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Additional Supporting Information may be found in the online version of this article.

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hep26551-sup-0001-suppfig1.tif2889KSupporting Figure 1. Liver size is reduced in GMP synthetases850 mutant larvae. Brightfield images of wild-type (A) and GMP synthetases850 mutant (B) larvae at 7 dpf are overlaid with Tg(fabp10:GFP-CAAX)lri1 expression (green). GFP expression is shown separately in A' and B'. (C) EdU incorporation rate in the liver of wild-type and GMP synthetases850 mutant larvae at 4 dpf. The cell proliferation rate, indicated by EdU incorporation, is lower in the liver of GMP synthetases850 mutant larvae at this stage. EdU incorporation was measured as previously described(s1).
hep26551-sup-0002-suppfig2.tif811KSupporting Figure 2. Positional cloning of the s850 locus. (A) Bulk segregant analyses using markers from the zebrafish genetic map placed the s850 locus on zebrafish chromosome 18 near z14694. We further defined a critical region surrounding the s850 locus, which was flanked by markers 0.05 cM (SCA-KB5) and 0.1 cM (SCA-GA4) from the s850 locus. (B) Sequencing genes in the critical region identified a single nucleotide change (T to A) in the GMP synthetase gene. (C) The s850 mutation changes T589 to A589 of the GMP synthetase gene. (D) The s850 mutation replaces histidine (H189) of GMP synthetase with glutamine (Q189).
hep26551-sup-0003-suppfig3.tif4895KSupporting Figure 3. Electron micrographs of the GMP synthetases850 mutant liver. Electron micrographs of the wild-type (E) and GMP synthetases850 mutant liver at 7 dpf are shown. In GMP synthetases850 mutant larvae, lipid droplets are evident in hepatocytes. l,lipid droplet; n, nucleus.
hep26551-sup-0004-suppfig4.tif937KSupporting Figure 4. Rac1 expression in the liver. Z-plane confocal image of the dissected liver of wild-type larvae at 7 dpf. (A) Rac1 expression (green) and DAPI staining (white) are visualized. The green channel is shown separately in A'. (B) Mouse IgG1 (geen), used as a negative control in immunofluorescence, and DAPI staining (white) are visualized. The green channel is shown separately in B'. There was no signal detected with mouse IgG1.
hep26551-sup-0005-suppfig5.tif1090KSupporting Figure 5. Tg(fabp10:GFP-DNRac1)lri4 larvae show no morphological defects. (A) Bright-field images of Tg(fabp10:GFP-DNRac1)lri4 larvae at 7 dpf are overlaid with Tg(fabp10:GFP-CAAX)lri1 expression (green). Tg(fabp10:GFP-DNRac1)lri4 larvae are viable and their physical appearance is normal. The liver size of Tg(fabp10:GFP-DNRac1)lri4 larvae is indistinguishable from that of wild-type larvae. (B) Quantification of liver steatosis measured by the percentage of hepatocytes containing Nile Red positive lipid droplets in wild-type sibling, Tg(fabp10:GFP-CAAX)lri1 and hydrogen peroxide-treated Tg(fabp10:GFP-CAAX)lri1 larvae. The percentage of hepatocytes containing lipid droplets is significantly increased in Tg(fabp10:GFP-CAAX)lri1 mutant larvae and is ameliorated by the hydrogen peroxide treatment.
hep26551-sup-0006-suppfig6.tif2583KSupporting Figure 6. The intrahepatic biliary and vascular networks exist in GMP synthetases850 mutant larvae. Wild-type (A) and GMP synthetases850 mutant (B) Tg(Tp1-MmHbb:EGFP)um14 Tg(kdrl:memCherry)s896 larvae visualized for GFP, mCherry, and DAPI expression. Z-plane confocal images of the liver at 7 dpf. GFP and mCherry expressions are shown separately in (A' and B') and (A'' and B''), respectively. Ventral views, anterior to the top. Although the liver size is smaller, the intrahepatic biliary and vascular networks, visualized by Tg(Tp1-MmHbb:EGFP)um14 and Tg(kdrl:memCherry)s896 expressions(s2, s3) respectively, appear to be formed in GMP synthetases850 mutant larvae.
