DEVELOPMENTAL GENETICS OF ASCIDIANS

海鞘为研究脊索动物发育过程中相关基因的功能提供了一个简单的实验系统。海鞘蝌蚪仅由约2600个细胞组成，这些细胞构成了包括表皮、中枢神经系统、脊索和尾肌在内的一小部分幼体器官。蝌蚪的这种形态代表了脊索动物的基本形体。研究最充分的海鞘和海鞘的胚胎发生速度快（约18小时），雌雄同体，自受精可行，整个生命周期不到3个月，便于诱变和遗传筛选。本研究的目的是介绍在这些海鞘中分离突变体的遗传方法。我们首先尝试用化学诱变剂诱变。用化学诱变剂ENU处理精子，建立了几个突变系。例如，chongmague影响脊索的形成，zebulon影响头部的形成。当突变是由单核苷酸取代引起时，鉴定受影响的基因并不简单。一种鉴定突变基因的策略包括借助转座因子的插入诱变。然而，以前在海洋无脊椎动物中进行插入诱变的尝试并不成功。我们发现Tc1/mariner家族转座子Minos，最初从果蝇hydei中分离出来，可以作为Ciona的转座子。这种植物的基因组富含at，而Minos靶向TA二核苷酸。这也许可以解释为什么米诺斯在乔娜胚胎中很活跃。通过仔细检查切除、缺失和转座活性，我们建立了许多增强子陷阱线。这一方法可能是研究Ciona基因功能的重大突破。目前，我们正在分离成年器官形成缺陷的突变体。因此，本研究是首次报道在海洋动物中可能的插入诱变。少

Ascidians provide an appealingly simple experimental system to investigate the function of developmentally relevant genes during chordate development. The ascidian tadpole is composed of only 〜2600 cells, which constitute a small set of larval organs including the epidermis, central nervous system, notochord and tail muscle. This configuration of the tadpole represents the basic chordate body plan. Embryogenesis of the best studied ascidians Ciona intestinalis and Ciona savignyi is rapid (about l8 hr), they are hermaphroditic, self-fertilization is feasible, and the entire life cycle takes less than 3 months, facilitating mutagenesis and genetic screens. The aim of the present study was to introduce forward genetic methods to isolate mutants in these ascidians.We first attempted mutagenesis with chemical mutagens. By treating sperm with the chemical mutagen ENU, several mutant lines were established. For example, chongmague affects notochord formation and zebulon affects head formation … More in Ciona intestinalis embryos.The identification of the affected genes is not simple when mutations are caused by single nucleotide substitutions. A strategy for identifying mutated genes involves insertional mutagenesis with the aid of transposable elements. However, previous attempts at insertional mutagenesis in marine invertebrates were not successful. We found that a Tc1/mariner family transposon Minos, originally isolated from Drosophila hydei, can function as a transposable element in Ciona. The Ciona intestinalis genome is AT-rich and Minos targets TA dinucleotides. This may explain why Minos is active in Ciona embryos. By careful examination of excision, deletion and transposable activity, we have established a number of enhancer trap lines in Ciona intestinalis. This method might represent a significant breakthrough for elucidating gene function in Ciona. At present, we are isolating mutants with deficiencies of adult organ formation.Thus, the present study is the first report of possible insertional mutagenesis in marine animals. Less