Loss of dystrophin expression in Duchenne muscular dystrophy (DMD) causes progressive degeneration of skeletal muscle, which is exacerbated by reduced self-renewing asymmetric divisions of muscle satellite cells. This, in turn, affects the production of myogenic precursors and impairs regeneration and suggests that increasing such divisions may be beneficial. Here, through a small-molecule screen, we identified epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurka) as regulators of asymmetric satellite cell divisions. Inhibiting EGFR causes a substantial shift from asymmetric to symmetric division modes, whereas EGF treatment increases asymmetric divisions. EGFR activation acts through Aurka to orient mitotic centrosomes, and inhibiting Aurka blocks EGF stimulation-induced asymmetric division. In vivo EGF treatment markedly activates asymmetric divisions of dystrophin-deficient satellite cells in mdx mice, increasing progenitor numbers, enhancing regeneration, and restoring muscle strength. Therefore, activating an EGFR-dependent polarity pathway promotes functional rescue of dystrophin-deficient satellite cells and enhances muscle force generation.

Here we report the identification of epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurka) pathways as determinants of asymmetric satellite stem cell divisions through an in-niche muscle stem cell screen. EGF stimulation activates EGFR localized at the basal surface of muscle stem cells and recruits the mitotic spindle assembly protein Aurka to induce apicobasal asymmetric divisions. Small interfering RNA (siRNA)-mediated knockdown of Aurka abolishes EGF-induced asymmetric divisions. Importantly, the EGFR polarity pathway acts independently of dystrophin and can rescue the deficit in asymmetric division in dystrophin-deficient satellite cells. Treatment with exogenous EGF in mdx mice, a mouse model of DMD, enhances the formation of new myofibers, resulting in better muscle function while delaying fibrotic accumulation. Therefore, we conclude that the EGFR pathway can be exploited to restore muscle stem cell polarity and function in DMD.

We recently discovered that deficits in muscle stem cell asymmetric divisions are part of the underlying mechanism that results in progressive wasting of skeletal muscle found in Duchenne muscular dystrophy (DMD), an X-linked genetic disease caused by mutations in the dystrophin gene (). Although dystrophin deficiency in muscle fibers makes them susceptible to membrane damage (), dystrophin deficiency in satellite stem cells results in loss of polarity determination and reduced asymmetric divisions, ultimately leading to diminished production of myogenic progenitors and hindered regeneration. The compounding effect of diminished regeneration with chronic degeneration of fragile myofibers accounts for the eventual replacement of muscle by adipose and fibrotic infiltrates in mouse () and human muscle ().

Stimulating satellite stem cell symmetric expansion results in augmented muscle regeneration with a dramatic increase in satellite cell numbers (). Conversely, promiscuous activation of JAK2-STAT3 mediates the decline of satellite cell self-renewal in aging by biasing satellite stem cells toward asymmetric divisions (). Moreover, cell-autonomous defects leading to loss of polarized p38MAPK signaling in aged cells attenuates self-renewal, whereby pharmacological rejuvenation of aged stem cells can restore muscle function (). Thus, regulation of satellite stem cell asymmetric division is a key control point that affects the efficiency of the muscle regenerative program.

Muscle stem cells, or satellite cells, are essential for the growth and regeneration of skeletal muscle (reviewed in). The majority of satellite cells represent a short-term repopulating cell (), whereas a subset is capable of long-term self-renewal and can give rise to committed progenitors through asymmetric cell divisions (). We term these cells satellite stem cells. A key feature of satellite stem cells is the lack of the myogenic transcription factor Myf5, which can be used to distinguish stem cells from committed progenitors ().

The balance between stem cell self-renewal and differentiation affects the kinetics and efficiency of tissue regeneration. Rather than directly undergoing differentiation, stem cells can give rise to progenitors through asymmetric cell divisions. This creates a layer of regulation that allows stem cells to self-renew as well as imprint the identity of their progeny by asymmetrically segregating fate determinants through polarity, protein trafficking, and cell cycle-dependent mechanisms (). Although many intrinsic mechanisms of asymmetric divisions are conserved across evolution and in different cell types, extrinsic determinants are dependent on the tissue organization and spatial localization of cell fate determinants ().

Together, these results indicate that EGF treatment provides long-term enhancement of muscle strength in mdx mice by slowing progression of the dystrophic phenotype. Thus, EGF stimulation of muscle stem cell asymmetric division results in increased generation of progenitors, improved regeneration potential, and amelioration of disease progression in a mouse model of DMD.

