Knockdown of a zebrafish aryl hydrocarbon receptor repressor (AHRRa) affects expression of genes related to photoreceptor development and hematopoiesis

Abstract

The aryl hydrocarbon receptor repressor (AHRR) is a transcriptional repressor of aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor (HIF) and is regulated by an AHR-dependent mechanism. Zebrafish (Danio rerio) possess two AHRR paralogs; AHRRa regulates constitutive AHR signaling during development, whereas AHRRb regulates polyaromatic hydrocarbon-induced gene expression. However, little is known about the endogenous roles and targets of AHRRs. The objective of this study was to elucidate the role of AHRRs during zebrafish development using a loss-of-function approach followed by gene expression analysis. Zebrafish embryos were microinjected with morpholino oligonucleotides against AHRRa or AHRRb to knockdown AHRR protein expression. At 72 h postfertilization (hpf), microarray analysis revealed that the expression of 279 and 116 genes was altered by knockdown of AHRRa and AHRRb, respectively. In AHRRa-morphant embryos, 97 genes were up-regulated and 182 genes were down-regulated. Among the down-regulated genes were several related to photoreceptor function, including cone-specific genes such as several opsins (opn1sw1, opn1sw2, opn1mw1, and opn1lw2), phosphodiesterases (pde6H and pde6C), retinol binding protein (rbp4l), phosducin, and arrestins. Down-regulation was confirmed by RT-PCR and with samples from an independent experiment. The four genes tested (opn1sw1, pde6H, pde6C, and arr3b) were not inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin. AHRRa knockdown also caused up-regulation of embryonic hemoglobin (hbbe3), suggesting a role for AHRR in regulating hematopoiesis. Knockdown of AHRRb caused up-regulation of 31 genes and down-regulation of 85 genes, without enrichment for any specific biological process. Overall, these results suggest that AHRRs may have important roles in development, in addition to their roles in regulating xenobiotic signaling.

Keywords: AHR; AHRR; TCDD; aryl hydrocarbon receptor; cones; development; dioxin; microarrays; morpholino oligonucleotides; opsins; repressor; zebrafish.

FIG. 1.

Altered gene expression in response to MO-induced knockdown of AHRR in zebrafish embryos. (A) Heat map representation of gene expression patterns significantly altered by AHRRa and AHRRb knockdown at 72 hpf, in comparison to a control morpholino-injected group. Hierarchial clustering of significant genes was performed. Adjusted p-value less than 0.01 is considered to be statistically significant. (B) Venn diagram showing the total number of up- and down-regulated genes that are uniquely altered by AHRRa and AHRRb MOs at 72 hpf. The set of genes that are differentially expressed by both MOs are shown in the intersection.

FIG. 2.

Biological network of genes involved in cone photoreceptor signaling. All the differentially expressed genes from the AHRRa-MO group were analyzed for functional association using the STRING database and statistically significant networks were identified using jActiveModules version 2.23. Only the network of genes involved in cone photoreceptor signaling is shown. The color of each node represents the fold change in gene expression. Different shades of green (light green to dark green) denote the magnitude of down-regulation. Unchanged nodes are colored white.

FIG. 3.

qRT-PCR confirmation of microarray results. Six genes that were significantly altered by AHRR knockdown as suggested by microarray analysis were selected for qRT-PCR. β-Actin was used as an internal standard. The ΔΔCt method was used to determine the fold change in gene expression. RNA was from larvae sampled at 72 hpf. Values represent mean + SD (one-way ANOVA; n = 3), *p ≤ 0.05.

FIG. 4.

Effect of TCDD on AHRR-MO induced gene expression changes. Injection of AHRR MOs and exposure to TCDD was carried out as described in Materials and Methods section. RNA was from larvae sampled at 72 hpf. β-Actin was used as an internal standard. The ΔΔCt method was used to determine the fold change in gene expression. Values represent mean + SD (two-way ANOVA; n = 3). Bonferroni's post hoc test was used for determining statistical significance. ^ap ≤ 0.05 TCDD versus DMSO; *p ≤ 0.05 AHRRa-MO group versus no-MO treatment. CYP1 expression data from these experiments was reported previously (Jenny et al., 2009).