CRISPR-Generated Nrf2a Loss- and Gain-of-Function Mutants Facilitate Mechanistic Analysis of Chemical Oxidative Stress-Mediated Toxicity in Zebrafish

Abstract

The transcription factor Nrf2a induces a cellular antioxidant response and provides protection against chemical-induced oxidative stress, as well as playing a critical role in development and disease. Zebrafish are a powerful model to study the role of Nrf2a in these processes but have been limited by reliance on transient gene knockdown techniques or mutants with only partial functional alteration. We developed several lines of zebrafish carrying different null (loss of function, LOF) or hyperactive (gain of function, GOF) mutations to facilitate our understanding of the Nrf2a pathway in protecting against oxidative stress. The mutants confirmed Nrf2a dependence for induction of the antioxidant genes gclc, gstp, prdx1, and gpx1a and identified a role for Nrf2a in the baseline expression of these genes, as well as for sod1. Specifically, the 4-fold induction of gstp by tert-butyl hydroperoxide (tBHP) in wild type fish was abolished in LOF mutants. In addition, baseline gstp expression in GOF mutants increased by 12.6-fold and in LOF mutants was 0.8-fold relative to wild type. Nrf2a LOF mutants showed increased sensitivity to the acute toxicity of cumene hydroperoxide (CHP) and tBHP throughout the first 4 days of development. Conversely, GOF mutants were less sensitive to CHP toxicity during the first 4 days of development and were protected against the toxicity of both hydroperoxides after 4 dpf. Neither gain nor loss of Nrf2a modulated the toxicity of R-(-)-carvone (CAR), despite the ability of this compound to potently induce Nrf2a-dependent antioxidant genes. Similar to other species, GOF zebrafish mutants exhibited significant growth and survival defects. In summary, these new genetic tools can be used to facilitate the identification of downstream gene targets of Nrf2a, better define the role of Nrf2a in the toxicity of environmental chemicals, and further the study of diseases involving altered Nrf2a function.

Figure 1.

Independently isolated nfe2l2a mutant alleles encode null (LOF) or hyperactive (GOF) proteins. (a) Schematic of the wild type (WT) Nrf2a protein showing all canonical Neh domains (light gray, numbers 1–7) and keap1 contact sites in Neh 2 (black arrowheads above); schematics of the proteins encoded by each mutant allele showing deletion sites (white arrowheads below) and remaining portions of the WT protein (see Figure S2 for complete sequences). (b) Expression of the known Nrf2a-dependent gene gstp confirms effect of nfe2l2a mutation state on Nrf2a protein function: gstp is induced in WT larvae exposed to tBHP from 5 hpf to 96 hpf with daily renewal; baseline expression of gstp is altered in nfe2l2a mutants (lower in putative LOF mutants and higher in putative GOF mutants) and exposure to tBHP has no effect on these altered levels of expression. Unexposed WT expression is set to 1.0 (red dashed line). All expression data represent the mean ± SEM (n = 5 biological replicates, 5 larvae per replicate for WT and LOF alleles, 1–3 larvae per replicate for GOF alleles). Asterisks indicate significantly different expression (p < 0.001) relative to WT control.

Figure 2.

Altered Nrf2a function correlates with significant differences in expression of some, but not all, oxidative stress response genes. (a) Baseline expression of homozygous LOF (nfe2l2a^w211) and homozygous GOF (nfe2l2a^dw213) relative to wild type (WT) larvae. (b) Expression of LOF, GOF, and WT following exposure to the same relative concentration (20% LC₅₀) of three gstp-inducing compounds from 5 hpf to 96 hpf with daily renewal, relative to untreated larvae of the same genotype. Control expression in all cases is set to 1.0. All data represent the mean ± SEM (n = 5 biological replicates). Asterisks indicate conditions that result in significantly different expression (p < 0.05) from WT (a) or untreated (b) controls. See Table S1 for detailed–fold change and statistical significance values.

Figure 3.

Mutation state of nfe2l2a correlates with chemical toxicity in larvae exposed to CHP and tBHP, but not those exposed to CAR. (a) LC₅₀ exposures between 5 hpf and 96 hpf with daily renewal. Symbols show survival of larvae after exposure to varying concentrations of each chemical; lines show calculated toxicity curves; arrows and numbers indicate calculated LC₅₀ values ± SEM (mg/L). (b) Survival of larvae in exposures starting at 4 dpf to CHP (for 48 h) and tBHP (for 24 h). In all experiments, genotypes are WT (gray), LOF (nfe2l2a^w211, light gray), and GOF/WT (heterozygous nfe2l2a^dw213, black). For CAR, n = 6 biological replicates, 10 fish per replicate; for CHP and tBHP, n = 3 biological replicates, 10 fish per replicate.