PNNL

Abstract

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.