A Twist2-dependent progenitor cell contributes to adult skeletal muscle

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.

Figure 1. Tw2-expressing and Pax7-expressing cells are distinct cell types in skeletal muscle

(a) Immunostaining of Tw2 (red) and Pax7 (green) on transverse sections of gastrocnemius muscle of 3-month old C57Bl6 WT mice. Myofibers were co-stained with wheat germ agglutinin (WGA) (white) and DAPI (blue). Arrows indicate Pax7+ cells and arrowheads indicate Tw2+ cells. Scale bar: 50 um. (b) Quantification of the number of Pax7+, Tw2+, and Pax7+/Tw2+ double positive cells per field in 3-month old C57Bl6 WT mice. Quantification was performed on the top panel images in Fig. 1a. For each muscle section, at least 6 different fields were quantified and averaged. Data are mean ± S.E.M. N=6 mice. (c) TA muscle of C57Bl6 WT mice was injured by CTX injection and harvested on Days 4, 7 and 10 post injury. Muscle sections were stained for Pax7 (green), Tw2 (red), DAPI (blue) and WGA (white). CTL: contralateral TA muscle. Scale bar: 100 um. (d) Quantification of the number of Pax7+, Tw2+, and Pax7+/Tw2+ double positive cells per field in C57Bl6 WT mice on days 4, 7 and 14 post-CTX injury. For each muscle section, at least 6 different fields were quantified and averaged. Data are mean ± S.E.M. N=3 mice. (e) Tw2 expression in TA muscle of Pax7-CreERT2; R26-tdTO mice after CTX injury. Adult Pax7-CreERT2; R26-tdTO mice were treated with TMX for 3 alternating days. One week after the first dose of TMX, CTX injury was performed on TA muscle. Muscles were harvested at Days 4 and 7 post injury and immunostained for Tw2 (green) and DAPI (blue). Scale bar: 100 um. (f) Quantification of the number of Pax7+ (tdTO+), Tw2+, and Pax7+/Tw2+ double positive cells per field in Pax7-CreERT2; R26-tdTO on days 4 and 7 post-CTX injury. Pax7+ cells were detected by tdTO+ signal. 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 b,d,f are provided in Supplementary Table 3.

Figure 2. Progressive contribution of Tw2+ cells to adult skeletal muscle

(a) Schematic of TMX treatment. Tw2-CreERT2; R26-tdTO mice on a mixed genetic background were injected with 1 mg TMX at 8 weeks of age on 3 alternating days. Mice were analyzed at various time points following initial TMX injection. (b) At 10 days post-TMX, Tw2+ cells marked by tdTO+ (red) were located outside of the basal lamina (green) (left panel). Middle panel is an enlarged image of the left panel showing the morphology of Tw2+ cells. In contrast, Pax7+ cells (stained with Pax7 antibody, green) were beneath the basal lamina. Arrow indicates a Pax7+ cell. Scale bar: 100 um. (c) Progressive tdTO labeling of myofibers of G/P muscle at the indicated times following TMX treatment. Transverse-sections of G/P muscle were co-stained with laminin (green) and DAPI (blue). Scale bar: 100 um. (d) Quantification of percentage of tdTO+ myofibers among all myofibers in each field in G/P muscle. Data are mean ± S.E.M. For each time point, N=3 mice were analyzed. For each mouse, between 1000 and 2000 myofibers of G/P muscle were quantified. (e) Transverse sections of different muscle groups obtained from Tw2-CreERT2; R26-tdTO mice at 4 months post-TMX were stained with various fiber type-specific antibodies (green as indicated). Scale bar: 100 um. Statistic source data for d are provided in Supplementary Table 3.

Figure 3. Ablation of the Tw2+ lineage causes type IIb myofiber atrophy

(a) Schematic of TMX treatment. Tw2-CreERT2; R26-DTA/+ (Cre+;DTA) and R26-DTA/+ (DTA) mice on a mixed genetic background were injected with 1 mg TMX at 4 weeks of age on 3 alternating days. Mice were kept on TMX-containing diet until the time of analysis. (b) Measurement of body weight (BW), heart weight (HW) and muscle mass normalized to tibia length (TL) of mice. Data are mean ± S.E.M; two sample t-test; *: P < 0.05. N =5 male mice for each genotype. (c) Type IIb (green) and laminin (red) immunostaining of transverse sections of quad, G/P, and masseter muscles of Cre+;DTA and DTA mice. Scale bar: 100 um. (d) Measurement of mean myofiber cross-sectional area (CSA) of type IIb fibers (left) and other fibers (right). Data are mean ± S.E.M; two sample t-test; *: P < 0.05. **: P < 0.01. ns: not significant. N =5 mice for each genotype. For each muscle, more than 300 fibers were measured and averaged. Statistic source data for b,d are provided in Supplementary Table 3.

Figure 4. Tw2+ cells contribute to skeletal muscle regeneration

(a) Schematic of TMX and CTX treatment on adult Tw2-CreERT2; R26-tdTO mice on a mixed genetic background. CTX was injected into the TA muscle at 1 week after the 1^st dose of TMX. Both contralateral (CTL) and CTX-injured TA muscles were harvested at 7 and 14 days post-CTX. (b) Immunostaining of transverse sections of TA muscles revealed that Tw2+ cells are activated and contribute to regenerating myofibers (indicated by centralized nuclei) on days 7 and 14 after CTX injury. Sections were co-stained with laminin (white) and DAPI (blue). Scale bar: 100 um. (c) Co-staining with IIb myosin (green) with newly regenerated tdTO+ myofibers on day 14 post-CTX. Scale bar: 100 um. (d) Quantification of the percentage of tdTO+ myofibers that are type IIb-positive among all tdTO+ myofibers on day 14 post-CTX. Data are mean ± S.E.M; N=5 mice. (e) Three days post-CTX, occasional small tdTO+ cells expressed desmin (green), indicating that these cells differentiated into desmin-positive myoblasts. Scale bar: 100 um. (f) Transverse-sections of TA muscle on Day 7 post-CTX from Tw2-CreERT2; R26-mT/mG mice on a mixed genetic background at 4 months post-TMX. Asterisk represents a GFP+ regenerated new myofiber that lost mT (tdTO) expression. Scale bar: 100 um. (g) Percent of de novo versus fusion events in Tw2-CreERT2; R26-mT/mG mice on Day 7 post-CTX. Data are mean ± S.E.M; N= 3 mice were analyzed. Statistic source data for d,g are provided in Supplementary Table 3.

Figure 5. Molecular profiling of the Tw2+ lineage in skeletal muscle

(a) Heat map of 4980 genes expressed in freshly sorted Tw2+, Tw2− Pax7+ and Pax7− cells identified by RNA-seq. Cells were isolated by FACS sorting from Tw2-CreERT2; R26-tdTO mice and Pax7-CreERT2; R26-tdTO mice at 10 days post-TMX, respectively. (b) Heat map of the top 23 genes enriched in Pax7+ cells compared to Pax7− cells. (c) CPM (counts per million) of Pax7, MyoD, Tw2, Peg3 expression by RNA-seq in Tw2+, Tw2−, Pax7+ and Pax7− cells. (d) Heat map of top 60 genes enriched in Tw2+ cells relative to Tw2− cells. (e) CPM (counts per million) of PDGFRα and PDGFRβ expression by RNA-seq in Tw2+, Tw2−, Pax7+ and Pax7− cells. (f) Pathways enriched in freshly sorted Tw2+ cells relative to Tw2− cells identified by Ingenuity Pathway Analysis (IPA).

Figure 6. Myogenic potential of Tw2+ cells in culture

(a) Myosin staining (My32, green) revealed the majority of Tw2+ (tdTO+) cells expressed myosin and formed multinucleated myotubes starting at 1 and 2 days in DM. Scale bar: 20 um. (b) Fusion index was calculated as the percentage of tdTO+ nuclei within multinucleated myotubes compared to the total number of tdTO+ nuclei in the culture. Data are mean ± S.E.M; N =3 independent experiments. (c) Real-time RT-PCR revealed that muscle genes, such as Myogenin, Ckm and Myh4 are strongly activated when Tw2+ cells were cultured in DM. Values were normalized to those of the GM sample, which is set as 1. Data are mean ± SEM. N =4 independent experiments. (d) Real-time RT-PCR revealed that Tw1 and Tw2 mRNA was enriched in freshly sorted Tw2+ cells (Sort). However, expression was extinguished once cells were plated in culture. Values were normalized to those of the GM sample, which is set as 1. Data are mean ± SEM. N =4 independent experiments. (e) Western blot analysis for Pax7 protein in Tw2+ and Pax7+ cells. Pax7 protein is expressed in both Tw2+ and Pax7+ cells in GM, but not in cells cultured for 2 days in DM. GAPDH protein was detected as loading control. Unprocessed original scans of blots are shown in Supplementary Fig. 9. (f) MyoD staining of Tw2+ (tdTO+) cells in GM revealed MyoD expression. Scale bar: 100 um. Statistic source data for b,c,d are provided in Supplementary Table 3.

Figure 7. Myogenic and osteogenic potential of tdTO+/CD34- cells in vitro

(a) Myosin staining (My32, green) revealed tdTO+/CD34− cells expressed myosin and formed multinucleated myotubes after 4 and 10 days in DM. Freshly sorted tdTO+/CD34− cells were cultured in GM for 48 hrs before induction of myogenesis. Scale bar: 20 um. (b) Fusion index was calculated as the percentage of tdTO+ nuclei within multinucleated myotubes compared to the total number of tdTO+ nuclei in the culture. Data are mean ± SEM. N =3 independent experiments. (c) Oil Red O staining revealed that only very few tdTO+ cells differentiated into adipocytes. Freshly sorted tdTO+/CD34- cells were cultured in GM for 48 hrs before induction of adipogenesis for 10 days. Cells were first stained with My32 and Hoechst before oil Red O staining. Scale bar: 20 um. (d) Percentage of oil Red O positive cells per field. Data are mean ± SEM. N =3 independent experiments. For each experiment, more than 500 nuclei per field and total of 3 fields were counted and averaged. (e) Alkaline phosphatase staining revealed tdTO+/CD34- cells can form osteoblasts when exposed to osteogenic medium. These cells did not express myosin. Scale bar: 20 um. (f) Clonal analysis of tdTO+/CD34 cells for myogenic potential. Five 96-well plates were seeded by FACS. Single tdTO+/CD34 clones were grown on inactivated MEF feeder layers and induced for myogenesis. Myogenic clones were identified by My32 staining. DAPI staining identified both Tw2+ cells (tdTO+/CD34-) and MEFs. Scale bar: 100 um. (g) Percent of myogenic clones (My32+) of the total surviving clones in the clonal analysis. Data are mean ± SEM. N=5 independent experiments. Statistic source data for b,d,g are provided in Supplementary Table 3.

Figure 8. Overexpression of Tw2 blocks myogenesis in vitro

(a) Tw2+ cells were infected with retroviruses expressing either GFP (retro -GFP) or Tw2 (Retro-Tw2-IRES-GFP) for 24 hours before switching to DM. After 5 days in DM, My32 staining (Purple) was performed to detect myotubes. Scale bar: 100 um. (b) Heat map of genes expressed in GFP− or Tw2-infected Tw2+ cells in GM and DM identified by RNA-Seq. (c) Heat maps of top genes enriched and repressed in GFP-DM vs. Tw2-DM. (d) Pathways of genes enriched in Tw2-DM relative to GFP-DM identified by IPA analysis. (e) Over-expression of Tw2 inhibits myotube formation of Pax7+ cells. Pax7+ cells were infected with retro-GFP or Retro-Tw2-IRES-GFP for 24 hours before switching to DM. After 5 days in DM, My32 staining (purple) was performed to detect myotubes. Scale bar: 100 um. (f) EdU labeling revealed increased DNA-synthesis in Tw2-infected Pax7 cells in DM. Infected Pax7 cells were treated with EdU (10 uM) for 24 hours in DM before harvesting and staining. Scale bar: 100 um.