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.
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