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.

  • Article
  • Published:

Prrx1 marks stem cells for bone, white adipose tissue and dermis in adult mice

Nature Genetics volume 54, pages 1946–1958 (2022)Cite this article

Subjects

Abstract

Specialized connective tissues, including bone and adipose tissues, control various physiological activities, including mineral and energy homeostasis. However, the identity of stem cells maintaining these tissues throughout adulthood remains elusive. By conducting genetic lineage tracing and cell depletion experiments in newly generated knock-in Cre/CreERT2 lines, we show here that rare Prrx1-expressing cells act as stem cells for bone, white adipose tissue and dermis in adult mice, which are indispensable for the homeostasis and repair of these tissues. Single-cell profiling reveals the cycling and multipotent nature of Prrx1-expressing cells and the stemness of these cells is further validated by transplantation assays. Moreover, we identify the cell surface markers for Prrx1-expressing stem cells and show that the activities of these stem cells are regulated by Wnt signaling. These findings expand our knowledge of connective tissue homeostasis/regeneration and may help improve stem-cell-based therapies.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: Tracing with knock-in Prrx1-CreERT2tdTomato reveals that Prrx1+ cells in the mesoderm contribute to three connective tissues.
Fig. 2: Prrx1+ cells drive the turnover of the three connective tissues in adult mice.
Fig. 3: Ablation of Prrx1+ stem cells leads to osteoporosis and atrophy of iWAT and dermis.
Fig. 4: Prrx1+ cells are required for bone fracture and skin wound healing.
Fig. 5: Single-cell profiling of Prrx1+ stem cells.
Fig. 6: Transplantation assays support the stemness of Prrx1-expressing cells.
Fig. 7: Tissue homeostasis is regulated by Wnt molecules secreted by Prrx1+ cells and their daughter cells.

Similar content being viewed by others

Data availability

Mice reference genome GRCm38.p6 was used for gene mapping. scRNA-seq data used in Supplementary Fig. 10 were from the GEO database with the access number of GSE172149. Only the cells from 2-month-old mice were used for analysis. The scRNA-seq data of Prrx1+ stem cells isolated from bone, iWAT and dermis have been deposited in the SRA database under accession code PRJNA695097. The bulk RNA-seq data of Prrx1+ stem cells, SSCs marked by various genes, APCs, fat tissue and dermal tissue have been deposited in the SRA database under accession codes PRJNA813686, PRJNA813685 and PRJNA813688, respectively. Source data are provided with this paper.

Code availability

All details of all analysis code used in the manuscript have been provided in the Methods.

References

  1. Shook, B. et al. The role of adipocytes in tissue regeneration and stem cell niches. Annu. Rev. Cell Dev. Biol. 32, 609–631 (2016).

    Article  CAS  Google Scholar 

  2. El Agha, E. et al. Mesenchymal stem cells in fibrotic disease. Cell Stem Cell 21, 166–177 (2017).

    Article  CAS  Google Scholar 

  3. Zwick, R. K., Guerrero-Juarez, C. F., Horsley, V. & Plikus, M. V. Anatomical, physiological, and functional diversity of adipose tissue. Cell Metab. 27, 68–83 (2018).

    Article  CAS  Google Scholar 

  4. Grayson, W. L. et al. Stromal cells and stem cells in clinical bone regeneration. Nat. Rev. Endocrinol. 11, 140–150 (2015).

    Article  CAS  Google Scholar 

  5. Caplan, A. I. New MSC: MSCs as pericytes are sentinels and gatekeepers. J. Orthop. Res. 35, 1151–1159 (2017).

    Article  Google Scholar 

  6. Galipeau, J. & Sensebe, L. Mesenchymal stromal cells: clinical challenges and therapeutic opportunities. Cell Stem Cell 22, 824–833 (2018).

    Article  CAS  Google Scholar 

  7. Vanakker, O., Callewaert, B., Malfait, F. & Coucke, P. The genetics of soft connective tissue disorders. Annu. Rev. Genom. Hum. Genet. 16, 229–255 (2015).

    Article  CAS  Google Scholar 

  8. Sipp, D., Robey, P. G. & Turner, L. Clear up this stem-cell mess. Nature 561, 455–457 (2018).

    Article  CAS  Google Scholar 

  9. Newton, P. T. et al. A radical switch in clonality reveals a stem cell niche in the epiphyseal growth plate. Nature 567, 234–238 (2019).

