Tissue-Restricted Expression of Nrf2 and Its Target Genes in Zebrafish with Gene-Specific Variations in the Induction Profiles

Abstract

The Keap1-Nrf2 system serves as a defense mechanism against oxidative stress and electrophilic toxicants by inducing more than one hundred cytoprotective proteins, including antioxidants and phase 2 detoxifying enzymes. Since induction profiles of Nrf2 target genes have been studied exclusively in cultured cells, and not in animal models, their tissue-specificity has not been well characterized. In this paper, we examined and compared the tissue-specific expression of several Nrf2 target genes in zebrafish larvae by whole-mount in situ hybridization (WISH). Seven zebrafish genes (gstp1, mgst3b, prdx1, frrs1c, fthl, gclc and hmox1a) suitable for WISH analysis were selected from candidates for Nrf2 targets identified by microarray analysis. Tissue-restricted induction was observed in the nose, gill, and/or liver for all seven genes in response to Nrf2-activating compounds, diethylmaleate (DEM) and sulforaphane. The Nrf2 gene itself was dominantly expressed in these three tissues, implying that tissue-restricted induction of Nrf2 target genes is defined by tissue-specific expression of Nrf2. Interestingly, the induction of frrs1c and gclc in liver and nose, respectively, was quite low and that of hmox1a was restricted in the liver. These results indicate the existence of gene-specific variations in the tissue specificity, which can be controlled by factors other than Nrf2.

Nrf2-dependent protection against acute sodium arsenite toxicity in zebrafish

“…A lineup of target genes was also demonstrated to be evolutionarily conserved. Their expression profiles were extensively analyzed in zebrafish larvae using whole mount in situ hybridization (Kobayashi et al, 2009;Nakajima et al, 2011) and a GFP-reporter assay (Suzuki et al, 2005;Tsujita et al, 2011). In this study, we carried out short-term survival assays using zebrafish Nrf2 mutants to evaluate the physiological role of the Keap1-Nrf2 system under the condition of high-dose arsenite exposure.…”

“…pKSabcc2 were linearized by BamHI and transcribed with T3 RNA polymerase (Roche Diagnostics, Indianapolis, IN) in the presence of DIG RNA labeling mix (Roche Diagnostics) to make an RNA probe. pKSgstp1N, pSKprdx1 and pSKgclc have been described elsewhere (Li et al, 2008;Nakajima et al, 2011). The larvae were photographed using an MZ16 microscope (Leica, Wetzlar, Germany) equipped with a DP73 digital camera (Olympus, Tokyo, Japan).…”

Genetic Evidence of an Evolutionarily Conserved Role for Nrf2 in the Protection against Oxidative Stress

“…Under basal conditions, Nrf2 is constantly degraded through the ubiquitin-proteasome pathway in a Keap1-dependent manner (28, 50). Upon exposure to electrophiles or oxidative stress, Nrf2 escapes from proteasomal degradation, accumulates in the nucleus, and transcriptionally activates its target genes.We previously isolated zebrafish homologs of Nrf2 and its regulator Keap1 genes (nrf2, keap1a, and keap1b) and demonstrated that the induction of cytoprotective genes by the Keap1-Nrf2 system is conserved among vertebrates (26,30,34). We also isolated zebrafish small Maf (mafg1, mafg2, mafk, and maft) and Pi-class GST (gstp1 and gstp2) genes and determined that the Nrf2-smallMaf heterodimer binds and transactivates gstp1 expression through a critical ARE sequence (41,44).…”

“…We also isolated zebrafish small Maf (mafg1, mafg2, mafk, and maft) and Pi-class GST (gstp1 and gstp2) genes and determined that the Nrf2-smallMaf heterodimer binds and transactivates gstp1 expression through a critical ARE sequence (41,44). Using this zebrafish system, we have provided new insights into the functions of Nrf2 (26,29,34,47), e.g., the identification of an evolutionarily conserved ETGE motif in Nrf2 that is necessary for its interaction with Keap1, the finding that there is more than one sensor cysteine residue in Keap1 that can sense different sorts of Nrf2-activating compounds, the isolation of several lines of mutant zebrafish that exhibit impaired responses to specific Nrf2-activating compounds, and the demonstration of tissue-specific expression of Nrf2 and its target genes. However, the physiological roles of Nrf2 in zebrafish remain unclear and need to be elucidated to understand Nrf2 functions from an evolutionary standpoint.…”

Sensory systems and ionocytes are targets for silver nanoparticle effects in fish

“…Whole mount in-situ hybridisation (WISH) as applied to zebrafish embryos and early life stages potentially offers a highly integrative, systems-wide approach to assess the toxicity of NPs through identifying target sites in the body where they effect gene expression. This approach in turn may help to predict their potential health effects (Nakajima et al, 2011b). Application of the technique of in situ hybridisation to assess for effects of toxicants on target genes, however, requires understanding on the ontogeny of the expression of the target genes, and this is known for very few genes of toxicological relevance.…”