Tissue-Restricted Induction of Nrf2 Target Genes Figure 4. Induction time profiles of Nrf2 target genes. 5-dpf larvae were treated with 100 mM DEM for indicated hours and expression of seven genes was analyzed by WISH. Lateral views. Red and white arrowheads indicate positive and negative expression, respectively, of each gene in the nose, gill and liver. Asterisks denote the basal expression in the intestine. doi:10.1371/journal.pone.0026884.g004 PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26884 Interestingly, hmox1a was only induced in the liver, suggesting the existence of gene-specific differences in tissue specificity. Expres- sion profiles of hmox1a and frrs1c in the liver were confirmed by section analysis in comparison with a liver-specific marker fabp10 (Figure S11) [26]. These results indicate that tissue-restricted induction is a common characteristic among Nrf2 target genes, not only for gstp1. The induction time profiles of seven genes were also analyzed in detail (Figure 4). All genes showed induction beginning three hours after DEM treatment. Interestingly, expression of hmox1a was rapidly reduced beginning six hours after DEM treatment to almost the same level as the non-induced condition, whereas expression levels of the remaining six genes were maintained until twelve hours after treatment. A negative feedback regulation may exist in the case of hmox1a. To determine whether tissue specificity and induction time profiles of Nrf2 target genes vary according to differences of Nrf2- activating compounds, expression profiles of seven genes by sulforaphane were analyzed (Figures S12). Induction profiles of all seven genes were basically identical to those in the case of DEM, suggesting that tissue-restricted induction of Nrf2 target genes is intrinsic properties of each gene. Gene regulation by Nrf2 Considering that all genes tested showed restricted-expression in the nose, gill, liver, and intestine, gene expression in these four tissues seemed to be a default state for Nrf2 target genes. This may suggest that Nrf2 or its activator dominantly exists in these tissues and directly transactivates the target genes. To test this hypothesis, Figure 5. Tissue-specific expression of the Nrf2 gene. Expression of Nrf2 gene in 24-hpf embryos and 5-dpf larvae were analyzed by WISH. Lateral (upper left, lower left), dorsal (upper right), and ventral (lower right) views. Arrowheads in light blue, red and orange indicate expression in the nose, gill and liver. doi:10.1371/journal.pone.0026884.g005