Development of a transgenic zebrafish model for the identification of hepatocyte-specific nascent gene expression (A) Schematic of larval SLAM-ITseq workflow. (B) Tg(lfabp:UPRT-T2A-mCherry) construct. (C) Fluorescent validation of hepatocyte-specific mCherry expression in 5 dpf zebrafish larvae. Yellow dashed line borders the mCherry+ liver. Scale bars, 50 ?m. (D) Percentage of T>C counts for hepatocyte-enriched nascent transcripts (fabp10a, apoa2, and fgb) in UPRT+ and UPRT? zebrafish labeled with 4TU for 6 h. p values were calculated using an unpaired two-tailed Student?s t test comparing each sample to the respective UPRT? control. (E) Hockey plot of nascent differentially expressed genes (DEGs) ranked by log-fold change comparing UPRT+ and UPRT? zebrafish labeled with 4TU for 6 h. Red genes indicate hepatocyte-enriched transcripts and blue genes indicate cholangiocyte-enriched transcripts obtained from the Human Protein Atlas (proteinatlas.org ). (F) Representative screenshot of the Integrative Genomics Viewer (IGV) genome browser within the 3? UTR locus of fgb derived from 3? RNA SLAM-ITseq. Blue asterisks indicate T>C conversions. See also Figure S1.

SLAM-ITseq reveals temporal dynamics of nascent gene expression in hepatocytes following liver injury (A) Representative multiphoton images of hepatocyte apoptosis (annexin V+ hepatocytes) in 5 dpf zebrafish livers treated with DMSO or APAP for 24 h. Cyan dashed line borders the zebrafish liver. White arrows represent apoptotic hepatocytes. Scale bars, 50 ?m. (B) Quantification of hepatocyte apoptosis in 5 dpf zebrafish livers treated with DMSO or APAP for 24 h. Data are shown as median and interquartile range, n = 7. p value was calculated using an unpaired two-tailed Student?s t test comparing each sample to the respective DMSO control. (C) Workflow for performing SLAM-ITseq in the larval zebrafish liver injury model. 4 dpf larvae were simultaneously treated with DMSO or APAP in the presence of 4TU for 1, 3, and 6 h. (D) Venn diagram depicting the overlap of total and nascent genes that were differentially upregulated in zebrafish larvae treated with APAP for 6 h. (E and F) Volcano plot (E) and HOMER motif analysis of genes (F) from SLAM-ITseq of zebrafish larvae treated with DMSO or APAP for 1 h. Genes within red area of volcano plots are significantly upregulated (log2FC > 0.6, p < 0.05), and genes within blue area of volcano plots are significantly downregulated (log2FC < 0.6, p < 0.05). Green symbols represent Nrf2 target genes, whereas purple symbols represent FOXO1 target genes. (G and H) Volcano plot (G) and HOMER motif analysis of genes (H) from SLAM-ITseq of zebrafish larvae treated with DMSO or APAP for 3 h. Genes within red area of volcano plots are significantly upregulated (log2FC > 0.6, p < 0.05), and genes within blue area of volcano plots are significantly downregulated (log2FC < 0.6, p < 0.05). Green symbols represent Nrf2 target genes, whereas purple symbols represent FOXO1 target genes. (I and J) Volcano plot (I) and HOMER motif analysis of genes (J) from SLAM-ITseq of zebrafish larvae treated with DMSO or APAP for 6 h. Genes within red area of volcano plots are significantly upregulated (log2FC > 0.6, p < 0.05), and genes within blue area of volcano plots are significantly downregulated (log2FC < 0.6, p < 0.05). Green symbols represent Nrf2 target genes, whereas purple symbols represent FOXO1 target genes. See also Figure S2 and Table S1.

Nrf2 is activated in hepatocytes following liver injury and is required for survival (A) Schematic of the compound transgenic line Tg(lfabp:NLS-mCherry; gstp1:EGFP) used to visualize hepatocyte nuclei and Nrf2 activity. (B) Representative multiphoton images of 5 dpf zebrafish treated with DMSO or APAP for 24 h. Cyan dashed line borders the liver. Scale bars, 50 ?M. (C) Quantification of Nrf2-activated hepatocytes (% GFP+ and mCherry+ cells) acquired from multiphoton imaging of 5 dpf zebrafish treated with DMSO or APAP for 24 h. p value was calculated using an unpaired two-tailed Student?s t test comparing each sample to the respective DMSO control, n = 4. (D) Quantification of Nrf2-activated hepatocytes (% GFP+ and mCherry+ events) as determined by flow cytometry of 5 dpf zebrafish treated with DMSO or APAP for 24 h. p value was calculated using an unpaired two-tailed Student?s t test comparing each sample to the respective DMSO control, n = 3. (E and F) Volcano plots of differentially expressed genes identified by comparing dissected livers of 5 dpf zebrafish treated with DMSO or APAP for 12 (E) and 24 h (F) by RNA-seq analysis. Green points represent Nrf2 target genes. Genes within red area of volcano plots are significantly upregulated (log2FC > 0.6, p < 0.05), and genes within blue area of volcano plots are significantly downregulated (log2FC < 0.6, p < 0.05). (G) Survival data of WT and Nrf2KO zebrafish (n = 120) treated with DMSO or APAP for 72 h from 4 dpf. p value was calculated using a Mantel-Cox test comparing the survival of Nrf2KO APAP to WT APAP. Data are shown as mean ± SEM. See also Figure S3.

