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A Twist2-dependent progenitor cell contributes to adult skeletal muscle

Nature Cell Biology volume 19, pages 202–213 (2017)Cite this article

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Abstract

Skeletal muscle possesses remarkable regenerative potential due to satellite cells, an injury-responsive stem cell population located beneath the muscle basal lamina that expresses Pax7. By lineage tracing of progenitor cells expressing the Twist2 (Tw2) transcription factor in mice, we discovered a myogenic lineage that resides outside the basal lamina of adult skeletal muscle. Tw2+ progenitors are molecularly and anatomically distinct from satellite cells, are highly myogenic in vitro, and can fuse with themselves and with satellite cells. Tw2+ progenitors contribute specifically to type IIb/x myofibres during adulthood and muscle regeneration, and their genetic ablation causes wasting of type IIb myofibres. We show that Tw2 expression maintains progenitor cells in an undifferentiated state that is poised to initiate myogenesis in response to appropriate cues that extinguish Tw2 expression. Tw2-expressing myogenic progenitors represent a previously unrecognized, fibre-type-specific stem cell involved in postnatal muscle growth and regeneration.

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Figure 1: Tw2-expressing and Pax7-expressing cells are distinct cell types in skeletal muscle.
Figure 2: Progressive contribution of Tw2+ cells to adult skeletal muscle.
Figure 3: Ablation of the Tw2+ lineage causes type-IIb-myofibre atrophy.
Figure 4: Tw2+ cells contribute to skeletal muscle regeneration.
Figure 5: Molecular profiling of the Tw2+ lineage in skeletal muscle.
Figure 6: Myogenic potential of Tw2+ cells in culture.
Figure 7: Myogenic and osteogenic potential of tdTO+/CD34− cells in vitro.
Figure 8: Overexpression of Tw2 blocks myogenesis in vitro.

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Acknowledgements

We are grateful to UT Southwestern Flow Cytometry Core Facility, the Moody Foundation Flow Cytometry Facility, and UTSW Genomics and Microarray Core Facility for technical help and service. We thank C. Nolen for technical assistance and D. Garry, M. Rudnicki, M. Buckingham and H. Sadek for constructive suggestions. This work was supported by grants from the NIH (HL-077439, AR-067294, HL-130253, DK-099653 and U01-HL-100401) and the Robert A. Welch Foundation (grant 1-0025 to E.N.O.). N.L. was supported by a Beginning-Grant-In-Aid (13BGIA17150004) from the American Heart Association. G.A.G. is a research Fellow supported by the Sarnoff Cardiovascular Research Foundation.

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Authors and Affiliations

  1. Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, Sen. Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA

    Ning Liu, Glynnis A. Garry, Stephen Li, Svetlana Bezprozvannaya, Efrain Sanchez-Ortiz, Priscilla Jaichander, Rhonda Bassel-Duby & Eric N. Olson

  2. Department of Clinical Sciences, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA

    Beibei Chen

  3. Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA

    John M. Shelton

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  1. Ning Liu
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Contributions

Experiments were designed by N.L., G.A.G. and S.L. Experiments were performed by N.L., G.A.G., S.L., S.B., E.S.-O., J.M.S. and P.J. Data were interpreted by N.L., G.A.G., S.L., B.C., R.B.-D. and E.N.O. The paper was written by N.L. and E.N.O.

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Correspondence to Ning Liu or Eric N. Olson.

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Integrated supplementary information

Supplementary Figure 1 Analysis of Tw2 expression in skeletal muscle.

