The proposed research is designed to establish zebrafish as a model system to better understand the molecular mechanism underlying the developmental toxicity of low dose dioxin exposure.
Specific aims i nclude:(I) Clone the aryl hydrocarbon receptor(AhR) and its dimerization partner AhR nuclear translocator protein (ARNT) from zebrafish. Bacterially expressed AhR and ARNT fusion proteins will be generated for the production and purification of affinity purified polyclonal antibodies. (II) Determine the temporal and spatial expression patterns of AhR and ARNT mRNAs and proteins during zebrafish development in the absence and presence of TCDD using in situ hybridization and immunohistochemical techniques. (III) Determine the timing and location of endogenous functionally active AhR-ARNT heterodimeric complexes during zebrafish development. Dioxin responsive promoters linked to reporter genes will be examined in transgenic zebrafish. (IV) The importance of AhR and ARNT in normal zebrafish development, will be determined by modifying the expression of these proteins. The effect of antisense repression and transgenic overexpression of AhR and ARNT during development in TCDD treated and untreated embryos will be evaluated. Completion of this work should provide the essential information needed to establish zebrafish as a model system to understand AhR mediated developmental toxicity. This work should also lead to a better understanding of AhR and ARNT function during vertebrate development.
