Tissue-Restricted Induction of Nrf2 Target Genes which are possible candidates for Nrf2 targets: SepW2 is a member of the selenoprotein family, which has been shown to possess anti-oxidative stress activity [23], and Frrs1 is an iron- metabolizing enzyme that reduces ferric ion [24]. Bcat1 is a metabolizing enzyme for branched-chain amino acids and plays important roles in ammonia metabolism [25]. A WISH analysis was carried out using eleven genes as probes (Figure 2). Among them, seven genes (gstp1, mgst3b, prdx1, frrs1c, fthl, gclc and hmox1a) showed clear and strong induction in response to DEM, suggesting they will be useful for studying tissue specificity of Nrf2 target genes. The remaining four genes showed either weak induction (sepw2b and gclm) or strong constitutive expression (gstal and bcat1), thus indicating that they were unsuited for gene expression studies. We, therefore, used the seven genes showing a strong induction for further analyses. Tissue-restricted induction of Nrf2 target genes Tissue-restricted induction of seven Nrf2 target genes was examined by WISH (Figures 3 and S11), using 5-dpf larvae, since the induction was much clearer compared with that in 4-dpf larvae. As a result, the induction of mgst3b, prdx1, frrs1c, fthl, and gclc was observed in nose, gill, and liver, similar to gstp1, although the expression of frrs1c and gclc in the liver and nose, respectively, was relatively weak. Induction in the intestine was observed in the case of mgst3b, prdx1, frrs1c and gclc, but we did not take them into account since a considerable level of basal expression was detected. Figure 3. Tissue-restricted induction of Nrf2 target genes. 5-dpf larvae were treated with or without 100 mM DEM for six hours (three hours only for hmox1a) and expression of indicated 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. doi:10.1371/journal.pone.0026884.g003 PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26884