Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055, STN CSC, Victoria, BC, Canada V8W 2Y2.
Department of Biological Sciences, Northern Arizona University, S. Beaver St., Flagstaff, AZ 86011, USA.
Aquat Toxicol. 2014 Mar;148:162-73. doi: 10.1016/j.aquatox.2014.01.008. Epub 2014 Jan 22.
Studies performed across diverse frog species have made substantial contributions to our understanding of basic vertebrate development and the natural or anthropogenic environmental factors impacting sensitive life stages. Because, anurans are developmental models, provide ecosystems services, and act as sentinels for the identification of environmental chemical contaminants that interfere with thyroid hormone (TH) action during postembryonic development, there is demand for flexible assessment techniques that can be applied to multiple species. As part of the "thyroid assays across indicator and sentinel species" (TAXISS) initiative, we have designed and validated a series of cross-species real time quantitative PCR (qPCR) primer sets that provide information on transcriptome components in evolutionarily distant anurans. Validation for fifteen gene transcripts involved a rigorous three-tiered quality control within tissue/development-specific contexts. Assay performance was confirmed on multiple tissues (tail fin, liver, brain, and intestine) of Rana catesbeiana and Xenopus laevis tadpoles enabling comparisons between tissues and generation of response profiles to exogenous TH. This revealed notable differences in TH-responsive gene transcripts including thra, thrb, thibz, klf9, col1a2, fn1, plp1, mmp2, timm50, otc, and dio2, suggesting differential regulation and susceptibility to contaminant effects. Evidence for the applicability of the TAXISS anuran qPCR assay across seven other species is also provided with five frog families represented and its utility in defining genome structure was demonstrated. This novel validated approach will enable meaningful comparative studies between frog species and aid in extending knowledge of developmental regulatory pathways and the impact of environmental factors on TH signaling in frog species for which little or no genetic information is currently available.
对多种青蛙物种进行的研究为我们理解基本的脊椎动物发育以及影响敏感生命阶段的自然或人为环境因素做出了重要贡献。由于无尾两栖动物是发育模型，提供生态系统服务，并充当识别环境化学污染物的哨兵，这些污染物会干扰胚胎后发育过程中的甲状腺激素 (TH) 作用，因此需要灵活的评估技术，可以应用于多种物种。作为“跨指示和哨兵物种的甲状腺测定”(TAXISS) 计划的一部分，我们设计并验证了一系列跨物种实时定量 PCR (qPCR) 引物组，这些引物组提供了有关进化上遥远的无尾两栖动物转录组成分的信息。对 15 个基因转录本的验证涉及在组织/发育特异性背景下进行严格的三级质量控制。在 Rana catesbeiana 和 Xenopus laevis 蝌蚪的多种组织（尾鳍、肝脏、大脑和肠）上进行了测定性能验证，从而可以比较组织并生成对外源性 TH 的反应谱。这揭示了 TH 反应性基因转录本之间的显著差异，包括 thra、thrb、thibz、klf9、col1a2、fn1、plp1、mmp2、timm50、otc 和 dio2，表明存在差异调控和对污染物效应的敏感性。还提供了跨其他七种物种的 TAXISS 无尾两栖动物 qPCR 测定的适用性证据，代表了五个青蛙科，并证明了其在定义基因组结构方面的效用。这种新颖的经过验证的方法将使青蛙物种之间进行有意义的比较研究成为可能，并有助于扩展对发育调节途径的认识以及环境因素对 TH 信号的影响，因为目前这些物种的遗传信息很少或根本没有。
