TCDD- INDUCED CARDIOTOXICITY IN DEVELOPING ZEBRAFISH

University Of Wisconsin-Madison, Madison WI

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Abstract

Growing epidemiological and laboratory evidence indicates that environmental contaminants such as TCDD affect vertebrate heart development. The cellular receptor for TCDD is the Aryl Hydrocarbon Receptor (AHR), a ligand-activated transcriptional activator. We have used zebrafish, an ideal system for studying heart development in vivo to document the cardiotoxic response to TCDD in embryos at the morphological, functional, and transcriptional levels. These results support a model in which TCDD alters gene regulation in the developing heart to disrupt normal development. An important challenge is identifying AHR target genes in zebrafish, and our recent microarray experiments have yielded a set of prospective genes that are induced in the developing heart by TCDD. The array experiment also revealed a cell cycle gene cluster that is turned off at 12 hr after TCDD exposure, a time that coincides with a halt in heart growth. This suggests a model in which exposure to TCDD halts cellular proliferation in the embryonic heart. Furthermore, the transcriptional response to TCDD in the embryonic heart does not match that observed in other tissues. It is surprising that AHR activation regulates a different set of genes in embryonic heart cells than in juvenile heart cells. This project is focused on the following questions: [unreadable] Are the cardiotoxic effects of TCDD mediated by AHR activation in the heart? This will be addressed by using transgenic zebrafish to manipulate AHR activity specifically in the heart. If AHR activation in the heart does cause cardiotoxicity, we will determine which of the genes induced by TCDD in the heart mediates the cardiotoxic responses. [unreadable] Does TCDD halt cell division in the heart? We will test this by staining dividing nuclei to count the number of proliferating cells in hearts treated with TCDD. We will also use chromatin immunoprecipitation (ChIP) and promoter mapping to understand how TCDD coordinately down regulates a cell cycle gene cluster in the embryonic heart. [unreadable] Why does TCDD activation of AHR affect a different set of genes in the embryonic heart than in the juvenile heart or the developing jaw? We will test the hypothesis that the AHR complex is different in different tissues, producing different DNA binding site specificity in different tissues. This will involve the use of in vitro DNA binding assays. As an alternative, we will test the hypothesis that binding sites for AHR are accessible in one tissue, but not in others. In this hypothesis the chromatin structure at a given gene may allow AHR binding in the embryonic heart, but in other tissues the chromatin structure at this gene would be different, preventing AHR binding. This will be tested using ChIP assays coupled to microarrays (ChIP-chip assays).