GENE EXPRESSION IN EARLY EMBRYONIC DEVELOPMENT

这项提议的主要目标是了解细胞如何相互作用， 发育中的胚胎产生决定和分化。 一个全能卵子的后代对各种 发育胚胎的分化途径是一个基本的生物学过程。 最重要的问题 细胞选择遵循不同的 路径是正常和异常胚胎发育的中心， 癌症，会修复，和老化。 实验将集中在海洋上 海胆胚胎，一个非常类似于脊椎动物的系统， 细胞相互作用作为一种方式， 早期发育中的细胞类型和谱系。 这个胚胎有各种各样的 卵裂球可以彼此分离， 各种方式 新的组织特异性标记物， 现在可以获得特异性分化途径的指标。 首先，这些标记将用于学习管理 特异性基因表达 这将涉及实验， 单独培养单个卵裂球或与其它卵裂球组合培养单个卵裂球 卵裂球类型，或用各种试剂（例如，Li离子、FGF）。 命运 和各种基因的表达将被测量， 原位杂交和免疫细胞化学。 是否有本地化的 卵的植物半球中的物质， 形成肠和骨针的谱系：是否有离子或激动剂， 可能会有效地导致动物细胞的植物分化？ 这些药物是否使用第二信使途径来影响它们的 行动？ 第二，开发和表征组织特异性的努力 将继续进行cDNA克隆。 这些努力将集中在克隆上 骨针基质基因，以及仅在 中粒 第三，骨针基质基因如何表达的细节 将进行研究。 这些基因的转录 研究，在体外，在核提取物，转录定义 模板 该功能测定将用于对 启动子活性所必需的DNA序列，并研究蛋白质在 与DNA相互作用的提取物。

The broad objective of this proposal is to learn how cells interact in the developing embryo to bring about determination and differentiation. the commitment of the progeny of a totipotent ovum to the various differentiation pathways of the developing embryo is a basic biological problem of prime importance. Choices made by cells to follow different paths are central to normal and abnormal embryonic development, to cancer, would repair, and aging. The experiments will focus on the sea urchin embryo, a system very similar to vertebrates in its reliance on cell interactions as a way of bringing about determination of various cell types and lineages in early development. This embryo has various blastomeres that can be separated form one another and recombined in various ways. New tissue specific markers that serve as reliable indicators of specific differentiation pathways are now available. first, these markers will be used to learn the rules that govern specific gene expression. This will involve experiments in which individual blastomeres are cultured alone or in combination with other blastomere types, or with various agents (e.g., Li ion, FGF). The fate of the blastomeres and expression of various genes will be measured by in situ hybridization and immunocytochemistry. Are there localized substances in the vegetal hemisphere of the egg that initiate the lineages that form gut and spicules: Are there ions or agonists that might be effective in causing vegetal differentiation of animal cells? Do these agents use secondary messenger pathways to effect their actions? Second, the effort to develop and characterize tissue specific cDNA clones will be continued. The efforts will concentrate on cloning spicule matrix genes, and on genes expressed solely in descendants of mesomeres. Third, the details of how expression of spicule matrix genes is regulated will be studied. Transcription of these genes will be studied, in vitro, in nuclear extracts that transcribe defined templates. This functional assay will be used to conduct studies on the DNA sequences necessary for promoter activity and to study proteins in the extracts that interact with the DNA.