    Article  CAS  Google Scholar 

  10. Worthley, D. L. et al. Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential. Cell 160, 269–284 (2015).

    Article  CAS  Google Scholar 

  11. Zhou, B. O., Yue, R., Murphy, M. M., Peyer, J. G. & Morrison, S. J. Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow. Cell Stem Cell 15, 154–168 (2014).

    Article  CAS  Google Scholar 

  12. Baron, R. & Kneissel, M. WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat. Med. 19, 179–192 (2013).

    Article  CAS  Google Scholar 

  13. Mizuhashi, K. et al. Resting zone of the growth plate houses a unique class of skeletal stem cells. Nature 563, 254–258 (2018).

    Article  CAS  Google Scholar 

  14. Debnath, S. et al. Discovery of a periosteal stem cell mediating intramembranous bone formation. Nature 562, 133–139 (2018).

    Article  CAS  Google Scholar 

  15. Ono, N., Ono, W., Nagasawa, T. & Kronenberg, H. M. A subset of chondrogenic cells provides early mesenchymal progenitors in growing bones. Nat. Cell Biol. 16, 1157–1167 (2014).

    Article  CAS  Google Scholar 

  16. Méndez-Ferrer, S. et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 466, 829–834 (2010).

    Article  Google Scholar 

  17. Seike, M., Omatsu, Y., Watanabe, H., Kondoh, G. & Nagasawa, T. Stem cell niche-specific Ebf3 maintains the bone marrow cavity. Genes Dev. 32, 359–372 (2018).

    Article  CAS  Google Scholar 

  18. Matsushita, Y. et al. A Wnt-mediated transformation of the bone marrow stromal cell identity orchestrates skeletal regeneration. Nat. Commun. 11, 332 (2020).

    Article  CAS  Google Scholar 

  19. Ambrosi, T. H. et al. Aged skeletal stem cells generate an inflammatory degenerative niche. Nature 597, 256–262 (2021).

    Article  CAS  Google Scholar 

  20. Chan, C. K. et al. Identification and specification of the mouse skeletal stem cell. Cell 160, 285–298 (2015).

    Article  CAS  Google Scholar 

  21. Hepler, C., Vishvanath, L. & Gupta, R. K. Sorting out adipocyte precursors and their role in physiology and disease. Genes Dev. 31, 127–140 (2017).

    Article  CAS  Google Scholar 

  22. Tang, W. et al. White fat progenitor cells reside in the adipose vasculature. Science 322, 583–586 (2008).

    Article  CAS  Google Scholar 

  23. Berry, R. & Rodeheffer, M. S. Characterization of the adipocyte cellular lineage in vivo. Nat. Cell Biol. 15, 302–308 (2013).

    Article  CAS  Google Scholar 

  24. Merrick, D. et al. Identification of a mesenchymal progenitor cell hierarchy in adipose tissue. Science 364, eaav2501 (2019).

    Article  CAS  Google Scholar 

  25. Rondini, E. A. & Granneman, J. G. Single cell approaches to address adipose tissue stromal cell heterogeneity. Biochem. J. 477, 583–600 (2020).

    Article  CAS  Google Scholar 

  26. Burl, R. B. et al. Deconstructing adipogenesis induced by beta3-adrenergic receptor activation with single-cell expression profiling. Cell Metab. 28, 300–309 (2018).

    Article  CAS  Google Scholar 

  27. Hepler, C. et al. Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice. ELife 7, e39636 (2018).

    Article  Google Scholar 

  28. Corvera, S. Cellular heterogeneity in adipose tissues. Annu. Rev. Physiol. 83, 257–278 (2021).

    Article  CAS  Google Scholar 

  29. Biernaskie, J. et al. SKPs derive from hair follicle precursors and exhibit properties of adult dermal stem cells. Cell Stem Cell 5, 610–623 (2009).

    Article  CAS  Google Scholar 

  30. Driskell, R. R. et al. Distinct fibroblast lineages determine dermal architecture in skin development and repair. Nature 504, 277–281 (2013).