To measure the effect of histological changes on function, we performed in situ measurements of muscle force. Strikingly, TA muscles electroporated with the EGFv generated 32% greater force at tetanus compared with Ev at 30 dpi ( Figure 7 K). Moreover, normalizing to the physiological cross-sectional area, TA muscles electroporated with EGFv exhibited a 25% higher specific force at 30 dpi that remained 17% higher than Ev muscles at 150 dpi ( Figures 7 L and S7 K). Normalizing to maximum force, there was no change in the force frequency response of either group at any time point, suggesting that EGF treatment does not alter fiber type composition ( Figure S7 L).

To determine whether the increased myofiber number was due to increased branching, single EDL myofibers from electroporated and non-electroporated muscles at 150 dpi were isolated. We observed no change in the proportion of non-branched fibers between non-electroporated or electroporated muscles ( Figure S7 F). However, EGFv muscles exhibited an increase in single-branched fibers but decreased numbers of double- and triple-branched fibers compared with Ev muscles ( Figure 7 F). Together, this supports that EGFv mdx muscle exhibits delayed progression of the dystrophic phenotype.

Similar to the effects we observed with short-term EGF treatment, electroporation with EGFv boosted the number of Myog-expressing progenitors at 30 dpi and maintained their numbers at 150 dpi, whereas fewer Myog-expressing cells were found in Ev controls ( Figure 7 D). EGFv increased the total number of myofibers by ∼30% consistently at 30 and 150 dpi ( Figures 7 E–7J) without a change in the distribution of fiber Feret, suggesting that they are not arising from the survival of hypertrophic fibers. Consistent with reduced dystrophic pathology, EGFv reduced the progressive increase in fibrosis and deposition of extracellular matrix proteins, as measured by wheat germ agglutinin staining ( Figures S7 C and S7D).

TA muscles were electroporated at 4 weeks of age, during onset of muscle degeneration, and collected 30 and 150 days post-intervention (dpi). Strikingly, mdx muscles electroporated with an EGF expression vector (EGFv) exhibited an 18% increase in mass by 30 dpi ( Figure 7 B). This increase in muscle mass is reflected in the overall cross-sectional area of muscles electroporated with EGFv compared with the empty vector (Ev) controls ( Figure 7 C).

Although acute damage can read out the capacity of muscle stem cells to facilitate regeneration, DMD is a progressive disease that requires long-term maintenance of muscle tissue against chronic myofiber damage. To address the effects of long-term EGF treatment, we electroporated an expression plasmid containing human EGF cDNA () into mdx TA muscles ( Figure 7 A). Secretion of EGF was validated in vitro through transfection in HEK293T cells ( Figure S7 A).

dpi, days post intervention. In (B)–(K), error bars represent means ± SEM; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005. n = 4 mice for each group at 30 dpi and 3 mice for each group at 150 dpi.

(K and L) Maximum tetanic force (K) and specific maximum force (L) of TA muscles of mdx mice 30 or 150 days after electroporation with Ev or EGFv.

(I and J) Representative mask generated from SMASH analysis of cross sections of TA muscles (I) and size distribution of myofibers from TA muscles (J) of mdx mice 150 days after electroporation with Ev or EGFv.

(G and H) Representative mask generated from SMASH (semi-automatic muscle analysis using segmentation of histology) analysis of cross-sections of TA muscles (G) and size distribution of myofibers from TA muscles (H) of mdx mice 30 days after electroporation with Ev or EGFv.

(F) Proportion of branched myofibers isolated from electroporated EDLs of mdx mice 150 days after electroporation with Ev or EGFv.

(B–E) Muscle mass (B), cross-sectional area (C), quantification of Myog-expressing cells (D), and quantification of myofibers (E) from TA muscles of mdx mice 30 or 150 days after electroporation with empty vector (Ev) or EGF expression vector (EGFv).

To assess the effect of EGF on dystrophin-deficient satellite cells in vivo, EGF protein was intramuscularly injected at the time of and 2 days after injury ( Figure 6 G). Notably, EGF-injected mdx muscles contained 26% more Pax7-expressing satellite cells and 50% more Myog-expressing cells ( Figures 6 J and 6K). Moreover, regenerating myofibers in EGF-treated muscles exhibited increased Feret diameters compared with vehicle-injected controls ( Figure S6 H). These results suggest that intramuscular supplementation with EGF restores generation of myogenic progenitors and enhances regeneration of mdx muscle.

