Deficiency of COPII-mediated trafficking in sec31anju221 mutants leads to the development of hepatic steatosis. (A) GFP expression pattern in the zebrafish trapping line NT-1254 at the indicated stages. Lateral views, head to left. I, intestine; L, liver; O, otic capsule. Images are representative of eight fish at each stage. Scale bars: 200 μm. (B) Upper panel: zebrafish sec31a genomic locus. The transposon is inserted into the 21st intron. Lower panel: schematic representations of the domain structure of the zebrafish Sec31a protein and the fusion protein in NT-1254 fish. (C) Live-cell imaging of shield stage zebrafish embryos injected with the trans-Golgi network reporter GalT-BFP (blue) and the plasma membrane reporter mYFP (magenta). Scale bars: 20 μm. (D) Quantification of GalT-BFP and mYFP fluorescence intensity (n=6). a.u., arbitrary units. ***P<0.001; ****P<0.0001 (unpaired two-tailed Student's t-test). (E) Left: bright-field images depicting the liver of live zebrafish larvae. Middle: Hematoxylin and Eosin (H&E) staining of liver from 7 dpf zebrafish larvae. Right: Oil Red O staining of 7 dpf zebrafish larvae. For bright-field and ORO staining images, livers are outlined with dashed lines. Scale bars: 100 μm (bright-field images); 20 μm (H&E and Oil Red O images). Images are representative of 20 fish within each group. (F) Quantitative PCR analysis of RNA samples extracted from livers of 7 dpf larvae. Data are mean±s.e.m. **P<0.01 (unpaired two-tailed Student's t-test).

Epistasis analysis indicates that the ATF4 pathway serves as the main mediator for liver steatosis in the sec31anju221 background. (A) Oil Red O (ORO) staining of 7 dpf zebrafish larvae. Livers are outlined with dashed lines. Scale bar: 100 μm. (B) Quantification of ORO staining in livers as shown in A. Each data point represents an individual animal with bars representing mean±s.e.m. Comparison between groups was undertaken using one-way ANOVA with Tukey's multiple comparisons test. For A,B, n=6 for the sec31anju221; atf4a/b DKO group, and n=12 for all other groups. a.u., arbitrary units. ns, not significant; *P<0.05; ****P<0.0001. (C) Gene set enrichment analysis (GSEA) of RNA-seq expression values from larvae livers showing distinct separation of different genotypes. NES, normalized enrichment scores. Statistical analysis was performed using an empirical phenotype-based permutation test embedded in GSEA. (D) Heatmap showing the expression levels of genes involved in lipogenesis and lipid droplet formation in livers from sec31anju221 and sec31anju221; atf4a/b DKO larvae. FC, fold change.

Adult sec31anju221 escapers display overt glycogenic hepatopathy. (A) Representative Kaplan–Meier plot for sec31anju221 fish and clutchmates from one of three independent experiments. n=80 for each group; P<0.0001 (Mantel–Cox test). (B) Live images of control and sec31anju221 zebrafish at 60 dpf. The livers are outlined with dashed lines. Lateral view, anterior to the left. Scale bars: 1 mm (left); 1 mm (right). (C) Ratio of the liver to body mass from 60 dpf sec31anju221 fish and clutchmates. n=11 for each genotype. (D) Representative photographs of H&E and ORO staining. Arrowheads indicate oil droplets. Scale bars: 20 μm. (E) Electron micrographic pictures of liver biopsy from control and sec31anju221 zebrafish. The distribution of intracellular glycogen granules is outlined with dashed lines. ER, endoplasmic reticulum; GG, glycogen granule; M, mitochondria; N, nucleus. The inset shows an enlarged view of the boxed area. Images are representative of three fish per group. Scale bars: 2 μm. (F) Representative photographs of PAS staining of liver and muscle sections. Images are representative of six fish per group. Scale bar: 50 μm. (G) Triglyceride (n=8), cholesterol (n=6) and glycogen (n=8) levels in fish livers were measured with enzymatic colorimetric assays. Each data point represents an individual animal with bars representing mean±s.e.m. Comparison between groups was performed using unpaired two-tailed Student's t-test. *P<0.05; **P<0.01; ****P<0.0001.

Proteomics analysis reveal that thyroid hormone signaling is dampened in sec31anju221 livers. (A) Principal component analysis (PCA) shows that the mass spectrometric data clearly segregate based on the sample genotype. Three replicates are shown for each genotype. (B) Volcano plot of proteomic data. Fold change in the expression of each protein is shown and the P-value was calculated by performing a unpaired two-tailed Welch's t-test. The horizontal dashed line indicates P=0.05; vertical dashed lines indicate ratios of 0.5 and 2, respectively. Proteins with significant increase in expression levels are shown in magenta, proteins with significant decrease in expression levels are in blue, and proteins with no significant change in expression levels are in gray. Proteins that are decreased downstream targets of thyroid hormone signaling are indicated. (C) Bubble plot showing the enrichment for Gene Ontology terms of differentially expressed proteins in the liver of adult sec31anju221 fish. The size of each dot represents the number of different genes in the corresponding biological process and molecular function term. (D) GSEA plot of thyroid hormone (T3)-regulated genes in the liver of adult sec31anju221 fish versus wild-type sibling. FDR, false discovery rate; NES, normalized enrichment score. (E) Heat map of lipid and glycogen metabolism pathway genes dysregulated in the liver of adult sec31anju221 fish.

The hepatic metabolic defects in sec31anju221 adult fish can be alleviated by thyroid hormone treatment. (A) Left: whole-mount RNA in situ hybridization for thyroglobulin in the thyroid gland of adult fish. Right: representative immunofluorescence of thyroxine (T4, purple) in the thyroid follicle lumen of wild-type and sec31anju221 fish (n=5 zebrafish per group). Thyrocyte identity is confirmed by DAPI counterstaining (white). Scale bars: 500 μm (left); 50 μm (right). (B) Serum thyroxine (T4) concentrations in wild-type and sec31anju221 fish (n=5). Data are mean±s.e.m. (C) Western blot analysis and relative quantification of Dio1 from adult wild-type and sec31anju221 fish liver (n=3). (D) Western blot analysis and relative quantification of Dio1 from adult wild-type, vehicle-treated sec31anju221 and T4-treated sec31anju221 fish liver (n=3). (E) T4, triglyceride and glycogen levels in fish liver were measured with enzymatic colorimetric assays. n=9, 9 and 8 for control, vehicle-treated sec31anju221 and T4-treated sec31anju221 fish, respectively. Each data point represents an individual animal, with bars representing mean±s.e.m. Comparisons between groups in B and C were performed using two-tailed unpaired Student's t-tests, and comparisons between groups in D and E were performed using one-way ANOVA with Tukey's multiple comparisons test. ns, not significant; *P<0.05; **P<0.01; ****P<0.0001. (F) Representative photographs of H&E, ORO and PAS staining of liver biopsy samples. Arrowheads indicate oil droplets. Images are representative of five fish per group. Scale bars: 50 μm.