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The RSPO–LGR4/5–ZNRF3/RNF43 module controls liver zonation and size

Nature Cell Biology volume 18, pages 467–479 (2016)Cite this article

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A Corrigendum to this article was published on 27 October 2016

This article has been updated

Abstract

LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/β-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic zonation requires a Wnt/β-catenin signalling gradient, but the instructive mechanism controlling its spatiotemporal regulation is not known. We have now identified the RSPO–LGR4/5–ZNRF3/RNF43 module as a master regulator of Wnt/β-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/β-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/β-catenin signalling gradient in a reversible and LGR4/5-dependent manner. Recombinant RSPO1 protein increased liver size and improved liver regeneration, whereas LGR4/5 LOF caused the opposite effects, resulting in hypoplastic livers. Furthermore, we show that LGR4+ hepatocytes throughout the lobule contribute to liver homeostasis without zonal dominance. Taken together, our results indicate that the RSPO–LGR4/5–ZNRF3/RNF43 module controls metabolic liver zonation and is a hepatic growth/size rheostat during development, homeostasis and regeneration.

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Figure 1: Lgr4 and Lgr5 are co-expressed in pericentral hepatocytes.
Figure 2: LGR4 and LGR5 lineage tracing during liver homeostasis.
Figure 3: LGR4 and LGR5 receptors control liver zonation.
Figure 4: RSPO blockade disrupts hepatic Wnt/β-catenin signalling and zonation.
Figure 5: ZNRF3 and RNF43 control liver zonation.
Figure 6: RSPO1 controls liver zonation through LGR4 and LGR5 receptors.
Figure 7: Loss of LGR4 and LGR5 results in impaired liver homeostasis and regeneration.
Figure 8: RSPO1 promotes hepatocyte proliferation and liver regeneration through LGR4 and LGR5 receptors.

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  • 27 September 2016

    In the version of this Article originally published, the authors inadvertently omitted a key reference. The following reference has been added: '48. Rocha, A. S. et al. The angiocrine factor Rspondin3 is a key determinant of liver zonation. Cell Rep. 13, 1757–1764 (2015).' References 48–56 have been renumbered accordingly. Furthermore, a new sentence citing this reference has been added at the end of the first paragraph in the Discussion: 'A recent report that was published during the final revision of our manuscript suggests a role for pericentrally confined RSPO3 in metabolic liver zonation48.' These changes have been made in the online versions of the Article.

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Acknowledgements

We thank J. Wirsching, C. Manneville, M. Lemaistre, M. Haffner, B. Leonhard, S. Ley, N. Guo, T. Lewis, M. Li, M. van de Velde, D. Breustedt, H. Lei, R. Boomer, S. Zurbruegg, L. Perrot, F. Cordoba, S. Schuierer, A. Arif and J. Gfeller for technical assistance. For helpful discussion and critical reading of the manuscript, we thank E. Wiellette, G. Hintzen, B. Dietrich, D. Liu, F. Bassilana, C. Parker, K. Seuwen and J. Porter. This work was supported by the Leverhulme Trust ECF-2012-262 to L.B., SNF grant 310030B_147089 to M.H.H., MRC Centre Grant and UKRMP Niche hub grant MR/K026666/1 to S.J.F. and the Novartis Institutes for BioMedical Research Postdoctoral Program.

Author information

Author notes

  1. Andreas Loew

    Present address: Present address: Elstar Therapeutics Inc., Cambridge, Massachusetts 02139, USA.,

  2. Lara Planas-Paz and Vanessa Orsini: These authors contributed equally to this work.

Authors and Affiliations

  1. Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland

    Lara Planas-Paz, Vanessa Orsini, Monika Pikiolek, Florian Nigsch, Guglielmo Roma, Patricia Marti, Nicolau Beckmann, Walter Carbone, Sebastian Bergling, Andrea Isken, Matthias Mueller, Bernd Kinzel, Tewis Bouwmeester, Heinz Ruffner & Jan S. Tchorz

  2. MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, EH4 2XU Edinburgh, UK

    Luke Boulter

  3. Division of Gastroenterology and Hepatology, University Hospital Basel, 4031 Basel, Switzerland

    Diego Calabrese, Michael T. Dill & Markus H. Heim

  4. Novartis Institutes for BioMedical Research, Novartis Pharma AG, Cambridge, Massachusetts 02139, USA

    Yang Xie, Adriana Donovan, Yi Yang, Xiaohong Mao, Thomas B. Nicholson, Raffaella Zamponi, Paola Capodieci, Reginald Valdez, Daniel Rivera, Andreas Loew, Chinweike Ukomadu & Feng Cong

  5. Institute for Pathology, University Hospital Basel, 4031 Basel, Switzerland

    Luigi M. Terracciano

  6. MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh EH16 4UU, UK

    Stuart J. Forbes

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Contributions

Experimental work and data analysis: L.P.-P., V.O., L.B., D.C., M.P., Y.X., A.D., P.M., N.B., M.T.D., W.C., R.Z. and P.C.; data analysis: F.N., G.R., S.B., R.V., L.M.T. and J.S.T.; mouse model generation: A.I., M.M., B.K., Y.Y., X.M. and T.B.N.; RSPO1 production: D.R. and A.L.; concept and design, and manuscript writing: L.P.-P., L.B., C.U., T.B., F.C., M.H.H., S.J.F., H.R. and J.S.T.

Corresponding author

Correspondence to Jan S. Tchorz.

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Competing interests

All authors except S.J.F., L.B., L.M.T., M.H.H., D.C. and M.T.D. are employed by and/or shareholders of Novartis Pharma AG.

Integrated supplementary information

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.

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 in consecutive liver sections of Lgr5ki/R26-tdTOM mice 2 days post-PH. Arrowheads point at 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 point at 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.

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.

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.

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.

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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Planas-Paz, L., Orsini, V., Boulter, L. et al. The RSPO–LGR4/5–ZNRF3/RNF43 module controls liver zonation and size. Nat Cell Biol 18, 467–479 (2016). https://doi.org/10.1038/ncb3337

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