Insect growth, development and metamorphosis are controlled endocrinologically, the endocrine system being modulated by environmental stimuli (e.g., photoperiod). The primary endocrine center is the insect brain which tranduces environmental information into the synthesis and release of a neuropeptide, prothoracicotropic hormone (PTTH) which in turn initiates a cascade of events culminating in molting. Although studies over the past 20 years, predominantly on the Lepidoptera, have yielded a great deal of information on the location of the prothoracicotropes, neurohemal organ and the means by which this cerebral neuropeptide stimulates the prothoracic gland to produce the pro-molting hormone, almost nothing is known of control mechanisms involved in PTTH synthesis and release, processing, etc. nor the molecular basis of any phenomena involving this critical cerebral neurohormone. It is the purpose of this proposal to provide the basic information on PTTH in Drosophila melanogaster so that genetic probes can be used to explore the regulation of PTTH synthesis, release and action. An in vitro assay will be developed for the PTTH of Drosophila melanogaster using the ability of extracts of this neuropeptide to stimulate ecdysone synthesis by larval and pupal ring glands (RG). A quantitative and qualitative analysis of ecdysteroids throughout development will be conducted by radioimmunoassay (RIA) and high pressure liquid chromatography (HPLC) and correlated with the in vitro activity of the RG and the PTTH titer of the brain. PTTH will be purified to homogeneity and utilized to provide antibodies to probe putative PTTH and RG temperature sensitive mutants, to identify both the prothoracicotropes in the Drosophila brain and the neurohemal organ. This will provide the tools and background to clone the gene of this neurohormone and develop cDNA probes. The gene locus will be identified by in situ hybridization and its regulation studied with the techniques of molecular genetics. These studies could provide the framework for the development of new growth regulators with which to control the numerous dipterans of biomedical and agricultural importance as well as furnish a model neuroendocrinological system for genetic analysis of endocrine interrelationships.
