ESTABLISHMENT OF THE DPP ACTIVITY GRADIENT IN DROSOPHILA

果蝇 DPP 活性梯度的建立

• 批准号: 2188958
• 负责人: EDWIN L FERGUSON
• 金额: $ 19.28万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2188958
• 关键词: Drosophilidae; chromosome walking; early embryonic stage; epitope mapping; gene deletion mutation; gene expression; gene interaction; genetic transcription; invertebrate embryology; laboratory rabbit; laboratory rat; molecular cloning; phenotype; polymerase chain reaction; posttranscriptional RNA processing; regulatory gene; restriction fragment length polymorphism; transcription factor; transforming growth factors

Although transforming growth factor beta (TGF-beta) was first identified based on its effect on the growth and differentiation of cultured cells, recent work has shown that members of this family of growth and differentiation factors play important roles in the specification of embryonic pattern in a large variety of organisms, including mice, Xenopus, chicken, and Drosophila. However, very little is known about the mechanisms used to regulate the activities of these proteins in vivo. In Drosophila, the TGF-beta family member decapentaplegic (dpp) acts as a morphogen to organize dorsalventral pattern within the ectoderm of the embyro; two-to four-fold increases in dpp activity are sufficient to specify progressively more dorsal cell fates. In vivo, the dpp gene is uniformly transcribed over the region in which it is expressed; thus a gradient of dpp activity must be established at a post-transcriptional level. Genetic studies indicate five genes differentially regulate dpp activity post-transcriptionally over the dorsal-ventral axis. The products of the tolloid, shrew, screw, and twisted gastrulation genes elevate dpp activity on the dorsal side of the embryo, while the product of the short gastrulation (sog) gene blocks dpp activity on the ventral side of the embryo. This proposal describes a series of experiments to determine how the products of these genes interact to establish a gradient of dpp activity during early embryogenesis. Because the proteins that regulate dpp activity are likely to be similar to the proteins that regulate the activities of other TGF-beta family members in vertebrates, these experiments could provide information about how the activities of TGF-beta family members are regulated to organize the fates of fields of cells during vertebrate development. Wild-type embryos respond asymmetrically to localized injection of dpp transcripts. Because this asymmetric response is likely to mirror the in vivo mechanisms used in the formation of the dpp gradient, the first specific aim is to use a variety of genetic, developmental, and biochemical manipulations to understand the mechanistic basis for the asymmetric response to dpp transcript injection. The second and third aims of this proposal are to undertake a molecular analysis of the sog and shrew genes. The sog gene will be cloned by using a preexisting transposon-induced allele of the gene as a tag to isolate genomic DNA encompassing the locus. The shrew gene will be cloned by undertaking a chromosomal walk from nearby cloned DNA. The DNA encoding these genes will be used to predict the protein product of each gene, to investigate the pattern of transcription of each gene in wild-type and mutant backgrounds, to make antibodies to the protein of each gene in order to assay subcellular localization. The fourth specific aim is to ascertain whether the dpp gradient represents a gradient of dpp protein or a gradient of an active form of the dpp protein. The technique of epitope tagging will be used to visualize the distribution of dpp protein in frozen sections of blastoderm embryos. The use of this technique will be expanded to explore the biochemical interactions that could be involved in the formation of the dpp gradient.

尽管首先确定了转化的生长因子β（TGF-β） 基于它对培养细胞的生长和分化的影响， 最近的工作表明，这个成长家庭的成员和 分化因素在规范中起着重要作用 多种生物中的胚胎模式，包括小鼠，爪蟾， 鸡肉和果蝇。 但是，关于 用于调节体内这些蛋白质活性的机制。 在果蝇中，TGF-BETA家庭成员脱皮术（DPP）充当 形态学以在外胚层内组织背侧面模式 Embyro; DPP活动的两到四倍就足以 指定更多的背细胞命运。 在体内，DPP基因是 在表达的区域上均匀抄录；因此 DPP活动的梯度必须在转录后建立 等级。 遗传研究表明五个基因差异调节DPP 在背腹轴上转录后的活动。 产品 Toloid，shrew，螺钉和扭曲的胃基因的升高 胚胎背侧的活性，而短的产物 胃（SOG）基因阻断了DPP的活性 胚胎。 该建议描述了一系列实验，以确定如何 这些基因的产物相互作用以建立DPP的梯度 早期胚胎发生期间的活性。 因为调节的蛋白质 DPP活性可能与调节的蛋白质相似 其他TGF-beta家庭成员在脊椎动物中的活动，这些 实验可以提供有关TGF-beta活动的信息 调节家庭成员以组织细胞领域的命运 在脊椎动物发育期间。 野生型胚胎对DPP的局部注入不对称地反应 成绩单。 因为这种不对称响应可能会反映 DPP梯度形成中使用的体内机制，第一个 具体目的是使用各种遗传，发育和生化 操纵不对称的机械基础 对DPP笔录注射的响应。 该提案的第二和第三目的是进行分子 SOG和SHREW基因的分析。 SOG基因将通过使用 先前存在的转座子诱导的基因等位基因作为分离的标签 涵盖基因座的基因组DNA。 Shrew Gene将被克隆 从附近的克隆DNA进行染色体步行。 DNA编码 这些基因将用于预测每个基因的蛋白质产物， 研究野生型和 突变背景，以对每个基因的蛋白质制作抗体 测定亚细胞定位。 第四个具体目的是确定DPP梯度是否代表 DPP蛋白的梯度或DPP活性形式的梯度 蛋白质。 表位标签的技术将用于可视化 DPP蛋白在胚泡胚胎的冷冻切片中的分布。 这 该技术的使用将扩展以探索生化 可能与DPP梯度形成有关的相互作用。