Sex hormones establish a reserve pool of adult muscle stem cells

An Erratum to this article was published on 28 September 2016

This article has been updated

Abstract

Quiescent satellite cells, known as adult muscle stem cells, possess a remarkable ability to regenerate skeletal muscle following injury throughout life. Although they mainly originate from multipotent stem/progenitor cells of the somite, the mechanism underlying the establishment of quiescent satellite cell populations is unknown. Here, we show that sex hormones induce Mind bomb 1 (Mib1) expression in myofibres at puberty, which activates Notch signalling in cycling juvenile satellite cells and causes them to be converted into adult quiescent satellite cells. Myofibres lacking Mib1 fail to send Notch signals to juvenile satellite cells, leading to impaired cell cycle exit and depletion. Our findings reveal that the hypothalamic–pituitary–gonadal axis drives Mib1 expression in the myofibre niche. Moreover, the same axis regulates the re-establishment of quiescent satellite cell populations following injury. Our data show that sex hormones establish adult quiescent satellite cell populations by regulating the myofibre niche at puberty and re-establish them during regeneration.

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Figure 1: Notch1 activation in cycling SCs at puberty.
Figure 2: Conversion of cycling juvenile SCs into quiescent SCs by Notch signalling.
Figure 3: Defective Notch activation and precocious depletion of SCs by Mib1-null MFs.
Figure 4: Induction of Mib1 in MFs by sex hormones.
Figure 5: Establishment of quiescent SCs via AR/ER-induced Mib1-expressing MFs.
Figure 6: Establishment of adult SC populations by the HPG axis.
Figure 7: Impaired re-establishment of SC populations in skeletal muscle regeneration.
Figure 8: Requirement of sex hormones in re-establishment of quiescent adult SC populations after muscle injury.

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Change history

  • 08 September 2016

    In the version of this Article originally published, in the key in Fig. 1b, the red and black data points were incorrectly labelled; the red data points should have been labelled ‘DHT’ and the black data points as 'Veh'. Furthermore, in the key in Fig. 2c, the red and black data points were also incorrectly labelled; the red data points should have been labelled 'N1ΔSC', and the black data points as 'N1WT'. These errors have been corrected in the online versions of the Article.

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Acknowledgements

We thank S. Kato and Y. Imai for providing Arf/y mice, J.-S. Kang for suggesting useful methods for this study, and S.-K. Lee for helpful comments for manuscript preparation. This work was supported by grants from the Basic Science Research Program (NRF-2014R1A2A1A10052675), the Bio and Medical Technology Development Program (NRF-2011-0019269) and the Korea Mouse Phenotyping Project (NRF-2014M3A9D5A01073930) of the Ministry of Science, ICT and Future of the National Research Foundation, funded by the Korean government, and by the SNU-Yonsei Research Cooperation Program through Seoul National University.

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J.-H.K. designed the study, performed the experiments, analysed the data, and wrote the manuscript. G.-C.H., J.-Y.S., I.K.P., W.J.P., H.-W.J., S.H.B., J.W.S., M.-K.Y. and S.H.H. conducted the experiments. S.H.L. and M.H.C. measured the serum E2 levels using LC–MS/MS and provided helpful comments. Y.-G.K. provided useful materials. D.K.S. performed statistical and bioinformatical analysis. Y.-Y.K. designed the research and wrote the manuscript.

Corresponding author

Correspondence to Young-Yun Kong.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Conversion of cycling satellite cells (SCs) into quiescent SCs by dihydrotestosterone (DHT).

