Expression and Function of a Regulatory Molecular Chaperone During Drosophila Development

组织和细胞如何以空间和时间协调的方式对发育线索做出反应仍然知之甚少。 存在许多信号转导途径，其组分通常是普遍存在的。 可以实现信号转导中特异性的一个水平是通过将信号转导分子（例如激素受体或某些激酶）包装成多组分复合物，所述多组分复合物起控制它们的细胞内定位和它们的活化的作用。 Hsp 90分子伴侣在脊椎动物和酵母中已被深入研究，并且已被证明是几种多组分信号传导复合物-类固醇激素受体、src家族激酶、raf激酶-的关键组分，其是转导分子的特异性定位、激活和/或功能所需的。 从这样的细胞生物学和生物化学分析的热休克蛋白90，一个重要的调节功能，在发展过程中可能会被预期。 然而，很少有人知道的任何分子伴侣，包括热休克蛋白90，在多细胞生物体的背景下的发育作用。 Hsp 90分子伴侣家族在果蝇中只有一个成员，称为Hsp 83。 本实验室的工作揭示了Hsp 83 RNA在卵子发生和胚胎发生过程中罕见的动态时空表达。 这导致了一种假设，即Hsp 83可能在发育过程中调节更普遍存在的伴侣蛋白的功能，仅在某些组织和特定阶段增强信号转导过程。 本研究旨在确定Hsp 83的特定发育功能。 母体合成的Hsp 83 RNA定位于后极胞质，被极细胞吸收，Hsp 83在原始生殖细胞和生殖系中通过大部分发育以高水平表达。 热休克蛋白83的合子转录最初仅限于胚胎的前三分之一，并且这种表达是在胚胎发育过程中进行的。 直接由前形态发生素bicoid调节。 随后，Hsp 83在胚胎的头部、神经母细胞和神经系统以及生殖系中表达。 控制这些组织中Hsp 83表达的顺式调节元件正在详细定义。 表位标记的转基因系已经产生，并被用于测定HSP 83蛋白表达模式。 Hsp 83的突变是致命的。 强等位基因是胚胎致死和死亡与新的头部缺陷与热休克蛋白83在协调头部发展的作用一致。 较弱的等位基因导致形态正常的第一龄幼虫不能蜕皮到第二龄，并最终在第一龄中度过几天后死亡。 这种表型与Hsp 83在协调幼虫蜕皮的激素信号转导中的作用一致。 将在胚胎、早期幼虫和生殖系中进行Hsp 83突变表型的详细分析。 缺失特定顺式调控元件的转基因Hsp 83构建体将被用于将表型靶向至感兴趣的时间和地点。 将进行相互作用位点的遗传测试，以确定特定组织和特定时间的HSP 83伴侣蛋白。 表位标记的HSP 83的免疫纯化和伴侣蛋白的共纯化测定将补充遗传相互作用测试。 两者合计，这些研究将提供第一次分析的Hsp 90家族的分子伴侣的发育功能。

How tissues and cells respond in a spatially and temporally coordinated fashion to developmental cues remains poorly understood. Numerous signal transduction pathways exist, the components of which are often ubiquitous. One level at which specificity in the transduction of a signal may be achieved is by packaging signal transduction molecules, such as hormone receptors or certain kinases, into multicomponent complexes which function to control their intracellular localization and their activation. The Hsp90 molecular chaperone has been studied intensively in vertebrates and in yeast, and has been shown to be a key component of several multicomponent signaling complexes - steroid hormone receptors, src-family kinases, raf kinase - that is required for specific localization, activation and/or function of the transducing molecules. From such cell biological and biochemical analyses of Hsp90, an important regulatory function during development might be anticipated. However little is known about the developmental role of any molecular chaperone, Hsp90 included, in the context of a multicellular organism. The Hsp90 family of molecular chaperones has a single member in Drosophila, called Hsp83. Work from this laboratory revealed an uncommonly dynamic spatial and temporal expression of Hsp83 RNA during oogenesis and embryogenesis. This led to the hypothesis that Hsp83 might regulate the function of more ubiquitous partner proteins during development, potentiating signal transduction processes only in certain tissues and at particular stages. The research proposed here seeks to define the specific developmental functions of Hsp83. Maternally synthesized Hsp83 RNA is localized to the posterior polar plasm, is taken up into the pole cells and Hsp83 is expressed at high levels in the primordial germ cells and the germline through most of development. Zygotic transcription of Hsp83 is restricted initially to the anterior third of the embryo, and this expression is regulated directly by the anterior morphogen, bicoid. Subsequently, Hsp83 is expressed in the head, the neuroblasts and nervous system, and the germline of the embryo. The cis-regulatory elements that control Hsp83 expression in these tissues are being defined in detail. Epitope-tagged transgenic lines have been produced and are being used to assay HSP83 protein expression patterns. Mutations in Hsp83 are lethal. Strong alleles are embryonic lethal and die with novel head defects consistent with a role for Hsp83 in coordinating head development. Weaker alleles result in morphologically normal first instar larvae that are unable to molt to the second instar and eventually die after spending several days in the first instar. This phenotype is consistent with a role for Hsp83 in the transduction of the hormonal signal that coordinates the larval molts. Detailed analyses of Hsp83 mutant phenotypes will be conducted in embryos, early larvae, and in the germline. Transgenic Hsp83 constructs that are deleted for particular cis-regulatory elements will be sued to target phenotypes to the times and places of interest. Genetic tests for interacting loci will be conducted in order to define HSP83's partner proteins in specific tissues and at particular times. Immunopurification of epitope-tagged HSP83 and assays for copurification of partner proteins will complement the genetic-interaction tests. Taken together, these studies will provide the first analysis of the developmental functions of the Hsp90 family of chaperones.