Overexpression of Dnajb1a-Prkacaa(zfDnaJa-Pkaca)can induce liver masses at adult stages. (A) Representative maximum-intensity projections (MIPs) of 7 day post-fertilization (dpf) FLC larvae [Tg(fabp10a:dnajb1a-prkacaa)/Tg(fabp10a:egfp-l10a)] and control siblings [Tg(fabp10a:egfp-l10a)]. (B) Chi-square graphs showing percentages of fish with masses (8 months, control N=5, FLC N=8; 12 months, control N=5, FLC N=7). (C) Representative images of H&E staining of the livers of 12-month-old fish at 10× and 40× magnification. Yellow outlines delineate masses, white arrowheads indicate prominent nucleoli and black arrowheads indicate lipid vacuoles. Scale bars: 60 μm (10× magnification) and 10 μm (40× magnification). M, mass; N, normal tissue.

Overexpression ofzfDnaJa-Pkacamodulates liver morphology. (A) Representative MIPs of 7 dpf FLC larvae [Tg(fabp10a:dnajb1a-prkacaa)/Tg(fabp10a:egfp-l10a)] and control siblings [Tg(fabp10a:egfp-l10a)]. Dotted red lines indicate liver area. (B,C) Graphs showing liver area (B) (control N=45, FLC N=53) and liver volume (C) (control N=22, FLC N=21). (D) Representative MIPs of 7 dpf FLC larvae [Tg(fabp10a:dnajb1a-prkacaa)/Tg(fabp10a:mCherry-kras)] and control siblings [Tg(fabp10a:mCherry-kras)]. Dashed red lines indicate hepatocyte area. (E-H) Graphs showing hepatocyte area (E,F) and diameter (G,H). In E and G, each dot represents one hepatocyte (control N=201, FLC N=261); in F and H, each dot represents one larva (control N=10, FLC N=13). Scale bars: 20 μm. Data are from at least three independent experiments. Analysis performed in EMM in R. Dot plots show mean±s.e.m.; P-values are shown on graphs.

Overexpression ofzfDnaJa-Pkacainduces liver inflammation. (A) Representative MIPs of 7 dpf FLC larvae [Tg(fabp10a:dnajb1a-prkacaa)/Tg(fabp10a:egfp-l10a)/Tg(lyzc:bfp)/Tg(mpeg1:mCherry-caax)] and control siblings [Tg(fabp10a:egfp-l10a)/Tg(lyzc:bfp)/Tg(mpeg1:mCherry-caax)]. Dotted red lines indicate liver area. (B,C) Graphs showing macrophage (B) (control N=44, FLC N=50) and neutrophil (C) (control N=40, FLC N=48) density in the liver area. Scale bars: 40 μm. Data are from at least three independent experiments. Analysis performed in EMM in R. Dot plots show mean±s.e.m.; P-values are shown on graphs.

FLC larvae show increased pro-inflammatory macrophages and increased caspase-a activityinthe liver area. (A) Representative MIPs of 7 dpf FLC larvae [Tg(fabp10a:dnajb1a-prkacaa_cryaa;Cerulean)/Tg(tnfa:egfp)/Tg(mpeg1:mCherry-caax)] and control siblings [Tg(tnfa:egfp)/Tg(mpeg1:mCherry-caax)]. Dotted red lines indicate liver area; red arrows indicate Tnfα-positive macrophages. (B) Graph showing ratio of Tnfα-positive macrophages to total macrophages in the liver area (control N=17, FLC N=19). (C) Representative MIPs of 7 dpf FLC larvae [Tg(fabp10a:dnajb1a-prkacaa_cryaa;Cerulean)] and control wild-type siblings. (D) Graph showing mean intensity fluorescent quantification in the liver area (control N=7, FLC N=16). Scale bars: 20 μm. Data are at least two independent experiments. Analysis performed in EMM in R. Dot plots show mean±s.e.m.; P-values are shown on graphs. a.u.f., arbitrary units of fluorescence.

Pharmacological inhibition of TNFα secretion and Caspase-a activity reduces inflammation and FLC progression. (A) Representative MIPs of 7 dpf FLC larvae [Tg(fabp10a:dnajb1a-prkacaa)/Tg(fabp10a:egfp-l10a)/Tg(lyzc:bfp)/Tg(mpeg1:mCherry-caax)] and control siblings [Tg(fabp10a:egfp-l10a)/Tg(lyzc:bfp)/Tg(mpeg1:mCherry-caax)] treated with 50 μM pentoxifylline (PTX), 50 μM metformin (MET) and 100 μM Ac-YVAD-CMK (C1INH). (B-D) Graphs showing liver area (B) (control N=32, PTX N=42, MET N=21, C1NH N=46), macrophage density (C) (control N=29, PTX N=42, MET N=19, C1NH N=42) and neutrophil density (D) (control N=32, PTX N=42, MET N=21, C1NH N=46). Scale bars: 40 μm. Data are from at least three independent experiments. Analysis performed in EMM in R. Dot plots show mean±s.e.m.; P-values are shown on graphs.