Department of Environmental and Molecular Toxicology, The Sinnhuber Aquatic Research Laboratory, Oregon State University, Corvallis, Oregon 97331, USA.
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Norris Cotton Cancer Center, Lebanon, New Hampshire 03756 USA.
Toxicol Sci. 2022 May 26;187(2):325-344. doi: 10.1093/toxsci/kfac037.
The aryl hydrocarbon receptor (AHR) is required for vertebrate development and is also activated by exogenous chemicals, including polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR activation is well-understood, but roles of downstream molecular signaling events are largely unknown. From previous transcriptomics in 48 h postfertilization (hpf) zebrafish exposed to several PAHs and TCDD, we found wfikkn1 was highly coexpressed with cyp1a (marker for AHR activation). Thus, we hypothesized wfikkn1's role in AHR signaling, and showed that wfikkn1 expression was Ahr2 (zebrafish ortholog of human AHR)-dependent in developing zebrafish exposed to TCDD. To functionally characterize wfikkn1, we made a CRISPR-Cas9 mutant line with a 16-bp deletion in wfikkn1's exon, and exposed wildtype and mutants to dimethyl sulfoxide or TCDD. 48-hpf mRNA sequencing revealed over 700 genes that were differentially expressed (p < .05, log2FC > 1) between each pair of treatment combinations, suggesting an important role for wfikkn1 in altering both the 48-hpf transcriptome and TCDD-induced expression changes. Mass spectrometry-based proteomics of 48-hpf wildtype and mutants revealed 325 significant differentially expressed proteins. Functional enrichment demonstrated wfikkn1 was involved in skeletal muscle development and played a role in neurological pathways after TCDD exposure. Mutant zebrafish appeared morphologically normal but had significant behavior deficiencies at all life stages, and absence of Wfikkn1 did not significantly alter TCDD-induced behavior effects at all life stages. In conclusion, wfikkn1 did not appear to be significantly involved in TCDD's overt toxicity but is likely a necessary functional member of the AHR signaling cascade.
芳香烃受体 (AHR) 是脊椎动物发育所必需的，也可被外源性化学物质激活，包括多环芳烃 (PAHs) 和 2,3,7,8-四氯二苯并对二恶英 (TCDD)。AHR 的激活已得到充分研究，但下游分子信号事件的作用在很大程度上仍不清楚。在先前的研究中，我们对暴露于几种 PAHs 和 TCDD 的 48 小时后孵化的斑马鱼进行了转录组学分析，发现 wfikkn1 与 cyp1a（AHR 激活的标志物）高度共表达。因此，我们假设 wfikkn1 在 AHR 信号转导中的作用，并表明在暴露于 TCDD 的发育中的斑马鱼中，wfikkn1 的表达依赖于 Ahr2（人类 AHR 的斑马鱼同源物）。为了对 wfikkn1 进行功能表征，我们构建了一个 CRISPR-Cas9 突变系，该突变系在 wfikkn1 的外显子中缺失了 16 个碱基对，并使野生型和突变型斑马鱼暴露于二甲基亚砜或 TCDD 中。48 小时时的 mRNA 测序揭示了超过 700 个基因在每对处理组合之间存在差异表达（p < .05，log2FC > 1），这表明 wfikkn1 在改变 48 小时时的转录组和 TCDD 诱导的表达变化方面发挥着重要作用。对 48 小时时野生型和突变型斑马鱼的基于质谱的蛋白质组学分析揭示了 325 个有显著差异表达的蛋白质。功能富集表明，wfikkn1 参与骨骼肌发育，并在 TCDD 暴露后在神经通路中发挥作用。突变型斑马鱼在形态上似乎正常，但在所有生命阶段都存在明显的行为缺陷，并且 Wfikkn1 的缺失并没有在所有生命阶段显著改变 TCDD 诱导的行为效应。总之，wfikkn1 似乎并没有明显参与 TCDD 的明显毒性，但很可能是 AHR 信号级联中的一个必要功能成员。
