Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Self-renewing diploid Axin2+ cells fuel homeostatic renewal of the liver

Abstract

The source of new hepatocytes in the uninjured liver has remained an open question. By lineage tracing using the Wnt-responsive gene Axin2 in mice, we identify a population of proliferating and self-renewing cells adjacent to the central vein in the liver lobule. These pericentral cells express the early liver progenitor marker Tbx3, are diploid, and thereby differ from mature hepatocytes, which are mostly polyploid. The descendants of pericentral cells differentiate into Tbx3-negative, polyploid hepatocytes, and can replace all hepatocytes along the liver lobule during homeostatic renewal. Adjacent central vein endothelial cells provide Wnt signals that maintain the pericentral cells, thereby constituting the niche. Thus, we identify a cell population in the liver that subserves homeostatic hepatocyte renewal, characterize its anatomical niche, and identify molecular signals that regulate its activity.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Axin2+ pericentral cells generate expanding clones of hepatocytes from the central vein towards the portal vein over time.
Figure 2: Axin2+ cells self-renew.
Figure 3: Axin2+ hepatocytes proliferate faster than other hepatocytes.
Figure 4: Axin2+ hepatocytes are mostly diploid.
Figure 5: Central vein endothelial cells produce Wnt proteins and act as a niche for pericentral cells.
Figure 6: Central-vein-derived Wnt proteins are required for pericentral cell proliferation.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE68806.

References

  1. Malato, Y. et al. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration. J. Clin. Invest. 121, 4850–4860 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Yanger, K. et al. Adult hepatocytes are generated by self-duplication rather than stem cell differentiation. Cell Stem Cell 15, 340–349 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Miyajima, A., Tanaka, M. & Itoh, T. Stem/progenitor cells in liver development, homeostasis, regeneration, and reprogramming. Cell Stem Cell 14, 561–574 (2014)

    Article  CAS  PubMed  Google Scholar 

  4. Jungermann, K. & Kietzmann, T. Zonation of parenchymal and nonparenchymal metabolism in liver. Annu. Rev. Nutr. 16, 179–203 (1996)

    Article  CAS  PubMed  Google Scholar 

  5. Ganem, N. J. & Pellman, D. Limiting the proliferation of polyploid cells. Cell 131, 437–440 (2007)

    Article  CAS  PubMed  Google Scholar 

  6. Sigal, S. H. et al. Partial hepatectomy-induced polyploidy attenuates hepatocyte replication and activates cell aging events. Am. J. Physiol. 276, G1260–G1272 (1999)

    CAS  PubMed  Google Scholar 

  7. DasGupta, R. & Fuchs, E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 126, 4557–4568 (1999)

    Article  CAS  PubMed  Google Scholar 

  8. Zeng, Y. A. & Nusse, R. Wnt proteins are self-renewal factors for mammary stem cells and promote their long-term expansion in culture. Cell Stem Cell 6, 568–577 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5 . Nature 449, 1003–1007 (2007)

    Article  CAS  PubMed  ADS  Google Scholar 

  10. Lim, X. et al. Interfollicular epidermal stem cells self-renew via autocrine Wnt signaling. Science 342, 1226–1230 (2013)

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  11. Clevers, H., Loh, K. M. & Nusse, R. Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science 346, 1248012 (2014)

    Article  PubMed  CAS  Google Scholar 

  12. Lustig, B. et al. Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors. Mol. Cell. Biol. 22, 1184–1193 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. van Amerongen, R., Bowman, A. N. & Nusse, R. Developmental stage and time dictate the fate of Wnt/β-catenin-responsive stem cells in the mammary gland. Cell Stem Cell 11, 387–400 (2012)

    Article  CAS  PubMed  Google Scholar 

  14. Benhamouche, S. et al. Apc tumor suppressor gene is the “zonation-keeper” of mouse liver. Dev. Cell 10, 759–770 (2006)

