In our analysis, all tested genes showed tissue-restricted induction. Furthermore, we found gene-specific variation in tissue specificity, e.g., a weak induction of frrs1c and gclc in the liver and nose, respectively, and no hmox1a induction in the nose and gill. The requirements of metabolizing enzymes encoded by each target gene may be different among tissues and the level of enzymes are controlled at a transcriptional level. An important finding in this study is that the expression level of Nrf2 itself is different among tissues, which will be the critical point to exert tissue-restricted induction of its target genes. We hypothesized that the gene expression profiles of the Nrf2 gene defines a default state of tissue specificity of target genes. In cases of frrs1c, gclc and hmox1a, some tissue- and gene-specific transcriptional repressors may be involved to exert their tissue-specific variations. Nrf2-dependent ARE gene regulation has been shown to be negatively regulated by other ARE- binding proteins such as Nrf1, Nrf3, Bach1, small Maf proteins, and c-Maf [31–35]. Since we have previously demonstrated that the target genes of Nrf-Maf proteins tend to differ somewhat among family proteins [36,37], it is possible that these ARE-acting factors may bind in a gene-specific manner and interfere with DNA binding of Nrf2. Furthermore, ATF3, c-Myc and p53 have also been reported to repress Nrf2-dependent gene activation [38–40]. Some of these transcription factors may bind near the ARE in a gene- and tissue-specific manner and inhibit the transcription activity of Nrf2. In contrast to Nrf2 regulation at a post translational step, molecular mechanisms of Nrf2 gene regulation have not been extensively studied. The exception is the upregulation of the Nrf2 gene by Nrf2 itself and aryl hydrocarbon receptor in response to their chemical activators [41,42]. We previously reported that the embryonic expression of the Nrf2 gene is quite low both in the mouse and zebrafish and it becomes elevated near birth [12,43]. The downregulation of Nrf2 was found in prostate cancer which may be related to the initiation of cellular transformation [44]. A low Nrf2 expression in the brain has been reported in humans, mice, and chickens [45–47], as was also the case in our present study. For the efficient medical applications of the Keap1-Nrf2 system for the treatment of neurodegenerative diseases [48,49], Nrf2 should be considerably expressed in the brain. However, we found the expression of Nrf2 in the brain to be low. It will thus be valuable to find new methods and procedures to elevate the Nrf2 expression in both brain and prostate cancer cells. Supporting Information Figure S1 Phylogenetic tree of Gsta family proteins. Amino acid sequences of full-length proteins were analyzed. The tree was constructed by the neighbor-joining method using the ClustalW program (http://clustalw.ddbj.nig.ac.jp/top-j.html). c, chicken; h, human; m, mouse; r, rat; xt, Xenopus tropicalis; z, zebrafish. (TIF) Figure S2 Phylogenetic tree of Mgst3 family proteins. Amino acid sequences of full-length proteins were analyzed. ci, Ciona intestinalis; x, Xenopus laevis. (TIF) Figure S3 Phylogenetic tree of SepW family proteins. Amino acid sequences of full-length proteins were analyzed. d, Drosophila melanogaster. (TIF) Figure S4 Phylogenetic tree of Bcat family proteins. Amino acid sequences of full-length proteins were analyzed. ce, Caenorhabditis elegans. (TIF) Tissue-Restricted Induction of Nrf2 Target Genes Figure S5 Phylogenetic tree of Prdx family proteins. Amino acid sequences of full-length proteins were analyzed. (TIF) Figure S6 Phylogenetic tree of Frrs family proteins. Amino acid sequences of full-length proteins were analyzed. (TIF) Figure S7 Phylogenetic tree of Fth family proteins. Amino acid sequences of full-length proteins were analyzed. (TIF) Figure S8 Phylogenetic tree of Gclc family proteins. Amino acid sequences of full-length proteins were analyzed. (TIF) Figure S9 Phylogenetic tree of Gclm family proteins. Amino acid sequences of full-length proteins were analyzed. (TIF) Figure S10 Phylogenetic tree of Hmox family proteins. Amino acid sequences of full-length proteins were analyzed. (TIF) Figure S11 Expression of frrs1c and hmox1c in the liver. Transverse sections of 5-dpf larvae through the trunk at the level of the liver (dotted line). Larvae were treated with (frrs1c, hmox1a) or without (fabp10) 100 mm DEM and analyzed by WISH before sectioning. Red and white arrowheads indicate positive and negative expression, respectively, of each gene in the liver. Asterisk denotes the basal expression in the intestine. Scale bar, 100 mm. (TIF) Figure S12 Induction of Nrf2 target genes by sulforaph- ane. 5-dpf larvae were treated with or without 40 mM sulforaphane for indicated hours and expression of seven Nrf2 target genes was analyzed by WISH. Lateral and ventral views. Red and white arrowheads indicate positive and negative expression, respectively, of each gene in the nose, gill and liver. Asterisks denote basal expression in the intestine. (TIF) Oligonucleotide primers used for RT-PCR Table S1 analyses. (DOC) Table S2 Oligonucleotide primers used for plasmid construction. (DOC) Table S3 Identification of DEM-inducible genes in zebrafish (1). (DOC) Table S4 Identification of DEM-inducible genes in zebrafish (2). (DOC) Acknowledgments We thank Y. Kikuchi (Hiroshima University) for the fabp10 probe, S. Hosoyamada, T. Kinoshita, H. Niu, T. Shimokoube, R. Sakurazawa and N. Tsuruta for help in fish maintenance, and L. Li for discussion. Author Contributions Conceived and designed the experiments: YT MY MK. Performed the experiments: HN YNT TT SA TW KM HK MK. Analyzed the data: HN YNT TT SA TW KM HK MK. Wrote the paper: MK. PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26884