The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic and carcinogenic effects of numerous environmental contaminants, including dioxin-like industrial waste compounds and aromatic hydrocarbons in petroleum and cigarette smoke. AHR also plays essential roles in normal developmental processes, including liver development, cardiovascular development and T-cell differentiation. Alterations in AHR activity thus underlie multiple human disease states. Humans have one Ahr gene. The mechanisms by which the single AHR carries out such diverse, seemingly unrelated functions are poorly understood. The African clawed frog, Xenopus laevis, is a widely used model of both basic vertebrate development and developmental toxicology. The frog model differs from humans in having two AHRs (AHR1? and AHR1?), which resulted from a taxonomically unique genome duplication ~40 mya. Their transcripts exhibit distinct expression patterns in adult frog, raising the possibility of subfunctionalization, or partitioningof multiple roles of a single ancestral protein into duplicate paralogs. This AREA grant will support an undergraduate lab group to test the hypothesis that AHR1? and AHR1? have non-redundant functions in frog development, transcriptional regulation, and/or toxicity. The proposed project has three Specific Aims.
In Aim 1, we will use in situ hybridization and quantitative RT-PCR to measure the relative expression of AHR1? and AHR1? mRNAs in embryonic and adult tissues, quantitatively verifying their differential expression. Studies under specific Aim 2 will test the hypothesis that AHR1? and AHR1? regulate distinct sets of target genes. Using transcription activator-like effector nucleases (TALENs) we will edit the genome of X. laevis cell line XLK-WG to knock out expression of one or both AHRs. Resulting mutant cell lines will be treated with the potent xenobiotic AHR ligand TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) or the endogenous AHR ligand FICZ (6-formylindolo[3,2b]carbazole), and resulting changes in mRNA expression will be measured and characterized on the genomic scale by RNAseq. Finally, in Aim 3 we will employ TALENs to generate mutant frogs lacking AHR1? and/or AHR1?. Knockout tadpoles will be used to test the hypothesis that each AHR paralog plays a distinct role in frog development or toxicity. In addition to measuring expression changes in transcripts identified in XLK-WG cells, we will examine TCDD and FICZ-treated embryos and tadpoles for common gross morphological defects (e.g. edema, spinal defects) as well as histological changes in specific tissues, including liver. Our comparative approach capitalizes on the opportunity presented by the two X. laevis AHRs to dissect the multiple, complex functions of the single human protein. Understanding the differences between frog and human AHR signaling will also aid toxicological risk assessment involving FETAX and similar developmental toxicity tests employing frog embryos. Finally, this AREA project will provide a technologically sophisticated, long-term research training experience for numerous undergraduate scientists.
This project examines the aryl hydrocarbon receptor (AHR), a protein that mediates the toxic effects of environmental contaminants such as dioxin. This protein also has important roles in embryonic and immune cell development. Unlike humans, frogs have two AHRs. We will compare the function of the two frog AHRs with the single, multi-functional AHR from humans. This comparative research will enable us to dissect and better understand the most important features of AHR function and how they relate to human disease and development. It will also aid in the understanding of important differences between frogs and humans and the interpretation of toxicology studies using frog embryos as a model system for measuring the developmental toxicity of chemicals and environmental samples.