    Article  CAS  Google Scholar 

  31. Rinkevich, Y. et al. Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential. Science 348, aaa2151 (2015).

    Article  Google Scholar 

  32. Berry, R., Jeffery, E. & Rodeheffer, M. S. Weighing in on adipocyte precursors. Cell Metab. 19, 8–20 (2014).

    Article  CAS  Google Scholar 

  33. Logan, M. et al. Expression of Cre recombinase in the developing mouse limb bud driven by a Prxl enhancer. Genesis 33, 77–80 (2002).

    Article  CAS  Google Scholar 

  34. Sanchez-Gurmaches, J., Hsiao, W. Y. & Guertin, D. A. Highly selective in vivo labeling of subcutaneous white adipocyte precursors with Prx1-Cre. Stem Cell Rep. 4, 541–550 (2015).

    Article  CAS  Google Scholar 

  35. Krueger, K. C., Costa, M. J., Du, H. & Feldman, B. J. Characterization of Cre recombinase activity for in vivo targeting of adipocyte precursor cells. Stem Cell Rep. 3, 1147–1158 (2014).

    Article  CAS  Google Scholar 

  36. Leavitt, T. et al. Prrx1 fibroblasts represent a pro-fibrotic lineage in the mouse ventral dermis. Cell Rep. 33, 108356 (2020).

    Article  CAS  Google Scholar 

  37. Wilk, K. et al. Postnatal calvarial skeletal stem cells expressing PRX1 reside exclusively in the calvarial sutures and are required for bone regeneration. Stem Cell Rep. 8, 933–946 (2017).

    Article  CAS  Google Scholar 

  38. Kawanami, A., Matsushita, T., Chan, Y. Y. & Murakami, S. Mice expressing GFP and CreER in osteochondro progenitor cells in the periosteum. Biochem. Biophys. Res. Commun. 386, 477–482 (2009).

    Article  CAS  Google Scholar 

  39. Chen, K. G., Johnson, K. R. & Robey, P. G. Mouse genetic analysis of bone marrow stem cell niches: technological pitfalls, challenges, and translational considerations. Stem Cell Rep. 9, 1343–1358 (2017).

    Article  CAS  Google Scholar 

  40. Leussink, B. et al. Expression patterns of the paired-related homeobox genes MHox/Prx1 and S8/Prx2 suggest roles in development of the heart and the forebrain. Mech. Dev. 52, 51–64 (1995).

    Article  CAS  Google Scholar 

  41. Martin, J. F., Bradley, A. & Olson, E. N. The paired-like homeo box gene MHox is required for early events of skeletogenesis in multiple lineages. Genes Dev. 9, 1237–1249 (1995).

    Article  CAS  Google Scholar 

  42. Buch, T. et al. A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration. Nat. Methods 2, 419–426 (2005).

    Article  CAS  Google Scholar 

  43. Haas, S., Trumpp, A. & Milsom, M. D. Causes and consequences of hematopoietic stem cell heterogeneity. Cell Stem Cell 22, 627–638 (2018).

    Article  CAS  Google Scholar 

  44. Shin, W. et al. Dysfunction of hair follicle mesenchymal progenitors contributes to age-associated hair loss. Dev. Cell 53, 185–198 (2020).

    Article  CAS  Google Scholar 

  45. Ferrero, R., Rainer, P. & Deplancke, B. Toward a consensus view of mammalian adipocyte stem and progenitor cell heterogeneity. Trends Cell Biol. 30, 937–950 (2020).

    Article  CAS  Google Scholar 

  46. Schwalie, P. C. et al. A stromal cell population that inhibits adipogenesis in mammalian fat depots. Nature 559, 103–108 (2018).

    Article  CAS  Google Scholar 

  47. Wu, H. et al. Bone size and quality regulation: concerted actions of mTOR in mesenchymal stromal cells and osteoclasts. Stem Cell Rep. 8, 1600–1616 (2017).

    Article  CAS  Google Scholar 

  48. Donati, G. et al. Epidermal Wnt/beta-catenin signaling regulates adipocyte differentiation via secretion of adipogenic factors. Proc. Natl Acad. Sci. USA 111, E1501–E1509 (2014).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  50. Brash, J. T. et al. Tamoxifen-activated CreERT impairs retinal angiogenesis independently of gene deletion. Circ. Res. 127, 849–850 (2020).

