Figure 6. Target gene induction in zebrafish embryos by Nrf2 overexpression. RT-PCR analysis using total RNA from the whole bodies of 30 embryos and specific primers of indicated genes. doi:10.1371/journal.pone.0026884.g006 we analyzed the expression of nrf2 in 5-dpf larvae by WISH (Figure 5). As expected, nrf2 is specifically expressed in the nose, gill, liver, and intestine, suggesting that tissue-restricted induction of Nrf2 target genes is based on the tissue-specific expression of nrf2 mRNA. Tissue-Restricted Induction of Nrf2 Target Genes We previously demonstrated that the expression of gstp1 and gclc (cgcsh) to be strongly induced when Nrf2 is overexpressed in zebrafish embryos [9]. We next investigated whether the other five Nrf2 targets can also be induced by Nrf2 overexpression. Nrf2 was overexpressed by injecting nrf2 mRNA into one-cell- stage embryos, and the expression of Nrf2 target genes was analyzed by RT-PCR seven hours after injection (Figure 6). The results indicate that all seven genes were induced by Nrf2 overexpression. Using the zebrafish genome database (http:// www.ensembl.org/Danio_rerio/Info/Index), we searched the ARE sequences in the 5-kb upstream regions of the deduced transcription initiation sites in these genes (Figure 7). All genes were found to have more than three ARE sites in the 5-kb regions, to which Nrf2 may bind and regulate. All of these results suggest that induction of these seven genes is directly regulated by Nrf2. Discussion The zebrafish is a good system to observe the tissue-specific expression of many genes, including gene induction, as described in this study. Indeed, tissue-specific induction in systems other than Keap1-Nrf2, such as the heat-shock genes, has been reported from studies in zebrafish [27,28]. These observations would be difficult to analyze using cultured cell lines, demonstrating a significant advantage for zebrafish system. Zebrafish is also good for drug toxicity screening and testing environmental toxicants [29,30]. Since Nrf2 is activated by a variety of toxic compounds and oxidative stress, it would be worthwhile for these screens and tests to analyze tissue-restricted induction of Nrf2 target genes. The seven target genes of Nrf2 selected in this paper would be useful for such studies. Figure 7. ARE sequences in upstream regions of Nrf2 target genes. ARE sequences (TGAG/CNNNGC) and AP1-type ARE (like) (TGAG/CTCAGN or TGAG/CTCANC) in the 5-kb region of deduced transcription initiation sites of indicated genes were searched using the zebrafish genome database. doi:10.1371/journal.pone.0026884.g007 PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26884