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

Abstract

The aryl hydrocarbon receptor (AHR) repressor (AHRR), an AHR-related basic helix-loop-helix/Per-AHR nuclear translocator-Sim 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 and AHRRb), but their functions in vivo are not well understood. We used antisense morpholino oligonucleotides (MOs) in zebrafish embryos and a zebrafish liver cell line (ZF-L) to characterize the interaction of AHRRs and AHRs in normal embryonic development, AHR signaling, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. Zebrafish embryos exposed to TCDD (2 and 8nM) during early development showed strong induction of CYP1A, AHRRa, and AHRRb at 48 and 72 hours post-fertilization (hpf). An 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 dimethyl sulfoxide (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.

FIG. 1.

Developmental time course of expression of AHR signaling pathway components in response to TCDD exposure. Real-time RT-PCR was used to quantify transcript expression in whole zebrafish embryos during development after exposure to moderate and high concentrations of TCDD. Standard curves derived from plasmid dilutions were used to calculate transcript abundance. To highlight changes in relative gene expression as a result of either changes in developmental time point or TCDD exposure, transcript abundance for each sample (time and exposure) was normalized to the 24 hpf DMSO control. Error bars represent one standard deviation; n = 3 replicates of 20 pooled embryos. Statistically significant difference between DMSO and TCDD at each time point is represented by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001).

FIG. 2.

Morpholino specificity and inhibition efficacy confirmed by in vitro translation. (A) In vitro transcription/translation of zebrafish AHR2, AHRRa, and AHRRb in the presence or absence of Ctrl MO, target-specific MO, or nonspecific MO. (B) Schematic of the proximal 5′ UTR of AHRRa and AHRRb highlighting the MO target sequences.

FIG. 3.

Effect of AHR2 knockdown on CYP1A, AHRRa, and AHRRb expression in response to TCDD exposure. AHR2 translation was blocked by an AHR2 MO injected at the two- to four-cell stage in zebrafish embryos. Real-time RT-PCR was used to quantify transcript abundance in whole zebrafish embryos at 48 hpf after exposure to 0.1% DMSO (carrier) or 2nM TCDD. Standard curves derived from plasmid dilutions were used to calculate transcript abundance. Relative fold induction was determined by comparing transcript abundance for each sample to the noninjected (No MO) DMSO control. Error bars represent one standard deviation; n = 3 replicates of 20 pooled embryos. Statistically significant difference in transcript expression compared to Ctrl MO is represented by asterisks (**p < 0.01, ***p < 0.001). Results presented are from one experiment, but data are representative of two separate experiments.

FIG. 4.

Enhancement of TCDD-induced expression of endogenous CYP1A by dual AHRRa/AHRRb MO knockdown in ZF-L cells. ZF-L cells were cotreated with Endo-Porter (MO transfection agent) and MOs targeting AHRRa and AHRRb for 48 h and then exposed to 0.1% DMSO or various concentrations of TCDD for 24 h. Real-time RT-PCR was used to quantify CYP1 transcript expression. Standard curves derived from plasmid dilutions were used to calculate transcript abundance. Relative fold induction was determined by comparing transcript abundance for each sample to the Ctrl MO DMSO. Error bars represent one standard deviation; n = 3 replicates of 6 pooled wells of cells from a 48-well plate. Statistically significant difference in CYP1 induction compared to DMSO for each individual MO treatment is represented by (+) sign (p < 0.05). Statistically significant difference in CYP1 induction between Ctrl MO and AHRRa MO1/AHRRb MO is represented by asterisks (**p < 0.01, ***p < 0.001).

FIG. 5.

Enhancement of TCDD-induced expression of endogenous CYP1 transcripts by individual AHRRa and AHRRb knockdowns in embryos. AHRRa or AHRRb translation was blocked by MO injection at the two- to four-cell stage in zebrafish embryos. Real-time RT-PCR was used to quantify CYP1 transcript abundance in whole zebrafish embryos at 72 hpf after exposure to 0.1% DMSO (carrier) or 2nM TCDD. Standard curves derived from plasmid dilutions were used to calculate transcript abundance. Relative fold expression was determined by comparing transcript abundance for each sample to the noninjected (No MO) DMSO control. Error bars represent one standard deviation; n = 3 replicates of 20 pooled embryos. Statistically significant difference in transcript expression compared to Ctrl MO is represented by asterisks (***p < 0.001). Results presented are from one experiment, but data are representative of two separate experiments.

FIG. 6.

Sox9b expression after AHRR knockdown or TCDD exposure. AHRRa or AHRRb translation was blocked by MO injection at the two- to four-cell stage in zebrafish embryos. Real-time RT-PCR was used to quantify Sox9b expression in whole zebrafish embryos at 72 hpf after exposure to 0.1% DMSO (carrier) or 2nM TCDD. Relative fold change was calculated by the 2^−ΔΔCt method by comparison to the Ctrl MO DMSO treatment. Error bars represent one standard deviation; n = 3 replicates of 20 pooled embryos. Statistically significant difference in transcript expression compared to Ctrl MO DMSO is represented by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001).

FIG. 7.

Recapitulation of TCDD-like developmental phenotypes by AHRRa knockdown in zebrafish embryos. AHRRa or AHRRb translation was blocked by MO injection at the two- to four-cell stage in zebrafish embryos. Common TCDD-induced developmental phenotypes, such as pericardial edema, craniofacial malformations, and cardiac deformities, were phenocopied by AHRRa knockdown in DMSO-treated embryos. Phenotype occurrence was confirmed by microinjection of two separate AHRRa MOs. In contrast, embryos microinjected with AHRRb MO did not display any abnormal developmental phenotypes in DMSO-treated embryos. Results presented are representative of at least three separate experiments.

FIG. 8.

PSS in embryos subjected to AHRR knockdown and exposed to DMSO or TCDD. AHRRa or AHRRb translation was blocked by MO injection at the two- to four-cell stage in zebrafish embryos. Image analysis was used to quantify PSS in 72 hpf embryos after exposure to 0.1% DMSO (carrier) or various concentrations of TCDD. PSS was normalized to the average sac size of noninjected, DMSO-treated embryos and expressed as a ratio of individual/mean of control. Error bars represent one standard deviation; n = 10 individual embryos. A two-way ANOVA was performed to determine the significance of combined TCDD exposure and MO knockdown on the severity of pericardial edema measured by changes in PSS. Statistically significant differences in relative PSS compared to Ctrl MO at each TCDD concentration are represented by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Results presented are representative of two separate experiments.

FIG. 9.

Enhancement of TCDD toxicity by AHRR knockdown as determined by severity of pericardial edema. Assessment of TCDD-induced pericardial edema for the various AHRR MO treatments are assumed to be the same as the average increase in PSS from TCDD-exposed embryos microinjected with Ctrl MO. Assessments of TCDD-induced pericardial edema in noninjected embryos (No MO) were based on comparison to control embryos (noninjected and DMSO treated). MO-induced changes in PSS were determined from DMSO-treated embryos. The sum of these two causative factors (MO and TCDD treatment) was used to determine the magnitude of TCDD-induced edema that is specifically enhanced (i.e., more than additive) as a result of AHRR knockdown. AHRRa knockdown alone resulted in significant pericardial edema that was further enhanced by TCDD exposure. In contrast, AHRRb knockdown alone caused minimal pericardial edema in control embryos but significantly enhanced the TCDD-induced pericardial edema.