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Altered basal and TCDD-induced gene expression in zebrafish embryos made AHRRa-null by ZFN-mediated gene targeting

ABSTRACT: The aryl hydrocarbon receptor (AHR) repressor (AHRR), a bHLH-PAS protein, is a transcriptional repressor of AHR and other transcription factors (HIF, ER) and is regulated by an AHR-dependent mechanism. However, the physiological and toxicological roles of AHRR are not well understood. We demonstrated earlier that knockdown of AHRRa (one of two AHRR paralogs) in zebrafish embryos using morpholino anti-sense oligonucleotides results in developmental phenotypes such as pericardial edema, craniofacial malformations, and cardiac deformities, similar to effects caused by exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHRRa morphants also exhibit down-regulation of genes associated with photoreceptor development. To characterize the AHRRa function further, we used ZFN (zinc finger nuclease) technology to generate a line of zebrafish with a 7-bp deletion in exon 3 of AHRRa, leading to a truncated AHRR (110 aa, of which 38 are from AHRRa, compared to the 550-aa wild-type (wt) AHRRa). The mutant AHRRa protein, which contains basic regions but lacks the HLH and PAS domains, did not repress AHR in vitro, suggesting that it is inactive (null). Unlike AHRRa morphants, AHRRa-null fish did not display a TCDD-like phenotype; exposure to TCDD caused defects similar to those in TCDD-exposed wt embryos. RNA-sequencing revealed that basal expression of 562 genes differed significantly between the null and wt embryos, including down-regulation of genes associated with photoreceptor development, as seen in AHRRa morphants. TCDD-induced gene expression patterns for the prototypic AHR target genes were similar for null and wt embryos. However, 22 genes were differentially regulated by TCDD in the AHRRa-null embryos as compared to wt embryos (>2-fold; 5% FDR). We are currently investigating the link between these differentially expressed genes and the function of AHRRa in development and AHR signaling. [Supported by NIH R01ES006272]

ORGANISM(S): Danio rerio

PROVIDER: GSE84420 | GEO | 2017/12/31

SECONDARY ACCESSION(S): PRJNA329125

REPOSITORIES: GEO

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Project description:The aryl hydrocarbon receptor repressor (AHRR) is a bHLH-PAS protein closely related to the aryl hydrocarbon receptor (AHR). AHRR is a transcriptional repressor of AHR and hypoxia-inducible factor (HIF) and is regulated by an AHR-dependent mechanism. Zebrafish possess two AHRR paralogs; AHRRa regulates constitutive AHR signaling during development, while AHRRb regulates polyaromatic hydrocarbon-induced gene expression. However, very little is known about the endogenous roles of AHRRs in development and physiology. The objective of this study was to elucidate the role of AHRRs during zebrafish development using a loss-of-function approach followed by microarray analysis. Zebrafish embryos were microinjected with morpholino oligonucleotides (MO) against AHRRa or AHRRb to disrupt endogenous signaling during development. At 72 hours post-fertilization (hpf), embryos were sampled for microarray analysis. Agilent microarray analysis revealed altered expression of 279 and 116 genes with knockdown of AHRRa and AHRRb, respectively. In AHRRa-morphant embryos, 97 genes were up-regulated and 182 genes were down-regulated. Among the differentially expressed genes were a large number related to photoreceptor development, including cone-specific genes such as short-wave, medium-wave and long-wave sensitive opsins (opn1sw1, opn1sw2, opn1mw1, opn1lw2), phosphodiesterases (pde6H, pde6C), retinol binding protein (rbp4l), phosducin, and arrestin. These results suggest that AHRRa may be involved in photoreceptor development. In addition, 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 of genes related to any specific biological process. Overall, these results suggest that AHRRs play multiple roles during development. Three different treatment conditions (Control-MO, AHRRa-MO and AHRRb-MO) and two replicates per treatment

Project description:Dioxin-like chemicals are well-known for their ability to upregulate expression of numerous genes via the AH receptor (AHR). However, recent transcriptomic analyses in several laboratories indicate that dioxin-like chemicals or AHR genotype itself also can downregulate levels of mRNAs encoded by numerous genes. The mechanism responsible for such downregulation is unknown. We hypothesized that microRNAs (miRNAs), which have emerged as powerful negative regulators of mRNA levels in several systems, might be responsible for mRNA downregulation in dioxin/AHR pathways. We conducted a thorough investigation to address the following questions: (1) does AHR genotype itself affect constitutive expression of microRNAs? (2) does TCDD affect microRNA levels and, if so, is this response dependent on the AHR? (3) does TCDD affect microRNA levels differently in animals that are sensitive to dioxin toxicity versus those that are dioxin-resistant? We used the Exiqon miRNA array platform as well as quantitative RT-PCR to measure miRNA levels in wildtype vs. Ahr-null mice. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo caused few changes in miRNA levels in mouse livers and those changes that were statistically significant were of modest magnitude. AHR genotype had little effect on hepatic miRNA levels, either in constitutive expression or in response to TCDD M-bM-^@M-^S only a few miRNAs differed in expression between Ahr-null mice compared to mice with wildtype AHR. It is unlikely that mRNA downregulation by dioxins is mediated by miRNAs, nor are miRNAs likely to play a significant role in dioxin toxicity in adult mouse liver. Manuscript Submitted: Moffat ID, Boutros PC, Celius T, Pohjanvirta R & Okey AB. Micro-RNAs in rodent liver are refractory to dioxin treatment. Toxicological Sciences May, 2007. Keywords: miRNA expression, gene knockout, response to xenobiotics, genetic modification This was a two-factor, two-level design, the factors being genotype Ahr+/+ (WT) or Ahr-/- (KO) and treatment TCDD-treated (T) or vehicle control (C). Each total-RNA sample was separately labeled either with a Hy3- or a Hy5-fluorophore (Exiqon). Hy3- and Hy5-labeled samples were co-hybridized to an array. Dye-reversal was performed to eliminate dye bias. We tested 3 TCDD-treated and 3 control mice in the Ahr-/- groups and 4 TCDD-treated and 4 control mice in the Ahr+/+ groups. Technical replication of 1 TCDD-treated and 1 control mice in the Ahr-/- groups was performed. A loop design was used.

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Altered basal and TCDD-induced gene expression in zebrafish embryos made AHRRa-null by ZFN-mediated gene targeting

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