(a) CPM (counts per million) of Tw2, MyoD and Myh4 expression by RNA-seq in G/P muscles of WT mice at 1, 2 and 4 months of age. Data are mean ± s.e.m.N = 3 mice for each time point. (b) Real-time RT-PCR demonstrates Tw2 mRNA is enriched in mononuclear cells of the adult muscle (mono) compared to whole quadriceps muscle (quad). Data are mean ± s.e.m.; N = 3 independent experiments. (c) Immunostaining of Tw2 (red) and Pax7 (green) on transverse sections of G/P muscle of 12-month old WT mice. Myofibers were co-stained with wheat germ agglutinin (white) and DAPI (blue). Arrows indicate Pax7+ cells and arrowheads indicate Tw2+ cells. Scale bar, 50 μm. (d) Quantification of the number of Pax7+ (tdTO+), Tw2+, and Pax7+/Tw2+ double positive cells per field in CTL TA muscles of Pax7-CreERT2; R26-tdTO mice. For each muscle section, at least 6 different fields were quantified and averaged. Data are mean ± S.E.M; N = 3 mice. Statistic source data for a, b, d are provided in Supplementary Table 3.

Supplementary Figure 2 Generation of Tw2-CreERT2 mice and analysis of the Tw2 lineage tracing.

(a) Targeting strategy of the Tw2-CreERT2 allele. Coding region of the Tw2 gene was replaced by a CreERT2-Frt-Neo-Frt cassette by homologous recombination in ES cells. Out of 500 ES cell clones 10% were correctly targeted. (b) Southern blot analysis of ES cells using 5′ probe and 3′ probes to demonstrate correct targeting. Unprocessed original scans of blots are shown in Supplementary Fig. 9. (c) In the absence of TMX treatment, tdTO is not detected in muscles of Tw2-CreERT2; R26-tdTO mice at 9 months of age. Scale bar, 100 μm. (d) Whole mount images showing tdTO+ muscles at 10 days (left) and 5 months (right) post-TMX. At 5 months, all muscles examined showed strong tdTO signals except for the tongue muscle. (e) Transverse-sections of tongue and masseter muscles from Tw2-CreERT2; R26- tdTO mice at 4 months post-TMX were co-stained with My32 (green) and DAPI (blue). Scale bar, 100 μm. (f) Longitudinal-sections of TA muscle from Tw2-CreERT2; R26-tdTO mice at 10 days and 4 months post-TMX were co-stained with My32 (green), Laminin (white) and DAPI (blue). tdTO signal expands through the entire myofibers. Scale bar, 100 μm. (g) Transverse sections of Tw2-CreERT2; R26-tdTO mice at indicated days post TMX were co-stained with wCD31 (green; left panel), an endothelial cell marker and vimentin (green; right panel), a fibroblast marker. Tw2+ cells do not give rise to endothelial cells or fibroblasts in adult skeletal muscle. Scale bar, 100 μm. (h) Type II myofiber specificity after prolonged labeling of Tw2-CreERT2; R26-tdTO mice. Transverse sections of indicated muscles of Tw2-CreERT2; R26-tdTO mice were obtained at 18 months post-TMX. Myosin staining using a type II specific antibody (My32, green) showed only a subset of Type II fibers were labeled by tdTO. Myofibers in tongue muscle were excluded from tdTO expression. Bottom panels show co-staining for type IIa myofibers. Scale bar, 100 μm. (i) Quantification of the percentage of tdTO+ myofibers among all myofibers in each field in G/P muscle at 18 months post-TMX. Data are mean ± S.E.M; N = 3 mice. (j) Seven-month old Tw2-CreERT2; R26-tdTO/+ mice were injected with 3 doses of TMX as described in Fig. 2a. Three months later, muscles were harvested and stained for type II myofibers. Tw2+ cells strongly labeled a subset of type II fibers in G/P, TA, masseter muscles, but not in tongue. Scale bar, 100 μm. (k) Soleus muscle of the same mice described in panel (b) were co-stained with antibodies against type I and type II myofibers. A subset of type II myofibers but not type I myofibers are labeled by tdTO. Scale bar, 100 μm. (l) Whole mount images showed intense tdTO signals in G/P, soleus, masseter and tongue of Pax7-CreERT2; R26-tdTO mice (right) compared to muscles of R26-tdTO mice (left) 8 weeks post-TMX. Mice were treated with the same regimen as shown in Fig. 2a. (m) Myosin staining using a type II specific antibody (My32, green) showed tdTO signals in the majority of myofibers of soleus, tongue and masseter muscle from Pax7-CreERT2; R26-tdTO mice at 8 weeks post-TMX. Scale bar, 100 μm. (n) Pax7-CreERT2; R26-tdTO mice were subjected to CTX injury and transverse sections of TA muscles were analyzed 7 and 14 days later. Contralateral uninjured muscle served as control (CTL). The results showed that Pax7+ cells contribute to all regenerating myofibers (indicated by centralized nuclei) on days 7 and 14 after CTX injury. Sections were co-stained with Laminin (green) and DAPI (blue). Scale bar, 100 μm. (o) Tw2+ cells do not contribute to embryonic myogenesis during development. Sections of Tw2-Cre; R26-tdTO embryos at E10.5, E15.5 and P1 were stained with α-actinin to detect somites (s) and muscle cells (m). Body wall muscles were shown for E15.5 and P1. Scale bar, 100 μm. Statistic source data for i are provided in Supplementary Table 3.

