Zebrafish cryab transcripts are regulated in a tissue-dependent manner. (A) Relative expression of cryaba and cryabb genes was compared between WT and nrf2^fh318/fh318 embryos at 4 dpf using qRT-PCR analysis. Data are expressed as mean ± SD from five independent measurements. The relative mRNA levels of cryaba(B) and cryabb(C) were measured between WT and nrf2^fh318/fh318 by qRT-PCR in the eyes, lens, heart, and brain tissues of 10-month-old zebrafish. Data are expressed as mean ± SD from at least n = 3 for brain, eye, lens, and heart. p-values were calculated using two-tailed t-test.

Nrf2 deficiency rescues lens defects in cryaba^−/− but not in cryabb^−/−. (A) Representative images of lens defects in zebrafish embryos at 4 day post fertilization (dpf). [(B), left panel] Percentage of embryos showing lens defects for WT, nrf2^fh318/fh318, cryaba^−/−, and cryaba^−/−; nrf2^fh318/fh318 was compared. [(B), right panel] To confirm the previous results, embryos from cryaba^+/−; nrf2^fh318/fh318 incross were collected, and the lens defect of each embryo was measured in a genotype-blinded at 4 dpf. Then, cryaba genotyping of individual embryos was determined to compare the percentage of lens defects between nrf2^fh318/fh318 and cryaba^−/−; nrf2^fh318/fh318. [(C), left panel] Percentage of lens defect between cryabb^−/− and cryabb^−/−; nrf2^fh318/fh318 embryos. [(C), right panel] Embryos from cryabb^−/−; nrf2^fh318/+ incross were collected, and lens abnormalities were examined before nrf2 genotyping. Then, the percentage of lens defects between cryabb^−/− and cryabb^−/−; nrf2^fh318/fh318 was analyzed. Data are expressed as mean ± SD from at least three independent experiments. n numbers indicate the total number of embryos across the independent experiments. p-values were calculated using one-way ANOVA and two-tailed t-test.

Cholesterol biosynthesis pathway is elevated in the lens of cryaba^−/−; nrf2^fh318/fh318(A) Lens RNA-seq data were analyzed through ingenuity pathway analysis (www.ingenuity.com). Orange bars that cross the threshold line (p < 0.05) indicate upregulated pathways in the lens of cryaba^−/−; nrf2^fh318/fh318 when compared to WT. (B) Heatmap of enriched genes in the superpathway of cholesterol biosynthesis. Z-scores were calculated for each gene, and these were plotted instead of the normalized expression values. (C) Bar charts represent the normalized count of each transcript in the cholesterol biosynthesis pathway in the lens tissues of WT, nrf2^fh318/fh318, cryaba^−/−, and cryaba^−/−; nrf2^fh318/fh318. *Indicates false discovery rate (FDR) < 0.05.

Increased penetrance of lens defects in cryaba^−/−; nrf2^fh318/fh318 in response to treatment of statins. (A) Zebrafish embryos were treated with either vehicle (1% DMSO) or 5 μM atorvastatin from 36 h post fertilization (hpf) until 4 dpf. Percentage of embryos showing lens defects between WT and cryaba^−/−; nrf2^fh318/fh318 were compared at 4 dpf. (B) Size of the lens of WT, cryaba^−/−, and cryaba^−/−; nrf2^fh318/fh318 embryos was measured in the presence and absence of 5 μM atorvastatin treatment. (C) WT and cryaba^−/−; nrf2^fh318/fh318 embryos were treated with 4 μM lovastatin for 16 h before examining lens abnormalities at 4 dpf. Data are expressed as mean ± SD from three independent experiments. n numbers indicate the total number of embryos across the independent experiments. Statistical significance was calculated using two-way ANOVA.

Nrf2 deficiency aggravates the heart edema phenotype of cryaba^−/− fish in response to dexamethasone (Dex). The heart area of nrf2^fh318/fh318 embryos was measured following treatment with 50 μM Dex and compared to (A) the WT embryos, (B)cryaba^−/− and cryaba^−/−; nrf2^fh318/fh318, and (C)cryabb^−/− and cryabb^−/−; nrf2^fh318/fh318 following the same treatment regimen. (D) Representative images of cardiac phenotypes in WT, cryaba^−/− and cryaba^−/−; nrf2^fh318/fh318 treated with either vehicle (DMSO) or Dex. (E) Relative changes in cryabb mRNA in 4 dpf embryos as measured by qRT-PCR. Data are expressed as mean ± SD obtained from three independent experiments. n numbers indicate the total number of embryos across the independent experiments. Statistical significance was calculated using two-way ANOVA.

Transcriptome analysis reveals changes in genes belonging to the extracellular region in the heart of cryaba^−/−; nrf2^fh318/fh318. The top-ranked significant GO clusters in heart tissues between (A) WT versus cryaba^−/−; nrf2^fh318/fh318 and (B)cryaba^−/− versus cryaba^−/−;nrf2^fh318/fh318. Heatmaps illustrate the transcripts in (C) the extracellular region GO term and (D) tight junction GO term. (E) Top terms in the Disease Ontology (DO) enrichment analysis highlight the number of genes enriched in each term (x-axis). The adjusted p-value of each term is indicated by color according to the legend. (F) The enriched genes in the cardiomyopathy DO cluster are illustrated by a heatmap. (G) Interaction between human homologs of transcripts in the cardiomyopathy DO term was assessed using STRING tool (Szklarczyk et al., 2021).