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.
