bTranscription factor Nrf2 is considered a master regulator of antioxidant defense in mammals. However, it is unclear whether this concept is applicable to nonmammalian vertebrates, because no animal model other than Nrf2 knockout mice has been generated to examine the effects of Nrf2 deficiency. Here, we characterized a recessive loss-of-function mutant of Nrf2 (nrf2 fh318 ) in a lower vertebrate, the zebrafish (Danio rerio). In keeping with the findings in the mouse model, nrf2 fh318 mutants exhibited reduced induction of the Nrf2 target genes in response to oxidative stress and electrophiles but were viable and fertile, and their embryos developed normally. The nrf2 fh318 larvae displayed enhanced sensitivity to oxidative stress and electrophiles, especially peroxides, and pretreatment with an Nrf2-activating compound, sulforaphane, decreased peroxide-induced lethality in the wild type but not nrf2 fh318 mutants, indicating that resistance to oxidative stress is highly dependent on Nrf2 functions. These results reveal an evolutionarily conserved role of vertebrate Nrf2 in protection against oxidative stress. Interestingly, there were no significant differences between wild-type and nrf2 fh318 larvae with regard to their sensitivity to superoxide and singlet oxygen generators, suggesting that the importance of Nrf2 in oxidative stress protection varies based on the type of reactive oxygen species (ROS). Oxidative stress causes damage to multiple cellular components, such as DNA, proteins, and lipids, and is implicated in various human pathological conditions, including cancer, neurodegeneration, and inflammatory diseases (37). Several proteins such as superoxide dismutase (SOD), catalase, glutathione peroxidase (Gpx), peroxiredoxin (Prdx), and the small thiol molecules glutathione (GSH) and thioredoxin (Txn), are directly involved in the removal of oxidative stress. Recent discoveries in the cellular antioxidant system gave rise to the novel concept of "indirect antioxidants," which act through the augmentation of cellular antioxidant capacity by enhancing the gene expression driven by the transcription factor Nrf2 (21, 22). Nrf2 is a basic-region leucine zipper (bZIP) transcription factor that heterodimerizes with small Maf proteins and binds to the antioxidant response element (ARE) within the regulatory region of its target genes (20,27). A variety of cytoprotective genes that encode phase 2 detoxifying enzymes and antioxidant proteins, such as glutathione S-transferases (GST), NAD(P)H:quinone oxidoreductase, and glutamate-cysteine ligase, are induced by Nrf2 via ARE sequences (14). Under basal conditions, Nrf2 is constantly degraded through the ubiquitin-proteasome pathway in a Keap1-dependent manner (28, 50). Upon exposure to electrophiles or oxidative stress, Nrf2 escapes from proteasomal degradation, accumulates in the nucleus, and transcriptionally activates its target genes.We previously isolated zebrafish homologs of Nrf2 and its regulator Keap1 genes (nrf2, keap1a, and keap1b) and demonstrated that ...

The Keap1-Nrf2 system serves as a defense mechanism against oxidative stress and electrophilic toxicants by inducing more than one hundred cytoprotective proteins, including antioxidants and phase 2 detoxifying enzymes. Since induction profiles of Nrf2 target genes have been studied exclusively in cultured cells, and not in animal models, their tissue-specificity has not been well characterized. In this paper, we examined and compared the tissue-specific expression of several Nrf2 target genes in zebrafish larvae by whole-mount in situ hybridization (WISH). Seven zebrafish genes (gstp1, mgst3b, prdx1, frrs1c, fthl, gclc and hmox1a) suitable for WISH analysis were selected from candidates for Nrf2 targets identified by microarray analysis. Tissue-restricted induction was observed in the nose, gill, and/or liver for all seven genes in response to Nrf2-activating compounds, diethylmaleate (DEM) and sulforaphane. The Nrf2 gene itself was dominantly expressed in these three tissues, implying that tissue-restricted induction of Nrf2 target genes is defined by tissue-specific expression of Nrf2. Interestingly, the induction of frrs1c and gclc in liver and nose, respectively, was quite low and that of hmox1a was restricted in the liver. These results indicate the existence of gene-specific variations in the tissue specificity, which can be controlled by factors other than Nrf2.

