GENE REGULATION IN THE ASCIDIAN CIONA INTESTINALIS

b .抽象。该研究的总体目标是确定海胆和海鞘早期胚胎中基因表达定位模式的分子机制。我们预计，这两个系统的详细比较将提供基本的见解基因网络负责指导谱系特异性的基因表达模式在后生动物胚胎。此外，这些研究应该提供关于脊索动物身体计划的进化起源的信息，因为海胆和海鞘是姐妹分支的成员，尽管它们具有完全不同的组织。莱文研究计划的组成部分包括四个具体目标。首先，我们将在Ci-sna和Ci- fkh的启动子区域找到相同的最小顺式调控模块，当这些组织在32细胞期胚胎中被指定时，这些启动子区域介导肌肉、肠道和表皮世系的表达。其次，利用最小顺式元素从琼脂草和海胆胚粗核提取物中分离母体调控因子。这些实验的目的是确定和表征与交流核心E盒元件相互作用的调节因子，这些元件似乎在初级肌肉规范期间激活Ci-sna表达中起关键作用。第三，母体决定因子的表达和活性将使用多种技术进行研究，包括原位杂交和免疫定位测定，以及在转基因胚胎中使用异源启动子进行异位表达测定。第四，我们将利用顺式调控模块的种间比较来研究海胆和海鞘体计划的关系。例如，将Ci-sna和Ci-fkh元件微注射到海胆胚胎中，并通过电穿孔在Ciona胚胎中表达相应的海胆元件。

B. ABSTRACT. The overall goal of the proposed study is to determine the molecular mechanisms underlying localized patterns of gene expression in the early embryos of sea urchins and ascidians. We anticipate that a detailed comparison of the two systems will provide basic insights into the gene networks responsible for directing lineage-specific patterns of gene expression in metazoan embryos. In addition, these studies should provide information regarding the evolutionary origins of the chordate body plan since sea urchins and ascidians are members of sister clades even though they possess radically divergent organizations. The Levine component of the research plan includes four specific aims. First, we will identical minimal cis-regulatory modules in the promoter regions of Ci-sna and Ci- fkh that mediate expression in the muscle, gut, and epidermal lineages at the time when these tissues are specified in 32-cell stge embryos. Second, minimal cis elements cis elements will be used to isolate maternal regulatory factors from crude nuclear extracts of Ciona and sea urchin embryos. The goal of these experiments is to identify and characterize regulatory factors that interact with AC-core E box elements, which appear to play a key role in the activation of Ci-sna expression during the specification of the primary muscles. Third, the expression and activities of maternal determinants will be investigated using a variety of techniques, including in situ hybridization and immunolocalization assays, and ectopic expression assays using heterologous promoters in transgenic embryos. Fourth, we will investigate the relationship of the sea urchin and ascidian body plans using inter-phyletic comparisons of cis regulatory modules. For example, Ci-sna and Ci-fkh elements will be microinjected into sea urchin embryos, and the corresponding sea urchin elements will be expressed in Ciona embryos via electroporation.