    Article  CAS  PubMed  Google Scholar 

  15. Huch, M. et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–250 (2013)

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  16. Cadoret, A. et al. New targets of β-catenin signaling in the liver are involved in the glutamine metabolism. Oncogene 21, 8293–8301 (2002)

    Article  CAS  PubMed  Google Scholar 

  17. Braeuning, A. et al. Differential gene expression in periportal and perivenous mouse hepatocytes. FEBS J. 273, 5051–5061 (2006)

    Article  CAS  PubMed  Google Scholar 

  18. Han, J. et al. Tbx3 improves the germ-line competency of induced pluripotent stem cells. Nature 463, 1096–1100 (2010)

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  19. Suzuki, A. et al. Tbx3 controls the fate of hepatic progenitor cells in liver development by suppressing p19ARF expression. Development 135, 1589–1595 (2008)

    Article  CAS  PubMed  Google Scholar 

  20. Moreira, P. I. et al. Estradiol affects liver mitochondrial function in ovariectomized and tamoxifen-treated ovariectomized female rats. Toxicol. Appl. Pharmacol. 221, 102–110 (2007)

    Article  CAS  PubMed  Google Scholar 

  21. Yu, H. M. et al. Impaired neural development caused by inducible expression of Axin in transgenic mice. Mech. Dev. 124, 146–156 (2007)

    Article  CAS  PubMed  Google Scholar 

  22. Tumbar, T. Defining the epithelial stem cell niche in skin. Science 303, 359–363 (2004)

    Article  CAS  PubMed  ADS  Google Scholar 

  23. Guidotti, J. E. et al. Liver cell polyploidization: a pivotal role for binuclear hepatocytes. J. Biol. Chem. 278, 19095–19101 (2003)

    Article  CAS  PubMed  Google Scholar 

  24. Comai, L. The advantages and disadvantages of being polyploid. Nature Rev. Genet. 6, 836–846 (2005)

    Article  CAS  PubMed  Google Scholar 

  25. Ohlstein, B. &. Spradling, A. The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature 439, 470–474 (2006)

    Article  CAS  PubMed  ADS  Google Scholar 

  26. Ding, B.-S. et al. Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration. Nature 468, 310–315 (2010)

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  27. Bänziger, C. et al. Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 125, 509–522 (2006)

    Article  PubMed  CAS  Google Scholar 

  28. Monvoisin, A. et al. VE-cadherin-CreERT2 transgenic mouse: a model for inducible recombination in the endothelium. Dev. Dyn. 235, 3413–3422 (2006)

    Article  CAS  PubMed  Google Scholar 

  29. Carpenter, A. C. et al. Generation of mice with a conditional null allele for Wntless. Genesis 48, 554–558 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Magami, Y. et al. Cell proliferation and renewal of normal hepatocytes and bile duct cells in adult mouse liver. Liver 22, 419–425 (2002)

    Article  PubMed  Google Scholar 

  31. Zajicek, G. & Schwartz-Arad, D. Streaming liver VII: DNA turnover in acinus zone-3. Liver 10, 137–140 (1990)

    Article  CAS  PubMed  Google Scholar 

  32. Duncan, A. W. et al. The ploidy conveyor of mature hepatocytes as a source of genetic variation. Nature 467, 707–710 (2010)

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  33. Niehrs, C. & Acebron, S. P. Mitotic and mitogenic Wnt signalling. EMBO J. 31, 2705–2713 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Vijayakumar, S. et al. High-frequency canonical Wnt activation in multiple sarcoma subtypes drives proliferation through a TCF/β-catenin target gene, CDC25A. Cancer Cell 19, 601–612 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Tan, X. et al. β-Catenin deletion in hepatoblasts disrupts hepatic morphogenesis and survival during mouse development. Hepatology 47, 1667–1679 (2008)

    Article  CAS  PubMed  Google Scholar 

  36. Zajicek, G., Oren, R. & Weinreb, M., Jr. The streaming liver. Liver 5, 293–300 (1985)

    Article  CAS  PubMed  Google Scholar 

  37. Lüdtke, T. H. et al. Tbx3 promotes liver bud expansion during mouse development by suppression of cholangiocyte differentiation. Hepatology 49, 969–978 (2009)