Nrf2 plays critical roles in animals' defense against electrophiles and oxidative stress by orchestrating the induction of cytoprotective genes. We previously isolated the zebrafish mutant , which displays up-regulated expression of Nrf2 target genes in an uninduced state. In this paper, we determine that the gene responsible for was the zebrafish homolog of phosphomannomutase 2 (Pmm2), which is a key enzyme in the initial steps of N-glycosylation, and its mutation in humans leads to PMM2-CDG (congenital disorders of glycosylation), the most frequent type of CDG. The larvae exhibited mild defects in N-glycosylation, indicating that the mutation is a hypomorph, as in human PMM2-CDG patients. A gene expression analysis showed that larvae display up-regulation of endoplasmic reticulum (ER) stress, suggesting that the activation of Nrf2 was induced by the ER stress. Indeed, the treatment with the ER stress-inducing compounds up-regulated the expression in an Nrf2-dependent manner. Furthermore, the up-regulation of by the inactivation was diminished by knocking down or out double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK), one of the main ER stress sensors, suggesting that Nrf2 was activated in response to the ER stress via the PERK pathway. ER stress-induced activation of Nrf2 was reported previously, but the results have been controversial. Our present study clearly demonstrated that ER stress can indeed activate Nrf2 and this regulation is evolutionarily conserved among vertebrates. Moreover, ER stress induced in mutants was ameliorated by the treatment of the Nrf2-activator sulforaphane, indicating that Nrf2 plays significant roles in the reduction of ER stress.

Tail resorption during amphibian metamorphosis has been thought to be controlled mainly by a cell-autonomous mechanism of programmed cell death triggered by thyroid hormone. However, we have proposed a role for the immune response in metamorphosis, based on the finding that syngeneic grafts of tadpole tail skin into adult Xenopus animals are rejected by T cells. To test this, we identified two tail antigen genes called ouro1 and ouro2 that encode keratin-related proteins. Recombinant Ouro1 and Ouro2 proteins generated proliferative responses in vitro in T cells isolated from naive adult Xenopus animals. These genes were expressed specifically in the tail skin at the climax of metamorphosis. Overexpression of ouro1 and ouro2 induced T-cell accumulation and precocious tail degeneration after full differentiation of adulttype T cells when overexpressed in the tail region. When the expression of ouro1 and ouro2 were knocked down, tail skin tissue remained even after metamorphosis was complete. Our findings indicate that Ouro proteins participate in the process of tail regression as immune antigens and highlight the possibility that the acquired immune system contributes not only to self-defense but also to remodeling processes in vertebrate morphogenesis.amphibian ͉ skin ͉ cell death ͉ T cell ͉ remodeling

Some nanoparticles (NPs) may induce adverse health effects in exposed organisms, but to date the evidence for this in wildlife is very limited. Silver nanoparticles (AgNPs) can be toxic to aquatic organisms, including fish, at concentrations relevant for some environmental exposures. We applied whole mount in-situ hybridisation (WISH) in zebrafish embryos and larvae for a suite of genes involved with detoxifying processes and oxidative stress, including metallothionein (mt2), glutathionine S-transferase pi (gstp), glutathionine S-transferase mu (gstm1), haem oxygenase (hmox1) and ferritin heavy chain 1 (fth1) to identify potential target tissues and effect mechanisms of AgNPs compared with a bulk counterpart and ionic silver (AgNO3). AgNPs caused upregulation in the expression of mt2, gstp and gstm1 and down regulation of expression of both hmox1 and fth1 and there were both life stage and tissue-specific responses. Responding tissues included olfactory bulbs, lateral line neuromasts and ionocytes in the skin with the potential for effects on olfaction, behaviour and maintenance of ion balance. Silver ions induced similar gene responses and affected the same target tissues as AgNPs. AgNPs invoked levels of target gene responses more similar to silver treatments compared to coated AgNPs indicating the responses seen were due to released silver ions. In the Nrf2 zebrafish mutant, expression of mt2 (24 hpf) and gstp (3 dpf) were either non-detectable or were at lower levels compared with wild type zebrafish for exposures to AgNPs, indicating that these gene responses are controlled through the Nrf2-Keap pathway.