The increased proportion of asymmetric divisions suggests that EGF stimulation has the potential to restore the rate of progenitor production in dystrophin-deficient satellite cells (). To assess whether EGF-driven asymmetric divisions could rescue the reduced generation of myogenic progenitors, single EDL myofibers from mdx mice were cultured for 72 h and immunostained for the expression of Myog. Notably, EGF stimulation of mdx satellite cells increased the number of Myog-expressing cells and total number of myogenic cells ( Figures 6 G and 6H).

To assess whether EGFR signaling can stimulate asymmetric division of dystrophin-deficient satellite stem cells, EDL myofibers were isolated from WT and mdx Myf5-Cre/R26R-eYFP mice and cultured for 42 h with or without EGF. The asymmetric division rate of mdx satellite cells was reduced relative to the wild-type (WT) ( Figure 6 E). Despite an expanded stem cell pool in mdx muscle, absolute numbers of asymmetric satellite stem cell divisions are less than half that of WT counterparts ( Figures 6 F, S6 B, and S6C). EGF treatment of WT satellite cells increased asymmetric division by 29% ( Figures 6 E and 6F), whereas EGF stimulation of mdx satellite cells increased the rate of asymmetric division by 67% ( Figure 6 E). Although EGF stimulation does not completely restore the symmetric and asymmetric division rates of mdx satellite stem cells, EGF treatment balances the absolute number of asymmetric stem cell divisions, similar to the numbers in untreated WT samples ( Figures 6 E and 6F).

Like WT satellite cells, EGF treatment of mdx satellite cells increased the number of apicobasally oriented mitotic centrosomes ( Figure 6 C) and polarized Pard3 ( Figure 6 D). However, the rate of abnormal division in mdx satellite cells, as evidenced by abnormal p-Aurk staining patterns, was unaffected by EGF stimulation. This implies that cell cycle dysregulation is not completely rescued by polarity signaling alone; however, EGF signaling through the EGFR-Aurka-Pard3 axis enforces polarity and facilitates productive asymmetric divisions in mdx satellite cells.

To establish whether EGFR activation is affected by the loss of dystrophin, single EDL myofibers from mdx mice were immunostained for p-EGFR after 1 h of EGF stimulation. p-EGFR was stimulated in mdx fibers treated with EGF ( Figures 6 A and 6B ) in streak-like domains on the basal surface of satellite cells ( Figure 5 A). This suggests that EGFR localization and activation occur normally in mdx satellite cells.

In (B)–(E) and (H)–(J), error bars represent means ± SEM; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005. In (B), n = 3 mice; in (C), (E), and (F), n = 3 WT mice and 7 mdx mice; in (D), n = 3 mice; in (G)–(H), n = 4 WT and 5 mdx mice; in (J) and (K), n = 4 mice for each group.

(J and K) Quantification of (J) Pax7-expressing and (K) Myog-expressing cells on sections from mdx TA muscles 10 days after injury treated with saline (vehicle) or recombinant EGF.

(G and H) Quantification of (G) Myog-expressing cells per mdx myofiber and (H) total myogenic cells (Pax7- or Myog-expressing cells) per mdx myofiber at 72 h of culture in vehicle or EGF-containing medium.

(F) Number of asymmetric divisions per myofiber in WT and mdx myofibers at 42 h of culture in vehicle or EGF-containing medium.

(E) Quantification of asymmetric divisions relative to total satellite stem cell divisions in WT and mdx myofibers at 42 h of culture in vehicle or EGF-containing medium.

(C and D) Quantification of (C) abnormal, planar, and apicobasally orientated mitotic spindles and (D) Pard3 localization in satellite cells on mdx myofibers at 36 h of culture in vehicle or EGF-containing medium.

(B) Quantification of p-EGFR staining in mdx satellite cells on EDL myofibers fixed at 1 h of culture in vehicle or EGF-containing medium.

(A) Signaling status of p-EGFR (green) in Pax7-expressing (red) and DAPI-positive (blue) cells on mdx EDL myofibers at 1 h of culture in vehicle or EGF-containing medium.

Loss of dystrophin in DMD causes a polarity deficit in satellite cells of mdx mice (). Satellite stem cells lacking dystrophin exhibit reduced asymmetric divisions, resulting in diminished generation of progenitors and delayed regeneration.

