Tissue-Restricted Expression of Nrf2 and Its Target Genes in Zebrafish with Gene-Specific Variations in the Induction Profiles Hitomi Nakajima 1 , Yaeko Nakajima-Takagi1, Tadayuki Tsujita1,2, Shin-Ichi Akiyama3, Takeshi Wakasa3, Katsuki Mukaigasa1, Hiroshi Kaneko1,4, Yutaka Tamaru3, Masayuki Yamamoto2,4, Makoto Kobayashi1,2* 1 Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan, 2 Environmental Response Project, Japan Science and Technology Agency, University of Tsukuba, Tsukuba, Japan, 3 Graduate School of Bioresources, Mie University, Tsu, Japan, 4 Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan 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. Citation: Nakajima H, Nakajima-Takagi Y, Tsujita T, Akiyama S-I, Wakasa T, et al. (2011) Tissue-Restricted Expression of Nrf2 and Its Target Genes in Zebrafish with Gene-Specific Variations in the Induction Profiles. PLoS ONE 6(10): e26884. doi:10.1371/journal.pone.0026884 Editor: Cormac T. Taylor, University College Dublin, Ireland Received July 28, 2011; Accepted October 5, 2011; Published October 25, 2011 Copyright: 2011 Nakajima et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (MK, MY) and the Japan Science and Technology Corporation (ERATO) (MY). Zebrafish microarrays were supported by Consortium R&D Project for Regional Revitalization from the Ministry of Economy, Trade and Industry of Japan (YT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: makobayash@md.tsukuba.ac.jp Introduction Nrf2 is a transcription factor that binds to the antioxidant response element (ARE) and transactivates cytoprotective genes [1,2]. In basal conditions, Nrf2 is degraded via the Keap1- dependent proteasome pathway, while it is stabilized after cells are exposed to electrophilic or oxidative stress, which transactivates its target genes. Many studies have identified the Keap1-Nrf2 system to have multiple sensor sites to a variety of stresses [3,4] and more than one hundred target genes [5,6]. Conservation of the Keap1- Nrf2 system has been demonstrated in vertebrates including zebrafish [7,8], which is a well-established research model. Through previous studies, we noticed that the expression of gstp1, a major target gene for zebrafish Nrf2, is not ubiquitous as expected, but is rather restricted in the nose, gill, and liver [8–13]. It is unclear whether this tissue-restricted induction is specific for gstp1 or a common feature for Nrf2 target genes, since gstp1 was the only gene available in our study that was suitable for WISH analysis. In addition, most studies related to Nrf2 target genes have been performed on cultured cells or a specific murine tissue, and therefore the tissue specificity of Nrf2 target genes has not yet been well characterized, even in other animals. In this paper, we identified seven zebrafish genes appropriate for WISH using microarray analysis and examined their tissue-specific expression in zebrafish larvae. Induced expression of all these genes was restricted to the nose, gill, and/or liver with gene-specific variations in tissue specificity. These results indicate tissue- restricted induction to therefore be a common feature of Nrf2 target genes, which can be a critical issue for both the pharmacological and clinical applications of Nrf2-activating compounds. Materials and Methods RT-PCR and WISH analyses Zebrafish embryos and larvae were obtained by natural mating. All experiments were carried out using a wild-type AB strain. For induction studies, embryos or larvae were placed in culture dishes containing 100 mM DEM (Wako, Osaka, Japan) or 40 mM sulforaphane (LKT laboratories, St. Paul, MN). RT-PCR analysis was performed as described previously [8] using PCR primers listed in Table S1. WISH analysis was carried out as described previously [14] with slight modifications in the fixation step. Briefly, the larvae were fixed with 4% paraformaldehyde (PFA) in PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26884