GENE REGULATION IN THE ASCIDIAN CIONA INTESTINALIS

B。摘要。这项研究的总体目标是确定海胆和海鞘早期胚胎中基因表达局部模式的分子机制。我们预计，这两个系统的详细比较将提供基本的见解，基因网络负责指导后生动物胚胎中的基因表达的谱系特异性模式。此外，这些研究应提供信息的进化起源的脊索动物的身体计划，因为海胆和海鞘的成员姐妹分支，即使他们拥有根本不同的组织。 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的启动子区域中相同的最小顺式调节模块，当这些组织在32细胞阶段胚胎中被指定时，所述启动子区域介导肌肉、肠和表皮谱系中的表达。第二，最小顺式元件顺式元件将被用来分离母源调节因子从粗核提取物的玻璃海鞘和海胆胚胎。这些实验的目的是鉴定和表征与AC核心E盒元件相互作用的调节因子，所述调节因子似乎在初级肌肉的特化期间在Ci-sna表达的激活中起关键作用。第三，母体决定因素的表达和活动将使用各种技术，包括原位杂交和免疫定位测定，以及异位表达测定使用异源启动子在转基因胚胎进行研究。第四，我们将调查海胆和海鞘的身体计划之间的关系，使用跨系比较顺式调节模块。例如，Ci-sna和Ci-fkh元件将被显微注射到海胆胚胎中，相应的海胆元件将通过电穿孔在玻璃海鞘胚胎中表达。

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.