(ac) Ten-day-old mice subcutaneously (s.c.) injected with corn oil (Veh) or DHT. (a) Quantification of Pax7+Ki67+ and Pax7+Ki67 cells per 100 myofibers (MFs). n = 5 animals for each group; data are mean ± s.d.; Bootstrap t-test; P < 0.05. Statistical source data for a is provided in Supplementary Table 2. (b,c) Representative IHC staining for Pax7, p57 (b) MyoD and Ki67 (c) in tibialis anterior (TA) muscles. Scale bar, 25 μm. (d,e) Microarray analysis of Veh or DHT-injected 10-day-old mice s.c. injected with Veh or DHT [GSE64454]. TA muscles were isolated 24 h after the injection. The significantly upregulated genes identified from an independent dataset are depicted as a Venn diagram (d). Selected microarray datasets: (1) Adult SCs in uninjured muscle compared to SCs in injured muscle [GSE471771], (2) quiescent SCs compared to activated SCs in culture conditions [GSE34832], and (3) TA muscles of 10-day-old male mice injected with DHT compared to Veh. Venn diagrams display the number of common genes obtained from the gene expression dataset. Integrative analysis (e). In total 1,031 genes were found to be significantly increased (>2-fold) or decreased (<-2-fold) in Veh and DHT-treated mice. These datasets were used as input for pathway analysis with the Ingenuity pathway analysis (IPA) software (Ingenuity Systems, http://www.ingenuity.com, Redwood City, CA, USA). The graph with the most significant P-values, and the highest-scoring functional pathways are shown. (f) The pathway analysis of Notch signalling and cell cycle-related genes that are expressed at a substantially higher or lower level in DHT-injected TA muscles than in Veh-injected TA muscles. The pathway analysis via IPA software is depicted as containing the majority of Notch signalling-regulated genes. The analysis was performed with the Ingenuity software package. Red and green circles represent upregulated and downregulated genes, respectively. (g) mRNA expressions of Ar, Mib1, Notch target genes (Hes1, Hey1, HeyL), cell cycle inhibitors (p21, p27, p57), Sprouty-1 (Spry1), and Cdc20 in TA muscles after 24 h of DHT treatment. n = 3 animals for each group; data are mean ± s.d.; Two-sample t-test; P < 0.05. (h) Relative expression levels of Notch target genes in myogenic cells (MCs) at the indicated ages. (i) IHC staining of Pax7 and Nicd in TA muscles at the indicated ages. Arrows and arrowheads indicate NicdPax7+ and Nicd+Pax7+ cells, respectively; scale bar, 25 μm.

Supplementary Figure 2 Notch activation converts cycling SCs to quiescent SCs.

(a,b) Relative muscle mass normalized to body weight (a) and mRNA expression in MCs (b). n = 3 animals for each group; data are mean ± s.d.; Two-sample t-test; P < 0.05. Relative muscle masses to body weights were comparable. (c) IHC staining of Pax7. Scale bar, 50 μm. (d,e) Seven-day-old N1WT and N1OE/SC mice were intraperitoneally (i.p.) injected daily with tamoxifen for 3 days. Nicd and nuclear GFP proteins are expressed in the Pax7+ cells of N1OE/SC mice. (d) IHC staining from 10-day-old N1WT and N1OE/SC TA muscles. Arrows and arrowheads indicate Pax7+GFPp57 and Pax7+GFP+p57+ cells, respectively; scale bar, 25 μm. (e) Quantification of Pax7, GFP, and p57 as in d; n = 3 animals for each group; data are mean ± s.d.

Supplementary Figure 3 Analysis of the hindlimb muscles lacking Mib1 in myofibers.

(ac) The expression of Mib1 (a; n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; P < 0.05), IHC staining for Mib1 (b; scale bar, 25 μm), and myonuclei number (c; n = 5 animals for each group; Data are mean ± s.d.; Poisson’s general linear model regression; N.S. not significant) in MFs from 8-week-old Mib1WT and Mib1ΔMFextensor digitorum longus (EDL) muscles. Myonuclei numbers in Mib1WT and Mib1ΔMF MFs were comparable. Statistical source data for a and c is provided in Supplementary Table 2. (d) Morphometric quantification of cross-sectional area validated in Laminin-stained TA muscles, which is comparable in Mib1WT and Mib1ΔMF mice;n = 5 animals for each group; data are mean ± s.d.; χ2 test. (e) IHC staining for MyoD and Ki67 in TA muscles from Mib1WT and Mib1ΔMF mice at the indicated ages. Arrows indicate MyoD+Ki67+ cells; scale bar, 25 μm. (f) Quantification of MyoD+ cells per 100 MFs in TA muscles of Mib1WT and Mib1ΔMF mice at indicated ages. n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; P < 0.05. (g,h) IHC staining (g; scale bar, 25 μm) and quantification (h; n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; N.D. not detected) for Pax7 and MyoG in TA muscles from 4- and 12-week-old Mib1WT mice. The Pax7+/MyoG+ cells were observed only in 4-week-old. Statistical source data for h is provided in Supplementary Table 2. (i) Immunoblotting of Myogenin in SCs isolated from hindlimb muscles of 4-week-old Mib1WT and Mib1ΔMF mice. Numbers represent the relative signal intensity of the designated antibody measured by densitometry. Unprocessed original scans of blots are shown in Supplementary Fig. 9. (j,k) IHC staining for Pax7 (j), Myogenin (k) and activated Caspase3 in TA muscles from 4-week-old Mib1WT and Mib1ΔMF mice. Scale bar, 25 μm. There was no significant increase of apoptotic cells in Mib1ΔMF mice. (l) mRNA expressions of Pax7, Notch target genes, differentiation markers, and stem cell markers in SCs purified from 4-week-old Mib1WT and Mib1ΔMF mice. n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; P < 0.05.

Supplementary Figure 4 MF-specific Mib1 ablation affects precocious depletion of SCs in hindlimb muscles and defective muscle regeneration.

(a) IHC staining for Pax7 and Ki67 in TA muscles from Mib1WT and Mib1ΔMF mice at the indicated ages. Arrows and arrowheads indicate Ki67+Pax7+ and Ki67Pax7+ cells, respectively; scale bar, 50 μm. (b) p21, p27 and Spry1 levels in the MCs from Mib1WT and Mib1ΔMF hindlimb muscles. n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; P < 0.05. (c,d) Optic (c; scale bar, 0.2 cm) and hematoxylin and eosin-stained (d; scale bar, 25 μm) images of BaCl2-injured TA muscles in 8-week-old mice. (e) IHC stained images for Laminin in TA muscles 5 or 10 days after BaCl2 injury. Scale bar, 25 μm. (f) Flow cytometric analysis of SC populations in 8-week-old ContWT (Mib1WT and N1WT), Mib1ΔMF, and N1ΔSC mice. n = 5 animals for each group; data are mean ± s.d.; Tukey’s pairwise comparison test; P < 0.01. Controls are designated as the WT of comparing mutant mice. Statistical source data for f is provided in Supplementary Table 2.

Supplementary Figure 5 DHT and E2 independently drive the conversion of cycling SCs into quiescent SCs.

(ad) The expression of Mib1 (a,c), androgen receptor (Ar, b), and estrogen receptor β (Esr2, d) in TA muscles. Ten-day-old Mib1WT and Mib1ΔMF mice were s.c. injected with Veh, DHT, or E2, and TA muscles were isolated 3 days after injection. Both DHT and E2 treatment induced the expression of the target genes Ar and Esr2, respectively, in Mib1WT and Mib1ΔMF TA muscles. n = 5 animals for each group; data are mean ± s.d.; two-sample t-test; P < 0.05. Statistical source data for ad is provided in Supplementary Table 2. (eh) IHC staining of Pax7 (e,f), MyoD (g,h) and Ki67 in TA muscles from 10-day-old mice injected with Veh, DHT (e,g) or E2 (f,h). TA muscles were isolated 3 days after injection. Most Pax7+ cells were still Ki67+ and the number of cycling MyoD+ cells was maintained by either DHT or E2 treatment in Mib1ΔMF TA muscles, while Pax7+ cells were Ki67 and the MyoD+ cells significantly decreased in Mib1WT TA muscles. Arrows and arrowheads indicate Ki67+ and Ki67 cells, respectively; scale bar, 20 μm. (i,j) Quantification of Pax7 and Ki67 in 10-day-old TA muscles treated with Veh, DHT (i) or E2 (j) as in e,f. n = 3 animals for each group; data are mean ± s.d.; Poisson’s general linear model regression; P < 0.05; N.S. not significant. Statistical source data for i,j is provided in Supplementary Table 2.

Supplementary Figure 6 E2 normally induces Mib1 in MFs lacking Ar.

(a,b) The Cre-mediated recombination was confirmed by PCR in ArΔMF mice at the indicated ages in the shown organs (a) and in myofiber but not in satellite cells (b). Unprocessed original scans of blots are shown in Supplementary Fig. 10. (c) The gross morphology in levator ani (LA) muscles from 4-week-old ArΔMF mice. Scale bars, 0.5 cm. (d,e) IHC staining (d; scale bars, 20 μm), and quantification (e; n = 3 animals for each group; data are mean ± s.d.; Bootstrap t-test; P < 0.05) for Pax7, Ki67, and Laminin in TA muscles from 10-day-old ArWT (upper) and ArΔMF (bottom) mice injected with Veh or E2. TA muscles were isolated from hindlimb muscles 3 days after treatment. Arrows and arrowheads indicate Ki67+Pax7+ and Ki67Pax7+ cells, respectively. Statistical source data for e is provided in Supplementary Table 2. (f,g) Muscle mass (f; n = 3 animals for each group; data are mean ± s.d.; two-sample t-test; P < 0.05), myonuclei numbers in MFs (g; n = 3 animals for each group; data are mean ± s.d.; Poisson’s general linear model regression; N.S. not significant) of 12-week-old ContWT and ArΔMFEsr2−/− mice. Muscle mass, myonuclei numbers were comparable. Statistical source data for f and g is provided in Supplementary Table 2. (h) Mib1 levels in 12-week-old EDL muscles from indicated genotypes. n = 3 animals for each group; data are mean ± s.d.; Tukey’s pairwise comparison test; P < 0.05. (ik) IHC staining for Mib1 (i; scale bar, 100 μm), the expression of Ar and Esr2 (j) in MFs, and the mRNA expression of Notch target genes in MCs (k). n = 3 animals for each group; data are mean ± s.d.; two-sample t-test; P < 0.05. Statistical source data for j and k is provided in Supplementary Table 2. The MFs were isolated from hindlimb muscles of 12-week-old ContWT and ArΔMFEsr2−/− mice.

