Aryl hydrocarbon receptor (AHR) deficiency in a frog model of dioxin toxicity

项目摘要。芳香烃受体蛋白(AHR)是多种人类疾病状态的基础。 AHR是一种配体激活的转录因子，介导多种环境污染物的毒性， 包括工业废物中的二恶英类化合物，如2，3，7，8四氯二苯并对二恶英(TCDD)和 风化原油和香烟烟雾中的多环芳烃。AHR还在以下方面发挥作用 肝脏、心血管系统和卵母细胞的正常发育，被有毒的激动剂或 突变可能会导致成年人患病。非洲爪蛙、假四倍体非洲爪蛙和二倍体非洲爪蛙。 热带，是被广泛研究的脊椎动物发育的水生模型。非洲爪哇还用于 发育毒性在两个生命阶段进行筛选。FETAX(青蛙胚胎致畸试验) 检测早期生命阶段接触化学物质造成的急性发育毒性。两栖动物 变态分析(AMA)模拟人类晚期胎儿发育，测量化学物质对 把小蝌蚪改造成小狐狸。AHR功能与发育毒性的人类健康相关性是 对青蛙的了解很少，限制了这些检测方法用于人类风险评估的有效性。一种理解 对青蛙AHR的基本发育和细胞功能的了解是使用该模型在 毒物学研究。总体目标是使用基因组编辑的、缺乏AHR的非洲爪哇模型，包括 AHR-空的热带X线虫动物和莱维氏X.laevis细胞系，以鉴定AHR在发育蛙中的特定功能 和培养的细胞。目标1下的研究将确定AHR在热带管圆线虫发育中的功能。我们会 使用FETAX方案比较AHR缺失型青蛙的频率和表型 Ahr缺失和野生型胚胎的典型发育缺陷的严重程度，包括存活、长度、轴向和 头部畸形和水肿症。我们还将测试ahr-空的青蛙显示肝脏和卵巢的假设。 AHR-/-小鼠的典型表型，比较形态和组织学。最后，我们将使用一套分析方法 以确定AHR的缺失如何改变变态过程中的组织重塑。我们将在形态上结合 与RNAseq研究的终点，以确定潜在的表型基因表达变化。在目标2中，我们 将使用在该物种中缺乏两个ahr类似基因之一的莱维氏X.laevis细胞系，检验以下假设 单个AHR类似物在细胞周期调节中发挥着不同的作用。我们将比较生长缓慢的ahr1a-Null 通过流式细胞仪检测ahr1b缺失突变体和野生型XLK-WG细胞，建立细胞周期分布 每个人。我们还将测试ahr1a缺失细胞容易发生凋亡的假设。初步数据显示 AHR1a和AHR1b调控不同的基因靶点。我们将在RNAseq研究中验证这一假设。 这一领域的项目将提供对AHR介导的发育毒性的途径的比较洞察。 在脊椎动物模型中共享。它还将为青蛙发育毒性提供机械性支持。 FETAX和AMA等筛查，提高了它们对人类风险评估的有效性。

Project Summary. The Aryl Hydrocarbon Receptor protein (AHR) underlies multiple human disease states. AHR is a ligand-activated transcription factor that mediates toxicity of numerous environmental contaminants, including dioxin-like compounds such as 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) from industrial waste and polynuclear aromatic hydrocarbons in weathered crude oil and cigarette smoke. AHR also plays roles in ordinary development of liver, cardiovascular system, and oocytes, disruption of which by toxic agonists or mutation can cause disease in adults. African clawed frogs, pseudotetraploid Xenopus laevis and diploid X. tropicalis, are widely studied aquatic models of vertebrate development. Xenopus is also used in developmental toxicity screens at two life stages. FETAX (Frog Embryo Teratogenesis Assay-Xenopus) detects acute developmental toxicity resulting from chemical exposure at early life stages. The Amphibian Metamorphosis Assay (AMA) models late fetal development in humans, measuring chemical disruption of remodeling of a tadpole to a froglet. The human-health relevance of AHR function in developmental toxicity is poorly understood in frogs, limiting the validity of these assays for human risk assessment. An understanding of the fundamental developmental and cellular functions of frog AHRs is essential for the use of this model in toxicological studies. The overall objective is to use genome-edited, AHR-deficient Xenopus models, including ahr-null X. tropicalis animals and X. laevis cell lines, to identify specific functions of AHRs in developing frogs and cultured cells. Studies under Aim 1 will identify functions of AHR in X. tropicalis development. We will characterize phenotypes displayed in ahr-null frogs, using the FETAX regimen to compare frequency and severity of typical developmental defects in ahr-null and wild-type embryos, including survival, length, axial and head deformities, and edemas. We will also test the hypothesis that ahr-null frogs display hepatic and ovarian phenotypes typical of AHR-/- mice, comparing morphology and histology. Finally, we will use a suite of assays to determine how loss of ahr alters tissue remodeling during metamorphosis. We will couple morphological endpoints with RNAseq studies to determine gene expression alterations underlying phentoypes. In Aim 2, we will use X. laevis cell lines lacking one of the two ahr paralogs in this species, testing the hypothesis that individual AHR paralogs play distinct role in cell cycle regulation. We will compare slow-growing ahr1a-null mutants with ahr1b-null and wild-type XLK-WG cells by flow-cytometry, establishing cell cycle distribution of each. We will also test the hypothesis that ahr1a-null cells are prone to apoptosis. Preliminary data suggest that AHR1a and AHR1b regulate distinct sets of gene targets. We will test this hypothesis in RNAseq studies. This AREA project will provide comparative insight into the pathways of AHR-mediated developmental toxicity shared among vertebrate models. It will also lend mechanistic underpinning to frog developmental toxicity screens such as FETAX and AMA, improving their validity for human risk assessment.