Aryl hydrocarbon receptor multiplicity in a frog model of dioxin toxicity

描述(申请人提供)：芳烃受体(AHR)是一种配体激活的转录因子，介导许多环境污染物的毒性和致癌作用，包括二恶英类工业废物化合物和石油和香烟烟雾中的芳香烃。AHR还在正常的发育过程中发挥重要作用，包括肝脏发育、心血管发育和T细胞分化。因此，AHR活性的改变是多种人类疾病状态的基础。人类只有一个ahr基因。单一的AHR执行这种不同的、看似无关的功能的机制鲜为人知。非洲爪蛙是一种广泛使用的基本脊椎动物发育和发育毒理学模型。青蛙模型与人类的不同之处在于有两个AHR(AHR1α和AHR1β)，这是由于分类学上唯一的基因组重复~40mya所致。它们的转录本在成年蛙中表现出不同的表达模式，增加了亚功能化的可能性，即将单个祖先蛋白的多种角色划分为重复的并列蛋白。这一领域的拨款将支持一个本科生实验室小组来测试这一假设，即ahr1α和ahr1β在青蛙发育、转录调控和/或毒性方面具有非冗余功能。拟议的项目有三个具体目标。在目标1中，我们将利用原位杂交和定量RT-PCR法检测AHR1α和AHR1mRNAs在胚胎和成人组织中的相对表达，定量验证它们的差异表达。在特定目标2下的研究将检验Ahr1α和ahr1β调控不同的靶基因集的假设。利用转录激活物样效应核酸酶(TALENS)，我们将编辑莱氏X.laevis细胞系XLK-WG的基因组，以敲除其中一个或两个AHR的表达。产生的突变细胞系将用有效的异源AHR配体TCDD(2，3，7，8-四氯二苯并-对二恶英)或内源性AHR配体FICZ(6-formyinddolo[3，2b]carbazole)处理，由此产生的mRNA表达的变化将通过RNAseq在基因组水平上进行测量和表征。最后，在目标3中，我们将使用TALEN来产生缺失Ahr1α和/或ahr1β的突变青蛙。敲除的蝌蚪将被用来检验这样的假设，即每一个AHR Paralog在青蛙的发育或毒性中扮演着不同的角色。除了测量XLK-WG细胞中确定的转录本的表达变化外，我们还将检查TCDD和FICZ处理的胚胎和蝌蚪是否存在常见的大体形态缺陷(例如，浮肿、脊髓缺陷)以及特定组织(包括肝脏)的组织学变化。我们的比较方法利用了两个莱维氏X.laevis AHR提供的机会来剖析单个人类蛋白质的多重、复杂的功能。了解青蛙和人类AHR信号之间的差异也将有助于使用FETAX进行毒理学风险评估，以及使用青蛙胚胎进行类似的发育毒性测试。最后，该领域项目将为众多本科生科学家提供技术先进的长期研究培训经验。

DESCRIPTION (provided by applicant): 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 (AHR1α and AHR1β), 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 AHR1α and AHR1β 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 AHR1α and AHR1β mRNAs in embryonic and adult tissues, quantitatively verifying their differential expression. Studies under specific Aim 2 will test the hypothesis that AHR1α and AHR1β 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 AHR1α and/or AHR1β. 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.