    Article  CAS  Google Scholar 

  51. Scott, C. C., Vossio, S., Rougemont, J. & Gruenberg, J. TFAP2 transcription factors are regulators of lipid droplet biogenesis. eLife 7, e36330 (2018).

    Article  Google Scholar 

  52. Cattaneo, P. et al. Parallel lineage-tracing studies establish fibroblasts as the prevailing in vivo adipocyte progenitor. Cell Rep. 30, 571–582 (2020).

    Article  CAS  Google Scholar 

  53. Han, X. et al. A suite of new Dre recombinase drivers markedly expands the ability to perform intersectional genetic targeting. Cell Stem Cell 28, 1160–1176 (2021).

    Article  CAS  Google Scholar 

  54. Wagner, D. E. & Klein, A. M. Lineage tracing meets single-cell omics: opportunities and challenges. Nat. Rev. Genet. 21, 410–427 (2020).

    Article  CAS  Google Scholar 

  55. Riazifar, M., Pone, E. J., Lotvall, J. & Zhao, W. Stem cell extracellular vesicles: extended messages of regeneration. Annu. Rev. Pharmacol. Toxicol. 57, 125–154 (2017).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Lu (East China Normal University) and A. Waisman (University of Cologne) for providing genetically-engineered mice. The work was supported by the National Key Research and Development Program of China (2018YFA0800803 to B.L.), the National Natural Science Foundation of China (91749201 to B.L. and 81830075 to L.C.), the Schaefer Research Scholarship (to B.L.) and Major Program of Development Fund for Shanghai Zhangjiang National Innovation Demonstration Zone (Stem Cell Strategic Biobank and Stem Cell Clinical Technology Transformation Platform; ZJ2018-ZD-004 to B.L.). We thank H. Jiang from the Core Facility and Technical Service Center for SLSB, School of Life Science and Biotechnology, Shanghai Jiao Tong University for technical support with FACS analysis.

Author information

Author notes

  1. These authors contributed equally: Huijuan Liu, Ping Li, Shaoyang Zhang.

Authors and Affiliations

  1. Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China

    Huijuan Liu, Ping Li, Shaoyang Zhang, Jinnan Xiang, Ruichen Yang, Jiajia Liu, Md Shafiquzzaman, Soma Biswas & Baojie Li

  2. Department of Osteoporosis and Bone Diseases, Shanghai Clinical Research Center of Bone Disease, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, China

    Huijuan Liu, Zhanying Wei & Zhenlin Zhang

  3. Institute of Traditional Chinese Medicine and Stem Cell Research, Chengdu University of Traditional Chinese Medicine, Chengdu, China

    Xin Zhou & Baojie Li

  4. Translational Medical Center for Stem Cell Therapy, Shanghai East Hospital, Tongji University, Shanghai, China

    Feng Yin & Baojie Li

  5. Department of Joint and Sports Medicine, East Hospital, Tongji University School of Medicine, Shanghai, China

    Feng Yin

  6. Department Of Wound Repair and Rehabilitation Medicine, Center of Bone Metabolism and Repair, State Key Laboratory of Trauma, Burns and Combined Injury, Trauma Center, Research Institute of Surgery, Daping Hospital, Army Medical University, Chongqing, China

    Yangli Xie & Lin Chen

  7. Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, and Department of Microbiology and Immunology, Columbia University, New York, NY, USA

    Stephen P. Goff

Authors
  1. Huijuan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ping Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shaoyang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jinnan Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ruichen Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jiajia Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Md Shafiquzzaman
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Soma Biswas
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Zhanying Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Zhenlin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Xin Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Feng Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Yangli Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Stephen P. Goff
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Lin Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Baojie Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.L., Z.Z. and L.C. designed the study. H.L., P.L., S.Z., J.X., J.L., S.B., Z.W., R.Y., M.S., Y.X. and X.Z. performed the research. H.L. and S.Z. analyzed the data. S.P.G., F.Y., L.C. and B.L. wrote the paper.

Corresponding authors

Correspondence to Lin Chen or Baojie Li.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Genetics thanks Bin Zhou and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Prrx1 is expressed in multiple mesoderm segments and the skeleton, WATs, and dermis in P1 and adult mice.

(a–d) In situ hybridization results showing detection of Prrx1 mRNA in the whole mesoderm of E9.5 embryos (a), skeleton and skin of E14.5 embryos (b), skeleton, iWAT, and dermis of P1 pups (c), and limited numbers of cells in adult connective tissues (d). Arrows indicate the signals. Scale bar: 100 μm. Each experiment was repeated three times independently with similar results obtained.

Extended Data Fig. 2 Prrx1+ cells contribute to 3 adult connective tissues.

(a) A schematic diagram depicting how Prrx1-Cre (KI) mouse was generated. (b) Illustrative images of E8.5 Prrx1-Cre; tdTomato embryos showed that Prrx1 lineage cells were present in the paraxial and intermediate mesoderm in addition to the lateral plate, while E7.5 embryos did not express Prrx1. nt: neural tube; pm: paraxial mesoderm; im: intermediate mesoderm; lp: lateral plate. Scale bar: 50 μm. (c, d) Illustrative immunofluorescent images showed that Prrx1 lineage generated most Col1α1+ osteoblasts and Col2+ chondrocytes (c), most Perilipin+ adipocytes in iWAT as well as other white adipose depots (d). Two-month-old Prrx1-Cre; tdTomato male mice were infused with PFA, and the organs were harvested, frozen sectioned, and stained with various antibodies. The numbers represent the percentages of Tomato+ cells in total tissue-specific cells. Scale bar: 50 μm. Arrows: double-labelled cells. gp: growth plate. (e) Prrx1 lineage generated Vimentin+ or PDGFRα+ dermal fibroblasts, Perilipin+ dermal adipocytes, and αSMA+ arrector pili in the back skin of 2-month-old Prrx1-Cre;tdTomato male mice. Scale bar: 50 μm. Arrows: double-labelled cells. (f) Immunostaining revealed that Prrx1 lineage cells in the bone, iWAT, and dermis of 2-month-old Prrx1-Cre;tdTomato mice did not express CD31. Scale bar: 50 μm. Each experiment was repeated three times independently with similar results obtained.

Source data

Extended Data Fig. 3 Prrx1+ stem cells replenish the special connective tissues in adult mice.

(a) Pulse-chase lineage tracing experiments showed that Prrx1 lineage cells replenished bone, iWAT, and dermis over time. Three-month-old Prrx1-CreERT2; tdTomato male mice received 3 consecutive daily doses of TAM. These mice were sacrificed at 1, 14, 30, 60, 180, or 360 days after the last TAM injection. Various organs were sectioned and stained with DAPI. Scale bar: 250 μm for the bone and 50 μm for the iWAT and dermis. (b) Pulse-chase tracing experiments showed that Tomato+ cells replenished dermal tissues of ventral, facial, and head skin. Scale bar: 50 μm. (c) Pulse-chase tracing experiments showed that Tomato+ cells replenished gonadal and mesenteric adipose tissues. Scale bar: 50 μm. Arrowheads: double-labelled cells. (d) Calcein labelling experiments showed that periosteal bone undergoes little bone formation compared to trabecular bones and endosteal bones in adult mice. Calcein was injected twice into 3-month-old male mice with an interval of 9 days. Arrows: calcein labelling at trabecular and endosteal surfaces not at periosteal surfaces. Scale bar: 250 μm. (e) Pulse-chase lineage tracing experiments showed that few Tomato+ cells were present in articular cartilage and growth plates. Scale bar: 50 μm. (f) Fifteen-month-old Prrx1-CreERT2;tdTomato male mice showed no Tomato+ cells in the skeleton, iWAT, or dermis without TAM administration. (g) Adult Prrx1-CreERT2;tdTomato male mice were injected with 1 dose of TAM and 1 month later, femur, iWAT, and skin were collected, sectioned and stained for Tomato, DAPI and lineage specific markers. Arrows: Tomato+ osteoblasts, adipocytes, or arrector pili; arrowhead: stromal/fibroblasts. Scale bar: 50 μm. Each experiment was repeated three times independently with similar results obtained.

Extended Data Fig. 4 Depletion of Prrx1+ cells leads to defects in homeostasis and repair of bone and skin.

(a, b) The numbers of Prrx1 lineage cells were decreased by TAM and DT injection into 3-month-old Prrx1-CreERT2;iDTR;tdTomato male mice. Stromal cells of iWAT and dermis were released by protease digestion and then analysed with flow cytometry (a), while bone sections of the mice were stained with DAPI (b). Scale bar: 250 μm. Each experiment was repeated three times independently with similar results obtained. (c) Representative images showed comparison of bone (μCT), iWAT, and dermis of Rosa-iDTR mice treated with DT or solvent (Veh). Scale bar: 250 μm for the bone and 50 μm for the iWAT and dermis. Each experiment was repeated three times independently with similar results obtained. (d) H/E staining showed that the growth plate and articular cartilage appeared normal in Prrx1-CreERT2; iDTR mice after depletion of Prrx1+ cells. Scale bar: 50 μm. The experiment was repeated with 6 mice for each group. (e) Depletion of Prrx1+ stem cells led to defects in gonadal and mesenteric adipose tissues. Scale bar: 50 μm. Each experiment was repeated with 3 mice for each group with similar results obtained. (f) A time-course study of the repair of skin puncture wounds when Prrx1+ stem cells were depleted. Bottom panel: quantitation data. Data are presented as mean ± S.D. N = 6 mice. P values were calculated by two-tailed Student’s t-tests. (g) Representative images showed comparison of skin wound healing of Rosa-iDTR mice treated with DT or Veh. (h) Representative images showed comparison of bone fracture healing of Rosa-iDTR mice treated with DT or Veh.

Source data

Extended Data Fig. 5 scRNA-seq analysis of Prrx1+ stem cells.

(a) Violin plots of Pecam1, Ptprc, and Epcam expression in Prrx1+ stem cells of BM, iWAT, and dermis. (b) Heatmap of top 500 genes expressed in the subgroups of BM Prrx1+ cells. (c) Violin plots of signature gene expression for iWAT Prrx1+ cells. (d) Violin plots of signature (DP and dermal fibroblasts) gene expression for dermal Prrx1+ cells.

Extended Data Fig. 6 scRNA-seq analysis of surface marker expression on Prrx1+ stem cells.

(a-c) Violin plots of common cell surface marker expression for BM (a), iWAT (b), and dermal (c) Prrx1+ cells.

Extended Data Fig. 7 Flow cytometric analysis of surface markers in Prrx1+ stem cells and MSCs.

(a) Gating strategies for Tomato+ cells from different tissues of 3-month-old Prrx1-CreERT2;tdTomato male mice (day 1 post TAM). Gating for Tomato+ cells of Prrx1-Cre;tdTomato mice were similar. (b) Flow cytometry results showed surface marker expression on freshly released Prrx1+ cells from BM, iWAT, and dermis of Prrx1-CreERT2;tdTomato mice (day 1 after 3 doses of TAM injection). A total of 106 events were used or BM cells while 105 events were used for iWAT or dermis. Quantitation data were shown in supplementary Table 4. (c) Flow cytometry results showed surface marker expression on freshly released all Tomato+ cells from BM, iWAT, and dermis of Prrx1-Cre;tdTomato mice. A total of 105 events were used or BM cells while 5×104 events were used for iWAT or dermis. Quantitation data were shown in supplementary Table 4. (d) Flow cytometric analysis showed that CD130 could be used to enrich Prrx1+ stem cells. CD31−CD45−CD29+CD130+ cells of BM or iWAT and CD31−CD45−ITGAV+CD130+ cells of dermis of Prrx1-CreERT2;tdTomato mice (day 1 after 3 doses of TAM injection) were analysed for Tomato signals.

Extended Data Fig. 8 Analysis of Thy1−CD105−6C3−ITGAV+CD200+ BM SSCs.

(a) tSNE analysis of the BM SSCs (Thy1−CD105−6C3−ITGAV+CD200+) scRNA-seq data, which were divided into root, osteoblast, chondrocyte, and stromal subgroups. Violin plots showed the expression of marker genes. (b) Heatmap showed expression of CD130 and Prrx1 in the 4 subgroups. (c) KEGG and GO analyses of CD130+ cells against CD130− cells. One-sided Fisher’s exact test was performed, and −log10(P values) are shown.

Extended Data Fig. 9 Flow cytometric analysis of surface marker expression in transplanted Prrx1+ cells.

(a) Representative μCT results showed that transplanted Prrx1+ (Tomato+ITGAV+CD130+) cells underwent ossification. The scaffold was marked in red while the ossified matrix was in grey. Scale bar: 250 μm. (b) Flow cytometric analysis of transplanted BM Tomato+ cells. Tomao+ cells were extracted from the scaffolds after 3 months and analysed for expression of CD130 and other markers. (c) Flow cytometric analysis of transplanted iWAT or dermal Prrx1+ cells. Tomato+ cells were extracted from the iWAT or reconstituted skin and analysed for expression of CD130 and other markers. (d) Skin reconstitution experiments showed that Tomato+ITGAV+CD130+ cells isolated from the transplants could differentiate into different dermal cell types when re-transplanted. Since retrieved Tomato+ cells were not sufficient, only a portion of Tomato+ITGAV+CD130+ cells were used. The reconstituted skin samples were sectioned and immunostained with various antibodies. Upper panel: diagram for skin reconstitution experiment. Scale bar: 50 μm. (e) Flow cytometric analysis of re-transplanted BM or iWAT Tomato+CD29+CD130+ cells or dermal Tomato+ITGAV+CD130+ cells. Tomato+ cells were extracted from the implants after 3 months (for BM) or one month (for iWAT and dermis) and analysed for expression of CD130 and other markers. Each experiment was repeated three times independently with similar results obtained.

Extended Data Fig. 10 Autocrine Wnt molecules regulate Prrx1+ stem cell activities and tissue homeostasis.

(a) Immunostaining results showed that Wls ablation led to decreased β-Catenin signalling in the bone, iWAT, and dermis. The sections of various tissues were immunostained for β-Catenin. Arrows: β-Catenin signals. Three-month-old male mice were used. Scale bar: 50 μm. Each experiment was repeated with 3 mice for each group with similar results. (b) Prrx1-CreERT2; Wlsf/f mice showed a decrease in BM CFU compared to control mice. Data are presented as mean ± S.E.M. N = 3. (c) BM Prrx1+ SSCs (isolated and pooled from 3 Prrx1-CreERT2; Wlsf/f;tdTomato mice, day 1 post TAM) showed decreased proliferation rate. Data are presented as mean ± S.E.M. N = 5 repeats. (d) BM Prrx1+ SSCs showed decreased osteoblast differentiation and enhanced adipogenic differentiation with normal chondrocyte differentiation. Left panels: histological staining; right panels: qPCR results showing expression of lineage specific genes. Data are presented as mean ± S.E.M. N = 3 repeats. (e) TRAP staining showed that ablation of Wls in Prrx1+ stem cells greatly reduced the number of osteoclasts on femur bones. Arrows: osteoclasts. Scale bar: 50 μm. (f) iWAT Prrx1+ cells (pooled from 3 Prrx1-CreERT2; Wlsf/f;tdTomato mice, day 1 post TAM) showed reduced cell proliferation. Data are presented as mean ± S.E.M. N = 5 repeats. (g) iWAT Prrx1+ cells showed enhanced adipogenic differentiation. Left panels: histological staining; right panels: qPCR results showing expression of lineage specific genes. Data are presented as mean ± S.E.M. N = 3 repeats. (h) Dermal Prrx1+ cells (pooled from 3 Prrx1-CreERT2; Wlsf/f;tdTomato mice, day 1 post TAM) showed reduced cell proliferation. Data are presented as mean ± S.E.M. N = 5 repeats. (i) Dermal Prrx1+ cells showed enhanced adipogenic differentiation. Left panels: histological staining; right panels: qPCR results showing expression of lineage specific genes. Data are presented as mean ± S.E.M. N = 3 repeats. P values were calculated by two-tailed Student’s t-tests.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–6 and Supplementary Tables 1–4.

Reporting summary

Peer Review File

Supplementary Data

Statistical supporting data for Supplementary Figs. 1, 3–5.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 10

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, H., Li, P., Zhang, S. et al. Prrx1 marks stem cells for bone, white adipose tissue and dermis in adult mice. Nat Genet 54, 1946–1958 (2022). https://doi.org/10.1038/s41588-022-01227-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41588-022-01227-4

This article is cited by

Search

Advanced 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