Understanding the molecular basis of behavior will undoubtedly require a fundamental knowledge of how the nervous system is affected at the level of gene expression by factors such as neuronal activity, hormones, and environment. In particular, steroid hormones regulate many aspects of development, reproduction, homeostasis, and behavior in both vertebrates and invertebrates. These ligands act through specific receptors which serve as trans-active transcriptional control factors. This project is concerned with the developmental regulation of expression of the genes coding for these receptors using the American lobster, Homarus americanus, as a model system. The use of Homarus is particularly beneficial to this study because of the relatively large amount of information available on the physiology and endocrinology of this animal, the large size of many nerve cells, and the ability to reliably identify cells which may be hormone sensitive. The goals of this proposal are as follows: (1) Members of the steroid receptor superfamily of genes will be identified and isolated from the lobster. This is being accomplished with hybridization and polymerase chain reaction (PCR) methodology using sequences derived from the steroid hormone receptor genes of other species. Special attention will be given to the identification of the receptor for the arthropod molting hormone, 20-hydroxyecdysone. (2) The tissue- and stage-specific expression of the receptor sequences will be mapped using Northern analysis, in situ hybridization, and PCR. (3) Identification of the ligands for members of the lobster steroid receptor superfamily will be accomplished with domain-swapping studies. (4) While 20-hydroxyecdysone titers are regulated cyclically within each molt cycle, the hormone affects different tissues in different ways during the overall course of development. This underscores the importance of cell- and stage-specific receptor regulation as potential determinative events during development. Receptor expression over the long-term course of development will be studied with methods indicated above. The hypothesis that 20-hydroxyecdysone receptor expression is regulated in a cyclical manner within the molt cycle will also be investigated. (5) Serotonin plays an important role in the lobster nervous system, and preliminary evidence suggests that it may be regulated by ecdysteroids. The gene for tryptophan hydroxylase, the rate limiting enzyme in serotonin synthesis, will be cloned in order to pursue the question of how serotonin is regulated. The proposed experiments will help to elucidate the question of how steroid receptors are regulated and in turn regulate gene expression in developing tissues at a high level of temporal and spacial resolution. These data will also prepare the way for studies on the effects of the hormones on the biochemistry and physiology of identified cells.
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