Distinct roles of two zebrafish AHR repressors (AHRRa and AHRRb) in embryonic development and regulating the response to TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)

The AHR repressor (AHRR), an AHR-related bHLH-PAS protein, is regulated by an AHR-dependent mechanism and acts as a transcriptional repressor of AHR function. Resulting from a teleost-specific genome duplication, zebrafish have two AHRR genes (AHRRa, AHRRb), but their functions in vivo are not well understood. We used antisense morpholino oligonucleotides (MO) in zebrafish embryos and a zebrafish cell line (ZF-L) to characterize the interaction of AHRRs and AHRs in normal embryonic development, AHR signaling, and TCDD toxicity. Zebrafish embryos exposed to TCDD (2 nM and 8 nM) during early development showed strong induction of CYP1A, AHRRa and AHRRb at 48 and 72 hpf. A MO targeting AHR2 inhibited TCDD-induced expression of CYP1A, AHRRa, and AHRRb by 84-95% in 48 hpf embryos, demonstrating a primary role for AHR2 in mediating AHRR induction. Dual MO knockdown of both AHRRs in ZF-L cells enhanced TCDD induction of CYP1A, but not other CYP1 genes. In embryos, dual knockdown of AHRRs, or knockdown of AHRRb alone, enhanced the induction of CYP1A, CYP1B1, and CYP1C1 by TCDD and decreased the constitutive expression of Sox9b. In contrast, knockdown of AHRRa did not affect Sox9b expression or CYP1 inducibility. Embryos microinjected with each of two different MOs targeting AHRRa and exposed to DMSO displayed developmental phenotypes resembling those typical of TCDD-exposed embryos (pericardial edema and lower jaw malformations). In contrast, no developmental phenotypes were observed in DMSO-exposed AHRRb morphants. These data demonstrate distinct roles of AHRRa and AHRRb in regulating AHR signaling in vivo and suggest that they have undergone subfunction partitioning since the teleost-specific genome duplication.