The principal investigator proposes five years of work to determine the sensitivity and nature of responses to estrogens and anti-estrogens in different tissues of zebrafish embryos and young fish. Methods will be employed to visualize estrogen-induced signal transduction, examine expression of estrogen receptor mRNA, and translate these results into 3-dimensional imaging of gene expression and mRNA localization in the intact organism. It is asserted that a need exists for a system where one can see estrogen signal transduction in the whole organism as it develops, because estrogens affect several different organ systems, and because it is important to determine the extent to which compounds acting as anti-estrogens in some tissues have estrogen-like activity in other tissues. This approach also can be extended to the examination of synthetic and natural estrogenic/anti-estrogenic compounds. The principal investigator poses several questions. Can estrogenic compounds affect where and when the estrogen receptor mRNA is expressed during development? Do estrogenic antagonists and agonists have different developmental target specificities, activating expression at different developmental times and places? Does """"""""artificial"""""""" estrogen exposure in early development affect reproductive capability and sexual phenotype of the resulting fish? These questions will be approached by a combination of molecular techniques including the use of fluorescent reporter genes and transgenic animal derivation, and laser scanning confocal microscopy followed by computer-assisted image analysis.
Five specific aims are identified. (1) Examine development in presence of beta-estradiol and other compounds identified as estrogenic antagonists or agonists in mammalian systems. (2) Isolate, characterize and computer archive the location of the zebrafish estrogen receptor during development from 24 hours post-fertilization through 3 weeks post-hatching. (3) Examine estrogen response in zebrafish to different estrogen-responsive elements (ERE) by co-injection of zebrafish estrogen receptor and ERE-thymidine kinase-promoter-green fluorescent protein (GFP) plasmid constructs to determine expression and sensitivity to different estrogens and anti-estrogens. (4) Generate transgenic zebrafish with chosen constructs to examine the estrogen response. (5) Examine regulation of the transgenic response and 3-dimensional expression of estrogen receptor mRNA in response to exposure to different estrogens and anti-estrogens.
