Disruption of the ypel5 gene causes liver enlargement. (A) Genetic inactivation of zebrafish ypel5 gene based on CRISPR/Cas9. Schematic representation of CRISPR/Cas9 target site at exon 2 as used in this study. The gRNA target site is highlighted in green, and the indel is indicated by a red dash. (B) Genotyping of the ypel5^−/− mutant by Sanger sequencing. (C) Western blot analysis of Ypel5 using lysates from wild-type (WT) and ypel5^−/− mutant embryos at 3 dpf. Gapdh was used as the loading control. (D) The survival of zebrafish larvae at different developmental stages. No homozygotes were identified at 10 dpf. (E–H) Brightfield images (E and F), relative liver size (G), and the ratio of the liver to whole body (H) of WT and ypel5^−/− mutant zebrafish at 5 dpf. The livers are outlined with green lines. Data shown are mean ± SEM. Student's t-test. **P < 0.01. (I and J) WISH assay of fabp10a at 8 dpf. Green lines circle the boundary of the liver. The ratio of embryos with the representative expression pattern is indicated at the right bottom. (K and L) Representative images of WT and ypel5^−/−/Tg(fabp10a:dsRed) zabrafish at 7.5 dpf. The liver was marked by red fluorescence.

ypel5 deficiency results in enhanced hepatic cell proliferation. (A–C) Representative images of H&E staining (A and B) and relative hepatocyte size (C) of WT and ypel5^−/− mutant zebrafish at 7.5 dpf. The livers are outlined with green lines. (D–F) FACS analysis (D and E) and quantification (F) of dsRed-positive hepatocytes from WT and Tg(fabp10a:dsRed) larvae at 7.5 dpf. (G–I) Representative images of pH3 staining (green) to label hepatic cell proliferation (G and H) and quantification of pH3-positive liver cells (I) in WT and ypel5^−/− mutant zebrafish at 7.5 dpf. The sections were counterstained with DAPI to label the nucleus (blue). White lines circle the boundary of the liver. Data shown are mean ± SEM. Student's t-test. n.s., not significant; **P < 0.01.

ypel5 depletion leads to impaired hepatic metabolism and function. (A–C) Volcano plot (A) and KEGG enrichment (C) of differential metabolites between control siblings and ypel5^−/− mutants at 7.5 dpf. (B) The 16 upregulated and 22 downregulated metabolites. (D and E) Volcano plot (D) and KEGG enrichment (E) of differentially expressed genes between control siblings and ypel5^−/− mutants at 3 dpf. (F and G) qPCR analysis of genes related to cholesterol metabolism and complement and coagulation cascades. Data shown are mean ± SEM. Student's t-test. *P < 0.05; **P < 0.01; ***P < 0.001.

Hnf4a acts as a crucial downstream effector of Ypel5. (A and B) ChEA3 transcription factor (TF) analysis was performed on differentially expressed genes. The top 10 proteins and their co-regulatory networks are shown. (C and D) Western blot analysis of HNF4A in Huh-7 cells. (E) qPCR analysis of hnf4a expression in control siblings and ypel5^−/− mutants at 3 dpf. (F and G) WISH assay of fabp10a (F) and relative liver size (G) of zebrafish larvae at 7.5 dpf. Green lines circle the boundary of the liver. (H) qPCR analysis of genes related to cholesterol metabolism and complement and coagulation cascades. Data shown are mean ± SEM. Student's t-test. **P < 0.01; ***P < 0.001.

PPARα signaling mediates the regulation of Hnf4a by Ypel5. (A) GSEA showing the enrichment of the PPAR signaling pathway in ypel5^−/− mutants. (B) Genomic browser view showing ChIP–seq signals of PPARα (GSM864671, GSM1163175) and H3K27ac (GSM2540257) at the Hnf4a gene locus in hepatocytes. The peaks are highlighted in yellow. (C) Luciferase activity assay in Huh-7 cells transfected with control vectors or PPARα and reporter constructs. The Renilla plasmid was used as an internal control. (D) Western blot analysis of Huh-7 cells treated for 48 h with control vehicle or GW6471. (E) qPCR analysis of hnf4a expression between control siblings and ypel5^−/− mutants treated for 48 h with bezafibrate. Data shown are mean ± SEM. Student's t-test. n.s., not significant; ***P < 0.001.