells of the tailbud embryos. As the results of northern blots and in situ hybridization, AP transcripts were not detected in the cytoplasm of unfertilized eggs and they were first detected at the neurula stage in endoderm precursor cells. These results indicate that at least in Halocynthia, embryonic AP activity exclusively depends on the activation of zygotic AP genes, presumably by cytoplasmic determinants present in eggs.