    Article  PubMed  CAS  Google Scholar 

  38. Laurent-Puig, P. & Zucman-Rossi, J. Genetics of hepatocellular tumors. Oncogene 25, 3778–3786 (2006)

    Article  CAS  PubMed  Google Scholar 

  39. Wang, R. et al. Activation of the Met receptor by cell attachment induces and sustains hepatocellular carcinomas in transgenic mice. J. Cell Biol. 153, 1023–1034 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Tward, A. D. et al. Distinct pathways of genomic progression to benign and malignant tumors of the liver. Proc. Natl Acad. Sci. USA 104, 14771–14776 (2007)

    Article  CAS  PubMed  ADS  PubMed Central  Google Scholar 

  41. Schwarze, P. E. et al. Emergence of a population of small, diploid hepatocytes during hepatocarcinogenesis. Carcinogenesis 5, 1267–1275 (1984)

    Article  CAS  PubMed  Google Scholar 

  42. Muzumdar, M. D. et al. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007)

    Article  CAS  PubMed  Google Scholar 

  43. Kreamer, B. L. et al. Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro Cell. Dev. Biol. 22, 201–211 (1986)

    Article  CAS  PubMed  Google Scholar 

  44. Wang, F. et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J. Mol. Diagn. 14, 22–29 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Goecks, J. et al. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 11, R86 (2010)

    Article  PubMed  PubMed Central  Google Scholar 

  46. Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Trapnell, C. et al. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nature Biotechnol. 31, 46–53 (2013)

    Article  CAS  Google Scholar 

  48. Edgar, R., Domrachev, M. & Lash, A. E. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30, 207–210 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

These studies were supported by the Howard Hughes Medical Institute and a grant from the Reed-Stinehart foundation. R.N. is an investigator with the Howard Hughes Medical Institute. B.W. was supported by F32DK091005. We thank D. M. Bissell and T. Desai for comments on the manuscript, V. Waehle for assistance in preparing RNA samples for RNA-seq, M. Britton for RNA-seq analysis, and P. Lovelace for assistance with FACS.

Author information

Authors and Affiliations

Authors

Contributions

B.W. carried out the experiments. D.Z. performed qRT–PCR analysis. M.F. performed in situ hybridization. C.Y.L. performed RNA-seq analysis. B.W. and R.N. designed the study, analysed data and wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Bruce Wang or Roel Nusse.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Leakiness in Axin2-CreERT2;Rosa26-mTmGflox mice is not observed in animals injected with corn oil.

a, c, No GFP labelling is seen in Axin2-CreERT2;Rosa26-mTmGflox mice after a single dose of corn oil and traced for 2 days (a) or 365 days (c). b, d, No GFP labelling is seen after five consecutive daily doses of corn oil and traced for 7 days (b) or 365 days (d). All animals were 8-week-old Axin2-CreERT2;Rosa26-mTmGflox mice. Images are representative images from n = 5 mice per condition and time point. CV, central vein; PV, portal vein. Scale bars, 100 μm.

Extended Data Figure 2 Axin2 expression remains restricted to pericentral cells.

ac, In situ hybridization for Axin2 in 120-day trace (a), 240-day trace (b) and 365-day trace (c) Axin2-CreERT2;Rosa26-mTmGflox mice. Representative in situ images are from n = 5 animals per time point. CV, central vein; PV, portal vein. Scale bars, 100 μm.

Extended Data Figure 3 Descendants of Axin2+ cells replaced 30% of the area of the liver.

Tiled image of entire liver section of a 365-day trace Axin2-CreERT2;Rosa26-mTmGflox mice. Image is representative of n = 5 animals at this time point. Scale bar, 2,500 μm.

Extended Data Figure 4 FACS sorting gates for GFP+ cells in Axin2-rtTA;TetO-H2B-GFP mice.

