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作出反应仍然知之甚少。存在许多信号转导途径，其组成部分通常是普遍存在的。信号转导的特异性可以通过将信号转导分子（如激素受体或某些激酶）包装成多组分复合物来实现，这些复合物的功能是控制其细胞内定位和激活。Hsp90分子伴侣已在脊椎动物和酵母中进行了深入研究，并已被证明是几种多组分信号复合物（类固醇激素受体、src家族激酶、raf激酶）的关键成分，这些信号复合物是转导分子特定定位、激活和/或功能所必需的。通过对Hsp90的细胞生物学和生化分析，可以预测其在发育过程中的重要调控功能。然而，对于包括Hsp90在内的任何分子伴侣在多细胞生物中的发育作用知之甚少。Hsp90分子伴侣家族在果蝇中只有一个成员，叫做Hsp83。本实验室的工作揭示了Hsp83 RNA在卵发生和胚胎发生过程中不寻常的动态空间和时间表达。这导致了一种假设，即Hsp83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。本研究旨在明确Hsp83的具体发育功能。母源合成的Hsp83 RNA定位于后极质，进入极细胞，Hsp83在原始生殖细胞和种系的大部分发育过程中都有高水平表达。Hsp83的合子转录最初局限于胚胎的前三分之一，这种表达直接受前形态原bicoid的调节。随后，Hsp83在胚胎的头部、神经母细胞和神经系统以及种系中表达。在这些组织中控制Hsp83表达的顺式调控元件正在被详细地定义。表位标记转基因系已经产生，并被用于检测HSP83蛋白表达模式。Hsp83的突变是致命的。强等位基因是胚胎致死性的，死亡时伴有新的头部缺陷，这与Hsp83在协调头部发育中的作用一致。较弱的等位基因导致形态正常的一龄幼虫不能蜕皮到二龄，最终在一龄中度过几天后死亡。这种表型与Hsp83在协调幼虫蜕皮的激素信号转导中的作用是一致的。Hsp83突变表型的详细分析将在胚胎、早期幼虫和种系中进行。对特定顺式调控元件进行删除的转基因Hsp83构建物将被用于在感兴趣的时间和地点靶向表型。为了确定HSP83在特定组织和特定时间的伴侣蛋白，将对相互作用位点进行基因检测。抗原表位标记的HSP83的免疫纯化和伴侣蛋白的共化试验将补充遗传相互作用试验。综上所述，这些研究将首次分析Hsp90伴侣蛋白家族的发育功能。

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.