Tissue-Restricted Induction of Nrf2 Target Genes using nrf2-specific morpholino oligonucleotide. As a result, the induction of eleven genes was clearly demonstrated to be Nrf2- dependent (Figure 1). We constructed phylogenetic trees of the 11 identified genes, with the exception of gstp1, using the zebrafish genome and cDNA information; we also renamed some genes (Figures S1–S10). Finally, the genes were identified as gstal (glutathione S-transferase alpha like), mgst3b (microsomal glutathione S-transferase 3b), sepw2b (selenoprotein W2b), bcat1 (branched chain aminotransfer- ase 1), prdx1 (peroxiredoxin 1), frrs1c (ferric-chelate reductase 1c), fthl (ferritin heavy chain like), gclc (glutamate-cysteine ligase catalytic subunit), gclm (glutamate-cysteine ligase modifier subunit) and hmox1a (heme oxygenase 1a). Gsta, Prdx1, Fth, Gclc, Gclm and Hmox1 have been identified as typical Nrf2 target genes in mammalian cells [15–19]. Mgst3 has also been suggested to be an Nrf2 target from several microarray studies [20–22]. These results indicated that target genes of Nrf2 are conserved among vertebrates. More interestingly, SepW2, Bcat1, and Frrs1 have never been indentified as Nrf2 targets, including from microarray analyses. Among these three, two are redox-regulating proteins Figure 2. WISH analysis of Nrf2 target genes. Expression of eleven Nrf2 target genes was analyzed by WISH. Embryos were injected with or without nrf2MO and treated with or without 100 mM DEM for six hours (three hours only for hmox1a) at 5 dpf. Lateral views. Numbers indicate the induction positive embryos/tested embryos. Red and white arrowheads indicate positive and negative expression, respectively. Asterisks denote basal expression in the intestine. doi:10.1371/journal.pone.0026884.g002 PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26884