Eight-week-old Axin2-rtTA;TetO-H2B-GFP mice were labelled with doxycycline for 7 days and chased for various lengths of time. Hepatocytes were enzymatically dispersed and sorted by FACS. ac, Successive gating shows sequential selection of all hepatocytes (a), single cells by forward scatter (b), and side scatter (c). d, Dead cells were excluded by propidium iodide labelling. e, GFP-positive cells were gated and either sorted for RNA-seq analysis or further graphed as histograms for GFP intensity analysis (see Fig. 3g).

Extended Data Figure 5 Axin2 gene dosage and tamoxifen have no effect on pericentral hepatocyte proliferation rate.

Wild-type and Axin2CreERT2+/− mice were given EdU daily for 7 days. A subset of wild-type and Axin2CreERT2+/− mice was given 4 mg of tamoxifen per 25 g body weight daily for 5 days. Pericentral hepatocytes were identified by Hnf4a+/GS+ staining. All other hepatocytes were identified by Hnf4a+/GS antibody staining. The EdU-positive rates within the two hepatocyte populations as a percentage of total HNF4a+ cells were essentially the same regardless of Axin2 gene dosage or tamoxifen administration. n = 5 animals per group. Data represent mean ± s.e.m. *P > 0.05.

Extended Data Figure 6 Axin2+ hepatocytes proliferate rapidly.

Axin2-rtTA;TetO-H2B-GFP mice were given doxycycline for 7 days. a, 56 days after cessation of doxycycline, very few GFP+ cells are seen around the central vein. b, After 84 days, no GFP+ cells are seen. Images are representative of n = 4 animals per time point.

Extended Data Figure 7 FACS sorting gates for GFP+ cells in Axin2-CreERT2;Rosa26-mTmGflox mice for ploidy analysis.

Eight-week-old Axin2-CreERT2;Rosa26-mTmGflox mice were labelled with five daily doses of tamoxifen and traced for 7 days. Hepatocytes were enzymatically dispersed and sorted by FACS. ac, Successive gating show sequential selection of all hepatocytes(a), single cells by forward scatter(b), and side scatter (c). d, Dead cells were excluded by propidium iodide labelling. e, GFP-positive cells were gated and graphed as histograms for Hoechst staining (see Fig. 4).

Extended Data Figure 8 FACS sorting gates for endothelial cells.

Eight-week-old wild-type C57B6 mice were used for endothelial cell isolation. Livers were enzymatically digested, hepatocytes were removed by centrifugation and nonparenchymal cells were antibody stained and sorted by FACS. ac, Successive gating showed sequential selection of non-parenchymal cells by size (a), single cells by forward scatter (b), and side scatter (c). d, Dead cells were excluded by DAPI labelling. e, endothelial cells were identified by CD31-phycoerythrin-positive staining. f, Sinusoidal endothelial cells (SEC) were identified as CD34-FITC+VEGFR3-APC+ while central vein endothelial cells (CEC) were identified as CD34-FITC+VEGFR3-APC.

Extended Data Figure 9 Histology of VE-cadherin-CreERT2;Wlsflox/flox animal versus control.

a, Control (VE-cadherin-CreERT2;Wlsflox/+) animals given five daily doses of tamoxifen and traced for 7 days after the last tamoxifen dose. Haematoxylin and eosin staining of the liver shows normal histology. b, Wls-knockout animals (VE-cadherin-CreERT2;Wlsflox/flox) also showed normal liver histology. Images are representative images from n = 5 animals per group. Insets show central veins. Scale bars, 100 μm.

Extended Data Table 1 Partial list of differentially expressed genes in Axin2+ vs Axin2 hepatocytes by RNA-seq analysis

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, B., Zhao, L., Fish, M. et al. Self-renewing diploid Axin2+ cells fuel homeostatic renewal of the liver. Nature 524, 180–185 (2015). https://doi.org/10.1038/nature14863

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature14863

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing