These studies address research objectives of this RFA including: mechanisms of regulation of metallothionein (MT) gene expression, metal induction during development, and organs specificity in transgenic models. The overall objective of theses studies is to understand the regulation and functions of MT in reproduction and embryonic development in mammals. This information, in turn, is likely to provide important clues as to possible roles of MT in carcinogenesis. Mt plays a pivotal role in essential metal homeostasis and in protection from toxic metals. Zinc (Zn), copper (Cu), and cadmium (Cd) have dramatic effects on embryogenesis. Insufficient dietary Zn or Cu leads to abnormal development and reproductive failure, whereas Cd, is embryotoxic, teratogenic, and carcinogenic. Our studies on mouse embryogenesis establish that the MT genes are actively expressed in specific cell-types that surround the developing embryo from the time of implantation to near parturition, as well as in metal-treated reimplantation blastocysts and in fetal hepatocyte at midgestation. This suggests that MT has important functions during embryogenesis.
The specific aims of the proposed studies are to: 1) determine mechanisms regulating the cell-type specific expression of these genes in the reproductive tract and embryo, and 2) delineate the functions of these proteins during embryogenesis.
Specific aim 1 will be approached by; a) examining the effects of well-documented MT gene transcriptional regulators on MT gene expression in vivo and/or in vitro in mouse decidual cells, visceral endoderm, reimplantation embryos and fetal hepatocytes, b) determining the in vivo interactions of proteins with the MT-I gene promoter in specific cell-types, using genomic footprinting techniques, and c) ultimately mapping cis-acting promoter sequences involved in MT gene expression in these cell-types using transgenic mouse strains that express reporter genes under the control of MT-I promoter deletion mutants. A testable hypothesis is that tissue-specific enhancer elements exist in the mouse MT-I promoter. Delineation of MTs functional roles in vivo is hampered by a lack of strains of mice that exhibit defects (mutations) effecting MT gene expression. Thus, specific aim 2 will be approached by creating strains of transgenic mice that; a) display constitutively heightened, or b) misexpression of the MT genes. Transgenic mice that constitutively over- express MT, due to integration multiple copies of the intact mouse MT-I gene, are currently under study. Transgenic strains that over-express the MT gene in the pancreas, liver and uterus due to enhancer-driven transgene expression will be created. Gene expression will be determined at the level of mRNA by Norther, in situ and solution hybridization, and the reverse transcriptase-polymerase chain reaction. MT protein synthesis and accumulation will be determined by the Cd-heme assay, pulse-labeling western blotting, immunocytochemistry, and anion exchange chromatography. Transgenic mice will be analyzed for effects of aberrant MT gene expression on normal embryogenesis as well as on the embryos response to exogenous metals. A testable hypothesis is that misexpression of MT will alter metal homeostasis during pregnancy causing increased sensitivity to dietary Zn deficiency and reduced sensitivity to Cd toxicity.
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