ahr2, But Not ahr1a or ahr1b, Is Required for Craniofacial and Fin Development and TCDD-dependent Cardiotoxicity in Zebrafish

Abstract

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds environmental toxicants and regulates gene expression. AHR also regulates developmental processes, like craniofacial development and hematopoiesis, in the absence of environmental exposures. Zebrafish have 3 paralogs of AHR: ahr1a, ahr1b, and ahr2. Adult zebrafish with mutations in ahr2 exhibited craniofacial and fin defects. However, the degree to which ahr1a and ahr1b influence ahr2 signaling and contribute to fin and craniofacial development are not known. We compared morphology of adult ahr2 mutants and ahr1a;ahr1b single and double mutant zebrafish. We found that ahr1a;ahr1b single and double mutants were morphologically normal whereas ahr2 mutant zebrafish demonstrated fin and craniofacial malformations. At 5 days post fertilization, both ahr1a;ahr1b and ahr2 mutant larvae were normal, suggesting that adult phenotypes are due to defects in maturation or maintenance. Next, we analyzed the function of zebrafish AHRs activated by environmental ligands. The prototypical AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces toxicity in humans and rodents via AHR and causes cardiotoxicity in zebrafish embryos. It has been shown that embryos with mutations in ahr2 are resistant to TCDD toxicity, yet it is unclear whether ahr1 receptors are required. Furthermore, though AHR was shown to interact with estrogen receptor alpha following TCDD treatment, it is not known whether this interaction is constitutive or context-dependent. To determine whether estrogen receptors are constitutive cofactors for AHR signaling, we used genetic and pharmacologic techniques to analyze TCDD-dependent toxicity in estrogen receptor and ahr mutant embryos. We found that embryos with mutations in ahr1a;ahr1b or estrogen receptor genes are susceptible to TCDD toxicity whereas ahr2 mutant embryos are TCDD-resistant. Moreover, pharmacologic blockade of nuclear estrogen receptors failed to prevent TCDD toxicity. These findings suggest that ahr1 genes do not have overlapping functions with ahr2 in fin and craniofacial development or TCDD-dependent toxicity, and that estrogen receptors are not constitutive partners of ahr2.

Molecular and Functional Properties of the Atlantic Cod (Gadus morhua) Aryl Hydrocarbon Receptors Ahr1a and Ahr2a

“…As discussed earlier, zfAhr2 has been shown to be involved in mediating toxicity of dioxinlike compounds and PAHs, as well as a suggested a role of in the development of the nervous system 43,44,95 . On the other hand, Karchner et al found high expression levels of zfahr1b in zebrafish embryos compared to the other two zfahrs, suggesting a role of zfAhr1b in embryonic development 24 .…”

“…Toxic responses to TCDD, PCB126 and B[a]P in zebrafish and mummichog have been demonstrated to be mediated by Ahr2 [37][38][39][40][41][42][43][44][45]88 . Other studies also suggested a role of Ahr2 in mediating TCDD toxicity in red seabream, medaka, and Atlantic salmon 30,33,89 .…”

“…Expression patterns of ahr genes vary among fishes, and ahr2 is often more abundant and has wider tissue distribution, whereas expression of ahr1 is mainly found in brain and heart [28][29][30]36 . Loss-of-function studies using morpholino-modified antisense oligonucleotides or genome editing have shown that Ahr2 has a primary role in mediating toxic responses to TCDD and dl-PCBs in some fishes [37][38][39][40][41][42][43][44][45] . The exact function of Ahr1 has not yet been elucidated.…”

“…The exact function of Ahr1 has not yet been elucidated. A possible physiological role in early development of zebrafish has been hypothesized 24 , but loss-offunction studies have not yet revealed such a role [44][45][46] .…”

Aryl Hydrocarbon Receptor Signaling Is Functional in Immune Cells of Rainbow Trout (Oncorhynchus mykiss)

“…We focus on the AhR2 clade, since the receptors of this clade mediate the effects of PHAHs and PAHs in fish [ 18 , 19 , 45 , 46 ]. The AhR2 isoforms were shown to play a key role in the developmental and cardiovascular toxicity of dioxin-like chemicals in several fish species including rainbow trout, and they contribute to the developmental toxicity of PAHs [ 45 , 47 , 48 ]. Beyond, there exists evidence that AhR2 signaling is involved in the toxic effects of HAHs and PAHs on the reproductive system of fish [ 11 , 49 ].…”