Supplementary Figure 3 Effect of Tw2+ cell ablation on non-muscle tissues.

(a) Cre+; DTA mice were smaller than control DTA mice at 9 months post-TMX. The difference in coat color is due to the mixed genetic background. (b) Whole mount images of G/P, Quad and heart of Cre+; DTA and control DTA mice at 9 months post-TMX. (c) Hematoxylin and eosin staining of liver, kidney, heart, white adipose tissue (WAT) and brown adipose tissue (BAT) from DTA and Cre+; DTA mice at 9 months post TMX. Black scale bar, 1 mm; White scale bar, 100 μm. (d) Quantification of type IIb myofibers per field and total myofibers per field in G/P, quad, mass and soleus muscles of DTA and Cre+; DTA mice. Data are mean ± S.E.M; N = 5 mice for each genotype. Two sample t-test;*: P < 0.05. Statistic source data for d are provided in Supplementary Table 3.

Supplementary Figure 4 Analysis of Tw2+ cells during regeneration following CTX injury and their engraftment capacity.

(a) Co-staining for type IIa myosin revealed regenerated tdTO+ myofibers are not type IIa myofibers. Scale bar, 100 μm. (b) Sections of TA muscle from Tw2-CreERT2; R26-tdTO mice were co-stained with desmin (green) on day 3 after CTX. The majority of tdTO+ cells are negative for desmin. Scale bar, 100 μm. (c) Sections of TA muscle from Tw2-CreERT2; R26-tdTO mice were co-stained with Pax7 (green) on day 7 after CTX. tdTO+ cells are negative for Pax7. Scale bar, 100 μm. (d) Quantification of the number of Pax7+, Tw2+ (tdTO+) and Pax7+/Tw2+ double positive cells per field in Tw2-CreERT2; R26-tdTO on days 7 and 14 post-CTX injury. CTL: contralateral TA muscle. For each muscle section, at least 6 different fields were quantified and averaged. Data are mean ± S.E.M; N = 3 mice. (e) Schematic of lineage tracing with Tw2-CreERT2; R26-mT/mG/+ mice. (f) Transverse-sections of G/P and masseter muscles from Tw2-CreERT2; R26-mT/mG mice at 4 months post-TMX. Tw2+ cells are labeled by GFP expression. All GFP+ myofibers remained TdTO+ in G/P and master, indicating Tw2+ cells fuse with existing myofibers. Scale bar, 100 μm. (g) Freshly isolated Tw2+ cells can engraft and form myofibers when transplanted into the TA muscle of mdx mice. 60,000 freshly isolated Tw2+ cells from Tw2-CreERT2; R26-tdTO mice at 10 days post-TMX were injected into TA muscle of 4 month-old mdx mice, which were injected with CTX 1 day before engraftment. TA muscles were harvested at 4 weeks post-injection and stained with laminin (green) and DAPI (blue) to visualize engrafted tdTO+ myofibers. Scale bar, 100 μm. (h) Quantification of the number of tdTO+ myofibers in the engraftment experiemnts. For each mouse, at least 6 fields of G/P muscle sections were quantified and averaged. Data are mean ± S.E.M; N = 3 mice. Statistic source data for d,h are provided in Supplementary Table 3.

