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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

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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.

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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.

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.

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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.

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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

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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.

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Correspondence to Lin Chen or Baojie Li.

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The authors declare no competing interests.

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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.

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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.

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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

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