ESTABLISHMENT OF DPP ACTIVITY GRADIENT IN DROSOPHILA

果蝇 DPP 活性梯度的建立

• 批准号: 6385870
• 负责人: EDWIN L FERGUSON
• 金额: $ 35.1万
• 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 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6385870
• 关键词: DNA footprinting; Drosophilidae; biological signal transduction; cellular polarity; early embryonic stage; epitope mapping; gel mobility shift assay; gene deletion mutation; gene expression; gene interaction; genetic transcription; invertebrate embryology; molecular cloning; phenotype; polymerase chain reaction; posttranscriptional RNA processing; regulatory gene; restriction fragment length polymorphism; transcription factor; transforming growth factors

The specification of positional information is a long standing question in developmental biology. The TGF-beta family member dpp plays a central role in patterning the dorsal-ventral axis of the Drosophila embryo: dpp is absolutely necessary for the production of all dorsal cell fates; moreover increasing concentrations of dpp are sufficient to specify all positional values within the embryonic ectoderm. While the dpp gene is uniformly expressed over the dorsal 40 percent of the embryo, genetic experiments indicated that dpp activity is subject to spatially-restricted, post-transcriptional regulation over the dorsal-ventral axis. The elucidation of the mechanisms underlying this post-transcriptional regulation will provide important insights into the nature of this patterning process. Furthermore, experiments that have been conducted over the past three years have indicated that the mechanism for dorsal-ventral patterning has been conserved between arthropods and vertebrates; thus, the knowledge gained from a detailed genetic and molecular analysis of dorsal-ventral patterning in Drosophila is likely to be directly applicable to the vertebrate embryo. Previous work had shown two TGF-beta type I receptors, TKV and SAX, are necessary for dorsal-ventral patterning. Injection of mRNA encoding the activated receptors into embryos lacking dpp signaling demonstrated that increasing levels of activation of TKV are sufficient to recapitulate embryonic dorsal-ventral pattern. Conversely, activation of SAX was without phenotypic consequences. However, sax activity synergizes with tkv activity to promote dorsal development. Functional experiments suggested that the receptors have different ligands: DPP acts through TKV, while a second TGF-beta needed for dorsal-ventral patterning, SCW, is a component of the SAX ligand. Furthermore SOG, a ventrally expressed negative regulator of dpp signaling, preferentially blocks SCW activity. These results have uncovered a novel mechanism for post-transcriptional regulation of dpp activity: modulation of DPP activity by the spatial regulation of SAX signaling through the action of SOG on SCW. The first two specific aims of the proposal focus on the signal transduction pathways downstream of the TKV and SAX receptors. The first aim is to characterize the synergy between the TKV and SAX receptors. We propose experiments to determine whether synergy occurs at the level of receptor activation, action on the MAD/MEDEA signaling complex, or formation of different transcriptional complexes upstream of specific target genes. The second aim is to identify intracellular negative regulatory components of dpp/scw signaling. The third and fourth specific aims focus on the extracellular components that regulate dpp/scw activity. In the third specific aim, we test the hypothesis that SOG facilitates the diffusion of ventrally-produced SCW to dorsal regions of the embryo, thereby elevating SCW signaling dorsally. The fourth specific aim is to determine the role of the twisted gastrulation gene, which encodes a novel secreted protein required for dorsal structures. The last specific aim is to address the question of specificity between the DPP and SCW ligands. We have assays for the activities of both ligands, and we will perform domain swaps to determine the amino acids that result in specificity, both for receptor activation and for interaction with SOG.

位置信息的规范是发育生物学中的一个长期问题。 TGF-beta家族成员DPP在对果蝇胚胎的背腹轴模式下起着核心作用：DPP对于产生所有背细胞命运是绝对必要的。此外，增加的DPP浓度足以指定胚胎外胚层内的所有位置值。 尽管DPP基因在背侧40％的胚胎上均匀表达，但基因实验表明，DPP活性受到背侧腹侧轴的空间限制，转录后调节的约束。 阐明本文后调节的机制将提供对这种模式过程性质的重要见解。此外，在过去三年中进行的实验表明，背腹模式的机制在节肢动物和脊椎动物之间得到了保守。因此，从果蝇背腹模式的详细遗传和分子分析中获得的知识可能直接适用于脊椎动物胚胎。先前的工作显示了两个TGF-beta I型受体TKV和SAX对于背腹图案是必需的。 注入编码活化受体的mRNA中缺乏DPP信号传导的胚胎表明，提高TKV的激活水平足以概括胚胎背腹模式。 相反，萨克斯的激活没有表型后果。 但是，萨克斯活动与TKV活性协同以促进背侧发育。 功能实验表明，受体具有不同的配体：DPP通过TKV起作用，而背腹图案SCW所需的第二个TGF-beta是SAX配体的组成部分。 此外，SOG是DPP信号传导的腹侧表达的负调节剂，优先阻止SCW活性。 这些结果发现了DPP活性转录后调控的新型机制：通过通过SOG对SCW的作用对SAX信号的空间调节对DPP活性的调节。该提案的前两个特定目的集中在TKV和SAX受体下游的信号转导途径上。第一个目的是表征TKV和SAX受体之间的协同作用。 我们提出的实验是确定协同作用是在受体激活水平，对MAD/MEDEA信号复合物的作用还是在特定靶基因上游上游的不同转录复合物的形成。 第二个目的是确定DPP/SCW信号的细胞内负调控组件。 第三和第四个特定目的集中于调节DPP/SCW活性的细胞外成分。 在第三个特定目的中，我们检验了SOG促进腹侧生产的SCW向胚胎背区域的扩散的假设，从而在背侧升高SCW信号。第四个具体目的是确定扭曲胃胃基因的作用，该基因编码了背侧结构所需的新型分泌蛋白质。 最后的特定目的是解决DPP和SCW配体之间的特异性问题。 我们对这两种配体的活性进行了测定，我们将执行域掉期，以确定导致受体激活和与SOG相互作用的特异性的氨基酸。