GENE REGULATION IN THE ASCIDIAN CIONA INTESTINALIS

B.摘要。这项拟议研究的总体目标是确定海胆和海鞘早期胚胎基因表达定位模式背后的分子机制。我们预计，对这两个系统的详细比较将提供对负责指导后生动物胚胎中特定血统基因表达模式的基因网络的基本见解。此外，这些研究应该提供有关脊索体计划的进化起源的信息，因为海胆和海鞘是姐妹支系的成员，尽管它们拥有截然不同的组织。研究计划的莱文部分包括四个具体目标。首先，我们将在Ci-SNA和Ci-Fkh的启动子区域相同最小的顺式调节模块，当这些组织在32-细胞STGE胚胎中被指定时，它们介导在肌肉、肠道和表皮谱系中的表达。其次，最低限度的顺式元件顺式元件将被用来从芝麻和海胆胚胎的粗核提取液中分离母体调节因子。这些实验的目的是识别和表征与AC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指定过程中激活CI-SNA表达方面发挥关键作用。第三，将使用多种技术来研究母体决定因素的表达和活性，包括原位杂交和免疫定位分析，以及使用异源启动子在转基因胚胎中的异位表达分析。第四，我们将使用顺式调控模块的类系间比较来研究海胆和海鞘身体计划的关系。例如，Ci-SNA和Ci-Fkh元素将被显微注射到海胆胚胎中，相应的海胆元素将通过电穿孔在Ciona胚胎中表达。

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.