hep26551-sup-0007-suppfig7.tif3850KSupporting Figure 7. Neutrophil infiltration to the liver is not observed in GMP synthetases850 mutant larvae. (A-F) Myeloid peroxidase (MPO) staining visualizing neutrophils. Wild-type (A and D), GMP synthetases850 mutant (B and E), and 150 µg/ml lipopolysaccharide (LPS) treated wild-type larvae at 7dpf with MPO-positive neutrophils visualized in dark brown in the livers (A-C) and posterior cardinal vein (D-F; arrows). There were no MPO-positive cells observed in the liver of wild-type and GMP synthetases850 mutant larvae, while MPO-positive cells do infiltrate the liver of LPS-treated larvae (arrowheads in C). These data suggest that inflammation, indicated by neutrophil infiltration, is not happening in the liver of GMP synthetases850 mutant larvae at this stage. MPO staining was performed as previously described (s4). (G and H) H&E staining of sections through the liver of 7 dpf wild-type (G) and GMP synthetases850 mutant (H) larvae. Hepatic cellular organization appeared to be normal in GMP synthetases850 mutant larvae at 7 dpf.
hep26551-sup-0008-suppfig8.tif160KSupporting Figure 8. MPA treatment increased the percentage of larvae showing hepatic steatosis in GMP synthetases850 mutant larvae. The percentage of wild-type, GMP synthetases850 mutant and MAP-treated GMP synthetases850 mutant larvae showing hepatic steatosis scored by whole-mount ORO staining at 7 dpf. Animals were treated with15 µg/ml MPA was treated from 3 to 7 dpf. MPA treatment increased the percentage of larvae showing hepatic steatosis in GMP synthetases850 mutant larvae at 7 dpf. **P<0.01; error bars indicate standard deviation.
hep26551-sup-0009-suppfig9.tif253KSupporting Figure 9. Small molecule inhibitor treatments of earlier stage larvae. The percentage of wild-type larvae treated with DMSO, Rac1 inhibitor or DPI from 3 to 5, from 3 to 6, or from 5 to 7 dpf, showing hepatic steatosis scored by whole-mount ORO staining at 5, 6, or 7 dpf, respectively, are shown. Rac1 inhibitor or DPI treatment from 3 to 5 dpf did not induce hepatic steatosis, while the treatment from 3 to 6 or from 5 to 7 dpf significantly increased the percentage of larvae showing hepatic steatosis. As a control, we also treated wild-type larvae with tunicamicin, and consistent with previous studies, tunicamicin-treated larvae significantly increased the percentage of larvae showing ORO staining in the liver at 7 dpf.
hep26551-sup-0010-suppfig10.tif192KSupporting Figure 10. Small molecule inhibitor treatments on fed-larvae. The percentage of starved wild-type larvae and fed wild-type larvae treated with DMSO, Rac1 inhibitor or DPI from 5 to 7 dpf, showing hepatic steatosis scored by whole-mount ORO staining at 7 dpf. Feeding increased the percentage of larvae showing hepatic steatosis in wild-type larvae, and both Rac1 inhibitor and DPI treatments further increased these percentages. ORO staining experiments with fed wild-type larvae were repeated five times with an average n = 9.8 larvae per experiment (total n = 49 larvae examined and total n = 23showed ORO signal in the liver). With Rac1 inhibitor-treated fed wild-type larvae, the experiments were repeated three times with an average n = 8 larvae per experiment (total n = 24 larvae examined and total n = 20 showed ORO signal in the liver). With DPI-treated fed wild-type larvae, the experiments were repeated three times with an average n = 8 larvae per experiment (total n = 24 larvae examined and total n = 20 showed ORO signal in the liver). *P<0.05, **P<0.01; error bars indicate standard deviation.
hep26551-sup-0011-suppfig11.tif191KSupporting Figure 11. qPCR analysis of mRNA expression levels in GMP synthetases850 mutant larvae at 6 dpf. The averages of at least three independent experiments are shown. In GMP synthetases850 mutant larvae, tgh mRNA expression level is significantly down-regulated but all other tested mRNA expressions are not significantly changed. ***P<0.001, n.s., not significant; error bars indicate standard deviation.
hep26551-sup-0012-supptab1.doc32KSupporting Table 1. List of primer sequences used in this study for qPCR.
hep26551-sup-0013-supptab2.doc39KSupporting Table 2. List of small molecule inhibitors used in this study.
hep26551-sup-0014-suppinfo.doc46KSupporting Information

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