Supplementary Figure 7 Surgically and pharmacologically disturbed hypothalamic and gonadal axis impair the establishment of SC population.

(a) A schematic diagram for Nal-Lys gonadotropin releasing-hormone antagonist (Antide) injection or orchiectomy (Orx). (b,c) Serum testosterone levels measured by ELISA (b) and estradiol levels measured by LC-MS/MS (c) at indicated ages. n = 3 animals for each group; data are mean ± s.d.; Bootstrap t-test; P < 0.05. (d,e) The expression of Ar and Esr2 in MFs (d) and Notch target genes in SCs (e) from 4-week-old sham and Orx mice. n = 3 animals for each group; data are mean ± s.d.; two-sample t-test; P < 0.05. (f) IHC staining of Pax7 and Ki67 in TA muscles from 4-week-old sham and Orx mice. Scale bar, 50 μm. Quantification data is presented in Fig. 6b left subfigure. (g,h) The expression of Ar and Esr2 in MFs (g) and Notch target genes in SCs (h) from 4-week-old Veh and Antide-treated mice. n = 3 animals for each group; data are mean ± s.d.; Two-sample t-test; P < 0.05. (i) IHC staining of Pax7 and Ki67 in TA muscles from 4-week-old Veh and Antide-treated mice. Quantification data is presented in Fig. 6b left subfigure. Arrows and arrowheads indicate Pax7+Ki67+ and Pax7+Ki67 cells, respectively; scale bar, 50 μm.

Supplementary Figure 8 Genetically disturbed hypothalamic and gonadal axis impairs the establishment of SC population.

(a,c) Gross morphology of seminal vesicles, testes and LA muscles (a; scale bar, 0.2 cm), muscle mass (b; n = 3 animals for each group; data are mean ± s.d.; Two-sample t-test; *P < 0.05) and myonuclei number of EDL muscles (c; n = 3 animals for each group; data are mean ± s.d.; Poisson’s general linear model regression; N.S. not significant) in 8-week-old Gnrh1+/+ and Gnrh1hpg/hpg mice. Muscle mass and myonuclei numbers were comparable in Gnrh1+/+ and Gnrh1hpg/hpg mice. (d,e) The expression of Ar and Esr2 in MFs (d) and Notch target genes in SCs (e). n = 3 animals for each group; data are mean ± s.d.; two-sample t-test; P < 0.05; P < 0.01. (f) IHC staining (f) for Pax7 and Laminin in TA muscles. MFs, SCs, and TA muscles were isolated from 8-week-old Gnrh1+/+ and Gnrh1hpg/hpg mice. Scale bar, 50 μm. (g) Flow cytometric analysis of mononuclear cells from 8-week-old control (sum data of Sham, Veh, and Gnrh1+/+), Orx, Antide-treated, and Gnrh1hpg/hpg hindlimb muscles. n = 3 animals for each group; data are mean ± s.d.; Bootstrap t-test; P < 0.05. (h) IHC staining of Pax7 in Vcam-1+Sca-1 cells sorted from mononuclear cells of Gnrh1+/+and Gnrh1hpg/hpg mice. Scale bar, 50 μm. (i) The representative chromatograms of E2 measurements by LC-MS/MS. n = 3 animals for each group; Data are mean ± s.d.

Supplementary Figure 9 Unprocessed original scans of blots.

Uncropped images of scanned western blots shown in Figures and Supplementary Figures are provided.

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Kim, JH., Han, GC., Seo, JY. et al. Sex hormones establish a reserve pool of adult muscle stem cells. Nat Cell Biol 18, 930–940 (2016). https://doi.org/10.1038/ncb3401

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