Lawrence Irwin Gilbert - US grants

Gilbert 9300164 The studies by Dr. Gilbert on the mechanism by which a hormone from the brain of the insect Manduca sexta acts upon specialized glands (prothoracic glands) to cause these glands to begin synthesizing a specific steroid hormone, an ecdysteroid. Ecdysteroids are hormones that cause the insect to molt. i.e. shed its exoskeleton, so that it can grow. They also elicit metamorphosis, i.e. the means by which a caterpillar is transformed into a pupa and then into an adult moth or butterfly. The brain neurohormone is synthesized and released as a result of environmental cues such as photoperiod and the synthesis and release of Ecdysteroids by the prothoracic glands must be extremely precise if the insect is to survive and be successful. He is studying the means by which this peptide hormone interacts with the glands, i.e. to stimulate the synthesis of a compound, a second messenger, which in turn indirectly mediates the phosphorylation (addition of a phosphate group) of specific proteins that control ecdysteroidogenesis. By utilizing the prothoracic glands which are composed of a single layer of homogeneous cells, he should ultimately gain insights into the mechanism of peptide hormone induced steroidogenesis that would not be possible with the more heterogeneous adrenal gland. ***

The means by which hormones interact and control growth and development will be examined using insert metamorphosis as a model system. Precisely timed tobacco hornworm larvae raised on an artificial diet will be the primary experimental organisms. We will attempt to purify and characterize the two forms of the brain neurohormone (PTTH) that initiate cellular reporgramming and the molting cycle, using chromatographic and electrophoretic methods and our newly devised specific and sensitive in vitro assay. Once purified, will be generated and an RIA will be developed so as to titer the hormone and immunocytochemically trace its transport from the site of synthesis (prothoracicotropes) to the neurohemal organ. The role of PTTH in stimulating the prothoracic glands to synthesize and secrete ecdysone, via cAMP, will be studied using organ culture techniques to quantify protein kinase activity (protein phosphorylation). ACtive and inactive prothoracic glands will be investigated as well as glands stimulated by crude extracts and purified PTTH. The role of PTTH in embryogenesis, pupal diapause, and diapause-break will also be explored. Studies will continue on the regulation of the mitochondrial cytochrome P450 ecdysone monooxygenase that converts the steroid prohormone, ecdysone, to the molting hormone, 20-hydroxyecdysone, with emphasison control by juvenile hormone and neural-factors, using our sensitive radioenzymological assay. We will investigate the relationship between the very specific juvenile hormone binding protein of insect hemolymph and target cell cytosol receptors utilizing competitive binding protein techniques developed in this laboratory. Using very high specific activity tritiated ecdysteroid and nominally high specific activity juvenile hormone, receptors for both hormone classes will be localized in endocrine gland so as to examine feedback relationships between the glands and the growth hormones. These studies should provide evidence for the means by which animals regulate and maintain hormone titers and how environment cues are transduced into endocrine mediated events. These phenomena could shed light on endocrinologically based diseases and may lead to new concepts for insect control, insects being vectors of numerous human diseases and the indirect cause of malnutrition.

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.

This proposal deals with Drosophila melanogaster as a model system in molecular endocrinology, and more specifically with a gene that encodes the Drosophila homolog to the mammalian H2RII binding protein. Both are members of the steroid hormone receptor gene superfamily. The mammalian protein participates in the transcriptional regulation of the major histocompatibility complex (MHC) class I genes which, in turn, plays a central role in T-cell lymphocyte recognition of certain carcinomas. Studies suggest that H2RII binding protein also plays a currently unexplored role in some aspect of neural regulation and may modify the transcriptional regulation of the estrogen receptor. The proposed experiments will exploit the numerous technical attributes of Drosophila melanogaster, primarily the wealth of genetic information, to investigate the role of this receptor-like gene product in development. The gene maps to the distal portion of the X chromosome (cytological position: 2C) and is expressed during embryogenesis. The specific aims are: (1) to examine the expression of the 2C gene through development; (2) to obtain a physical map of the 2C gene locus; (3) to identify genes transcriptionally regulated by the 2C gene product; (4) to isolate and characterize mutations of the 2C gene and mutations of loci which interact with the 2C gene product; (5) to test the 2C gene product for its cognate ligand (e.g. ecdysteroid, juvenile hormones, retinoic acid, etc.) and (6) to characterize other candidate receptor clones isolated in our initial genomic screen. The results of these experiments should provide significant insights concerning the role of endocrine factors in mediating the orderly progression of development in Drosophila and perhaps other eukaryotic organisms. More generally, these findings will lead to specific lines of investigation concerning the regulatory role of the H2RII binding protein in mammalian systems.