supplementary figure 1 - media.nature.com 0 cv1 cv2 cv3 pa pv3 pv2 pv1 60 20 parenchyma parenchyma...
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S U P P L E M E N TA RY I N F O R M AT I O N
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DOI: 10.1038/ncb3337
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Supplementary Figure 1Planas-Paz & Orsini et al.
Parenchyma
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dLgr4 promoter Lgr4 locus ATG
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AlbCre mice
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Lgr4 promoter Lgr4 locus
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Rnf43 promoter BAC transgenic
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Exon ExonLoxP
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Supplementary Figure 1 LGR4 and LGR5 expression and mouse model generation. a, Lgr4 and Lgr5 ISH in liver parenchyma of control mice. The images are representative for 4 mice each. b, Percent of hepatocytes expressing Lgr4 and Lgr5 in the indicated liver zones. n = 4 mice. These data involved
assessment of 3346 cells from the indicated mice. c, Scheme depicting the generation of Rnf43-LacZ mice. d, Scheme depicting the generation of liver-specific Lgr4/5dLKO mice by crossing with AlbCre mice. Data represent mean ± s.d. Scale bars, (a) 20 µm and (magnification in a) 10 µm.
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Supplementary Figure 2Planas-Paz & Orsini et al.
Lgr4ki/R26-LacZ mice(10 days)
Nuclear Fast Red LacZ
b cLgr4ki/R26-LacZ mice
(10 months)Lgr5ki/R26-LacZ mice
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Supplementary Figure 2 LGR4 and LGR5 lineage tracing. a, Scheme depicting lineage tracing in Lgr4ki/R26-LacZ and Lgr5ki/R26-LacZ mice. b, LacZ staining in Lgr4ki/R26-LacZ mice showing LGR4+ hepatocytes in the liver (magnified area: parenchyma) after 10 days or 10 months of tracing. c, LacZ staining in Lgr5ki/R26-LacZ mice showing no LGR5+ hepatocytes in the liver (magnified area: parenchyma) after 10 days or 18 months of tracing. d, Distribution of Lgr5ki/R26-LacZ+ hepatocytes after 10 days or 18 months of tracing. n = 3 mice per group. These data involved assessment of 210 cells (10 days) and 330 cells (18 months) from the indicated mice. e, Axin2 ISH and β-Gal staining in Lgr5ki/R26-LacZ mice after 10 days of tracing showing Axin2+/LacZ+ hepatocytes. f, LacZ staining, Axin2 and Lgr5 co-ISH in consecutive liver sections of Lgr5ki/R26-LacZ mice after 10 days of tracing. g, Percent of hepatocytes expressing Axin2 and Lgr5 in Lgr5ki/R26-LacZ+ and Lgr5ki/R26-LacZ- hepatocytes. n = 4 mice. h, Scheme depicting lineage tracing in Lgr5ki/tdTOM mice 2 days post-PH. i, tdTOM
and Ki67 staining on consecutive liver sections of Lgr5ki/R26-tdTOM mice 2 days post-PH. Arrowheads indicate tdTOM+/Ki67+ hepatocytes. j, tdTOM+/Ki67+ hepatocytes quantified in Lgr5ki/R26-tdTOM mice 2 days post-PH. n = 4 mice. k, Scheme depicting EdU injections in WT mice. l, GS and EdU co-staining in control mice. Arrowheads indicate EdU+ hepatocytes. m, EdU+ hepatocytes quantified in liver zones of control mice. n = 5 mice. n, Distribution of EdU+ hepatocytes in the indicated liver zones. n = 5 mice. These data involved assessment of (d) 210 cells (10 days) and 330 cells (18 months); (g) 1970 LacZ- and 87 LacZ+ cells; (j) 14681 tdTOM- and 49 tdTOM+ cells; (m) 2650 cells; and (n) 577 cells from the indicated mice. CV, central vein; PV, portal vein. The images in (b, c) and (e, f, i, l) are representative for 3 and 4 mice each, respectively. Data represent mean ± s.d. ns, not significant; two-tailed unpaired t-test (j) and one-way ANOVA with Tukey’s test (m) were used. Scale bars, (b, c, f) 100 µm, (magnifications in b, c) 50 µm, (e, l, magnification in f) 20 µm and (i) 50 µm.
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Supplementary Figure 3Planas-Paz & Orsini et al.
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Supplementary Figure 3 Role of LGR4 and LGR5 during hepatocyte maturation and differentiation. a, GS and CK19 co-staining in P2, P10 and P30 control and Lgr4/5dLKO mice. b, GS+ staining quantified in the indicated mice. n = 4 mice (P10 control and P30 control) and 5 mice (all other groups). c, HNF4α and CK19 co-staining in P2, P10 and P30 control
and Lgr4/5dLKO mice. d, SOX9 staining in P2, P10 and P30 control and Lgr4/5dLKO mice. CV, central vein; PV, portal vein. The images in (a, c, d) are representative for 29 mice, and the stainings were repeated 2 times. Data represent mean ± s.d. **, P<0.01; ***, P<0.001; ****, P<0.0001; two-tailed unpaired t-test (b) was used. Scale bars, 20 µm.