Supplementary Figure 5 FACS analysis of Tw2+ cells.

(a) Representative FACS plots of Tw2+ (tdTO+) cells. Mononuclear cells from Tw2-CreERT2; R26-tdTO mice at 10 days post-TMX were sorted based on expression of tdTO. Approximately 3.3% of all mononuclear cells were positive for tdTO+. Sorting gates were drawn as indicated for both tdTO+ and tdTO- cell populations. (b) FACS plots of cell surface marker expression of tdTO+ cells from the Tw2-CreERT2; R26-tdTO mice at 10 days post-TMX.

Supplementary Figure 6 Properties of Tw2+ cells in culture.

(a) Tw2+ cells isolated by FACS sorting proliferated efficiently in growth medium (GM) and differentiated into multinucleated myotubes in differentiation medium (DM). These cells remain tdTO+ in GM and DM. Bright field images are presented for comparison. Scale bar, 20 μm. (b) Western blotting analysis for Tw2 protein of Tw2+ cells and Pax7+ cells in GM and DM. Ad-Ctl represents protein samples from neonatal rat cardiomyocytes infected with adenoviruses expressing either Twist1 (Tw1) or Twist2 (Tw2). The upper band present in all samples represents a non-specific band. GAPDH protein is detected as loading control. Unprocessed original scans of blots are shown in Supplementary Fig. 9. (c) Heat map of genes expressed in Pax7+ cells and Tw2+ cells in GM and DM identified by RNA-seq analysis (left panel). Heat map of the top 39 genes enriched in Pax7-DM versus Pax7-GM are shown in the middle panel, and the heat map of top 35 genes enriched in Pax7-GM versus Pax7-DM are list on the right panel. Importantly, these genes showed the same trend of enrichment and repression in Tw2-DM versus T w2-GM samples. (d) Tw2+ cells and SCs can fuse with each other to form multinucleated myotubes. Tw2+ cells were isolated by FACS sorting from adult Tw2-CreERT2; R26-tdTO mice 10 days post-TMX, which are labeled by tdTO expression. SCs, which are labeled by GFP expression, were isolated by FACS sorting from CAG-eGFP mice. Equal numbers of Tw2+ cells and SCs were mixed and grown in GM, followed by differentiation in DM. Cells were visualized by direct fluorescence. (e) Myosin immunostaining (My32) revealed formation of multi-nucleated myotubes that express both GFP and tdTO. Scale bar, 100 μm.

Supplementary Figure 7 Growth and myogenesis of tdTO+/CD34− cells in culture.

Freshly sorted tdTO+/CD34− cells were grown in GM for 48 h before being switched to DM to induce myogenesis. Scale bar, 20 μm.

Supplementary Figure 8 Model of Tw2 maintains stemness and blocks myogenesis.

Pax7 expression is not detectable in Tw2+ cells in vivo. However, when removed from their native milieu, Tw2+ cells rapidly down-regulate Twist expression and enter a Pax7+ state en route to a myogenic pathway.

Supplementary Figure 9 Unprocessed original scans of for Fig. 6e, Supplementary Fig. 2b, and Supplementary Fig. 6b.

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Liu, N., Garry, G., Li, S. et al. A Twist2-dependent progenitor cell contributes to adult skeletal muscle. Nat Cell Biol 19, 202–213 (2017). https://doi.org/10.1038/ncb3477

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