Treatment with methionine and cysteine can rescue liver development defects in Cnr mutants. (A) Scatter plot of liver morphometric measurements of cnr1-/- and cnr2-/- mutants after 100µM methionine or cysteine versus DMSO exposure from 24-72hpf. Results shown represent one independent experiment of triplicate experiments yielding similar results. Mean±s.e.m.; n>15. One-way ANOVA. ***P<0.001, cnr1-/- methionine versus control; **P<0.01, cnr2-/- methionine versus control; *P<0.05, cnr2-/- cysteine versus control. (B) fabp10a expression at 72hpf in cnr1-/- embryos after DMSO, methionine or cysteine exposure from 24-72hpf. (C) H&E staining of transverse sections of 120hpf larvae shows that treatment of cnr1-/- and cnr2-/- mutants with methionine partially restores WT liver histology. cnr1-/-, 6 abnormal/9; cnr1-/-+methionine, 5 normal/7; P=0.065 (Chi-square). cnr2-/-, 6 abnormal/9; cnr2-/-+methionine, 6 normal/8; P=0.043 (Chi-square). (D) Confocal microscopy images of livers in cnr2-/- larvae after BODIPY C5 incorporation with and without methionine treatment. Extensive biliary branching is observed in WT larvae, and decreased branching with lipid and cholestatic deposition in cnr2-/- mutants (7 abnormal/10). Treatment of cnr2-/- mutants with methionine causes increased biliary branching and decreased lipid accumulation (2 abnormal/6). (E) Percentage of larvae with fatty liver after ethanol-induced liver injury and with methionine or cysteine treatment for cnr1-/- and cnr2-/- larvae at 120hpf. n>30 samples. Fisher′s exact test. *P<0.05 and **P<0.01 for methionine and cysteine versus DMSO. (F) ORO stain in cnr2-/- embryos at 120hpf after 2% ethanol exposure alone or co-treated with 100µM methionine or cysteine from 96-120hpf. Liver region is outlined. (G) H&E staining of liver sections in 2-month-old male zebrafish. cnr2 morphant liver histology appears similar to that of WT; cnr2-/- and cnr1-/-; cnr2-/- double mutants have decreased cytoplasmic hepatocyte staining. Early methionine treatment of cnr2-/- mutants had no effect on liver histology; however, daily methionine altered histological appearance towards WT features, with increased cytoplasmic staining and decreased cytoplasm-to-nucleus ratios. n=5/group. Scale bars: 0.2mm in B,F; 0.1mm in C; 20µm in D; 50µm in G.
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