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Supplementary Figure 4Planas-Paz & Orsini et al.
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Supplementary Figure 4 Lgr4 and Lgr5 control liver zonation. a, Volcano plot showing pericentral genes (in green), periportal genes (in blue) and genes involved in Wnt signalling (in purple) that are differentially expressed in livers of the indicated mice. n = 5 mice per group. b, Gene sets downregulated in livers of Lgr4/5dLKO mice compared to control mice. n = 5 mice per group.
c, d, ARG1 (c) and PCK1 (d) staining in the indicated mice. CV, central vein; PV, portal vein. The images in (c, d) are representative for 10 mice, and the stainings were repeated 3 times. Empirical Bayes with Benjamini-Hochberg test (a), and weighted Kolmogorov-Smirnov and Mann-Whitney U-test with Benjamini-Hochberg test (b) were used. Scale bars, 100 µm.
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Supplementary Figure 5Planas-Paz & Orsini et al.
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Supplementary Figure 5 Role of LGR4 and LGR5 in the control of liver growth. a, b, c, GS, CK19 and Ki67 co-staining in P2 (a), P10 (b) and P30 (c) control and Lgr4/5dLKO mice, showing proliferating hepatocytes (arrowheads). Bottom: Hepatocyte proliferation quantified in liver zones of the indicated mice. n = 4 mice (P30 controls), 5 mice (P2 and P10), and 6 mice (P30 Lgr4/5dLKO). These data involved assessment of 2582 cells (P2 control), 2705 cells (P2 Lgr4/5dLKO), 4217 cells (P10 control), 3911 cells (P10 Lgr4/5dLKO), 2882 cells (P30 control) and 3171 cells (P30 Lgr4/5dLKO) from the indicated mice. d, Relative
liver weight of the indicated mice. n = 5 mice (P2 control) and 6 mice (all other groups). e, Ki67 staining of the indicated mice at embryonic day (E)16.5. f, Liver cell proliferation quantified in the indicated mice at E16.5. n = 5 mice (control, Lgr5LKO and Lgr4/5dLKO) and 6 mice (Lgr4LKO). CV, central vein; PV, portal vein. The images in (a-c) and (e) are representative for 30 and 21 mice, respectively, and the stainings were repeated 2 times. Data represent mean ± s.d. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant; two-tailed unpaired t-test (a-d, f) was used. Scale bars, 50 µm.
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5 days 7 days
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Supplementary Figure 6Planas-Paz & Orsini et al.
Wild type d2 post-PHWild typea
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Supplementary Figure 6 Role of LGR4 and LGR5 during liver regeneration. a, Axin2 ISH in wild type mice +/- PH. b, Axin2 ISH quantified in wild type mice +/- PH in the indicated liver zones. n = 5 mice (WT) and 3 mice (WT d2 post-PH). These data involved assessment of 4225 cells (WT) and 1905 cells (WT d2 post-PH) from the indicated mice. c, Volcano plot showing genes differentially expressed in livers of the indicated mice. n = 5 mice per group. d, Lgr4 and Lgr5 ISH in wild type mice +/- PH. e, f, Lgr4 (e) and Lgr5 (f) ISH quantified in wild type mice +/- PH in the indicated liver zones. n = 5 mice (WT, Lgr5), 4 mice (WT, Lgr4) and 3 mice (WT d2 post-PH). g, Volcano plot showing genes differentially
expressed in livers of the indicated mice. n = 5 mice per group. h, Scheme depicting lineage tracing in Lgr5ki/R26-LacZ mice 7 days post-PH. i, LacZ staining showing that LGR5+ hepatocytes (arrowheads) did not overtly proliferate during liver regeneration following PH. CV, central vein; PV, portal vein. The images in (a, d) and (i) are representative for 8 and 6 mice, respectively. Data represent mean ± s.d. *, P<0.05; ****, P<0.0001; two-way ANOVA with Sidak’s test (b, e, f) and Empirical Bayes with Benjamini-Hochberg test (c, g) were used. Scale bars, (a, d) 10 µm and (i) 100 µm. Statistics source data for (b, e, f) can be found in Supplementary Table 4.
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Supplementary Table Legends
Supplementary Table 1 Differential gene expression analysis. Differentially expressed genes in Lgr4/5dLKO vs control mouse livers +/- PH.
Supplementary Table 2 Gene set enrichment analysis. Differentially expressed gene sets in Lgr4/5dLKO vs control mouse livers +/- PH.
Supplementary Table 3 Probes used for Taqman RT-PCR.
Supplementary Table 4 Statistics source data.
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