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

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 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 < 0.05, 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.

Concentration-response gene expression analysis in zebrafish reveals phenotypically-anchored transcriptional responses to retene

“…While there is strong evidentiary support in the literature for which genes are induced by AHR activation, it is not clear if teratogenicity at high exposure concentrations of AHR activators is caused by 1) increased/decreased expression of direct AHR-regulated genes or 2) other cascading transcriptional responses which are only induced at concentrations at which teratogenicity is observed. The AHR battery contains a large number of genes, some of which have been studied significantly, while others have been only recently been identified and investigated ( Timme-Laragy et al, 2007 ; Timme-Laragy et al, 2008 ; Planchart and Mattingly, 2010 ; Shankar et al, 2022 ). AHR activators can also induce a variety of toxic effects, not all of which involve the same AHR-regulated genes, highlighting the need to better understand which transcriptional changes can be causally associated with specific toxicity endpoints ( Walisser et al, 2005 ; Watabe et al, 2010 ; Yoshioka et al, 2011 ; Wang et al, 2015 ; Zhang et al, 2016 ; Wright et al, 2017 ).…”

Coupling Environmental Whole Mixture Toxicity Screening with Unbiased RNA-Seq Reveals Site-Specific Biological Responses in Zebrafish

“…It then breaks free from its chaperones and enters the nucleus where it canonically partners with AHR nuclear translocator (ARNT) to bind xenobiotic response elements within the genome. This induces the transcription of numerous genes involved in the xenobiotic response including p450s, wfkkn1n, foxq1a, and nrf2 [ 18 , 19 , 20 , 21 , 22 ].…”