September 21, 2022
Journal Article

The Ahr2-Dependent wfikkn1 Gene Influences Zebrafish Transcriptome, Proteome, and Behavior


The aryl hydrocarbon receptor (AHR) is required for vertebrate development and is also activated by exogenous chemicals including polycyclic aromatic hydrocarbons (PAHs) and TCDD. AHR activation is well-understood, but roles of downstream molecular signaling events are largely unknown. From previous transcriptomics in 48-hours post fertilization (hpf) zebrafish exposed to several PAHs and TCDD, we found wfikkn1 (WAP, Kazal, immunoglobulin, Kunitz, and NTR domain-containing protein) was highly co-expressed 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 DMSO or TCDD. 48-hpf mRNA sequencing revealed over 700 genes that were differentially expressed (p 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 significantly altered TCDD-induced behavior effects in larval zebrafish. 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.

Published: September 21, 2022


Shankar P., G.R. Garcia, J.K. LaDu, C.M. Sullivan, C. Dunham, B. Goodale, and K.M. Waters, et al. 2022. The Ahr2-Dependent wfikkn1 Gene Influences Zebrafish Transcriptome, Proteome, and Behavior. Toxicological Sciences 187, no. 2:325–344. PNNL-SA-169011. doi:10.1093/toxsci/kfac037

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