Nrf2-dependent activation of the PPP is evolutionarily conserved and required to stimulate regeneration (A) Volcano plot of differentially expressed genes and Nrf2 target genes (green) identified by comparing WT and Nrf2KO animals treated with APAP at 4 dpf for 24 h. Genes within red area of volcano plots are significantly upregulated (log2FC > 0.6, p < 0.05), and genes within blue area of volcano plots are significantly downregulated (log2FC < 0.6, p < 0.05). (B) Heatmap depicting the differentially expressed genes in the pentose phosphate pathway following 24 h of DMSO or APAP treatment in WT and Nrf2KO zebrafish. (C) ChIP-qPCR analysis of NRF2 binding to the PRDX1, G6PD, and chromosome 2 (chr 2) gene desert loci in untreated HepG2 cells and HepG2 cells treated with 10 mM APAP for 6 or 24 h, n = 3. p values were calculated using an unpaired two-tailed Student?s t test comparing each sample to the respective untreated control. Data are shown as mean ± SEM. (D) Survival of WT zebrafish exposed to DMSO, APAP, G6PDi-1, or APAP in the presence of G6PDi-1 for 72 h from 4 dpf. p value was calculated using a Mantel-Cox test comparing the survival of APAP+G6PDi-1 to APAP alone. Data are shown as mean ± SEM. (E) Representative multiphoton images of hepatocytes in G1 phase (magenta) and S/G2-M phase (cyan) in livers of 4 dpf lf:FastFUCCI animals treated with DMSO, APAP, G6PDi-1, or APAP in the presence of G6PDi-1 for 24 h. (F) Quantification of mitotic hepatocytes (% S/G2-M) derived from lf:fastFUCCI analysis. p value was calculated using an unpaired two-tailed Student?s t test comparing each sample to its respective control. Data are shown as median and interquartile range, n = 15. (G) Representative images of BrdU incorporation (yellow) in hepatocytes (magenta) derived from lf:NLS-mCherry zebrafish exposed to DMSO, APAP, G6PDi-1, or APAP in the presence of G6PDi-1 for 24 h. (H) Quantification of BrdU incorporation (% BrdU+ hepatocytes) in livers of WT zebrafish following 24 h exposure to DMSO, APAP, G6PDi-1, or APAP in the presence of G6PDi-1. p value was calculated using an unpaired two-tailed Student's t test. Data are shown as median and interquartile range, n = 15. See also Figure S4.

Nucleoside supplementation rescues defects in liver regeneration and survival that are observed in Nrf2KO zebrafish (A) Representative multiphoton images of BrdU incorporation (yellow) in hepatocytes (magenta) derived from WT zebrafish exposed to DMSO, APAP, APAP + N-acetyl cysteine (NAC), or APAP + nucleoside (NS) cocktail (deoxyadenosine [dA], deoxyguanosine [dG], deoxycytidine [dC], and thymidine [dT]). Scale bars, 50 ?M. (B) Quantification of BrdU incorporation into hepatocytes (% mCherry+/BrdU+ cells) in WT zebrafish during liver regeneration following exposure to DMSO, APAP, APAP + NAC, or APAP + NS. p value was calculated using an unpaired two-tailed Student's t test. Data are shown as median and interquartile range, n = 15. (C) Representative multiphoton images of BrdU incorporation (yellow) in hepatocytes (magenta) derived from Nrf2KO zebrafish exposed to DMSO, APAP, APAP + NAC, or APAP + NS. Scale bars, 50 ?M. (D) Quantification of BrdU incorporation into hepatocytes (% mCherry+/BrdU+ cells) in Nrf2KO zebrafish during liver regeneration following exposure to DMSO, APAP, APAP + NAC, or APAP + NS. p value was calculated using an unpaired two-tailed Student's t test. Data are shown as median and interquartile range, n = 15. (E) Survival of WT zebrafish following exposure to DMSO, APAP, APAP + NAC, or APAP + NS for 72 h from 4 dpf. p values were calculated using a Mantel-Cox test comparing the survival of APAP + NS to APAP alone. Data are shown as mean ± SEM, n = 80. (F) Survival of Nrf2KO zebrafish following exposure to DMSO, APAP, APAP + NAC, or APAP + NS for 72 h from 4 dpf. p values were calculated using a Mantel-Cox test comparing survival of APAP + NS to APAP alone. Data are shown as mean ± SEM, n = 80. (G) Summary illustrating the temporal regulation of metabolic reprogramming during liver regeneration.