Similar to pharmacological inhibition of Aurka, knockdown of Aurka by siRNA (siAurka) decreased asymmetric divisions by 59% and increased satellite stem cell numbers by 36% ( Figures 5 D–5F and S5 B). Notably, siAurka did not decrease the rate of the cell cycle, as determined by Ki67 staining (data not shown), or the number of total satellite cells ( Figure S5 C). Importantly, EGF stimulation of satellite cell asymmetric division was abolished following siAurka transfection ( Figures 5 G, S5 D, and S5E). Therefore, we conclude that Aurka is a key effector of EGFR regulation of asymmetric division.

In support of our hypothesis, Aurka has been identified as an EGF-dependent interactor of EGFR in lung cancer cells (). We observed, by reciprocal co-immunoprecipitation (coIP) western blot, EGF-dependent binding between p-EGFR and Aurka in myoblasts ( Figure 5 B). In addition, a proximity ligation assay (PLA) detected a strong interaction between p-EGFR and Aurka in cultured myoblasts that was increased by EGF ( Figure 5 C; Figure S5 A).

Protein phosphorylation profiling using an in situ proximity ligation assay: phosphorylation of AURKA-elicited EGFR-Thr654 and EGFR-Ser1046 in lung cancer cells.

Aurora kinases are a family of kinases that regulate mitosis (). Aurka and/or Aurkb have been suggested to act with upstream regulators in determination of mitotic orientation of symmetric and asymmetric cell divisions (). Consistent with involvement in organizing mitotic centrosomes, Aurka protein is localized at centrosomes in M phase myoblasts ( Figure 5 A). Challenging the idea that Aurka is essential for mitosis, the presence of Aurka at the centrosomes of cycling myoblasts is heterogeneous ( Figure 5 A).

(G) Number of asymmetric satellite stem cell divisions per myofiber at 42 h of culture in EGF-containing medium after transfection with siSCR or siAurka normalized to control medium after transfection with siSCR.

(F) Number of eYFP Neg satellite stem cells per myofiber at 42 h of culture after transfection with siAurka normalized to siSCR.

(D and E) Number of (D) asymmetric and (E) symmetric satellite stem cell divisions per myofiber at 42 h of culture after transfection of siRNA against Aurka (siAurka) normalized to siSCR.

(C) Proximity ligation assay for interactions between Aurka and p-EGFR (red) in serum-starved Pax7-nGFP (green) myoblasts refed for 1 h in vehicle or EGF-containing growth medium. DAPI is shown in blue.

(B) Immunoblotting of reciprocal co-immunoprecipitation of Aurka and p-EGFR in serum-starved myoblasts refed for 1 h in vehicle or EGF-containing growth medium.

(A) Immunofluorescence localization of Aurka (red) at centrosomes in mitotic metaphase (left) and anaphase (right) myoblasts. Pax7 is shown in green; DAPI is shown in blue.

Our observation that EGF regulates mitotic orientation of satellite cells suggests that EGFR signaling induces recruitment of centrosome regulators along the apicobasal axis. We hypothesized that aurora kinases, identified as regulators of asymmetric stem cell divisions in our screen, are effectors of EGFR signaling.

To assess the effect of EGF treatment on other cell types in skeletal muscle, we performed immunofluorescence staining for α-smooth muscle actin (αSMA) and VEGFR2 in EGF-treated TA (tibialis anterior) muscles to quantify changes in muscle vasculature. Neither αSMA nor VEGFR2 showed any change with EGF treatment or when comparing EGFR cKO mice or Pax7-CreERT2 littermates (Figures S3F and S3G). These findings support that differences in Pax7-expressing cells and Myog-expressing cells observed with EGF treatment are not due to EGF promoting vascularization within the muscle.

To understand the effect of EGFR signaling in satellite cells and other cell types that express EGFR in muscle, we developed a satellite cell-specific EGFR conditional knockout (EGFR cKO) mouse model by crossing the Pax7-CreERT2 allele with floxed alleles of EGFR (). Excision of EGFR in satellite cells by tamoxifen treatment did not change the number of Pax7-expressing satellite cells in resting muscle; however, we observed a decreased number of Pax7-expressing cells 10 days after injury compared with tamoxifen-treated Pax7-CreERT2 littermates ( Figure 4 F). Correspondingly, treatment with EGF at early time points after injury (day 0 and 2) increased the number of Pax7-expressing cells and Myog-expressing cells in muscles of Pax7-CreERT2 mice on day 10 post-injury ( Figures 4 G and 4H), whereas EGFR cKO satellite cells did not respond to exogenous EGF stimulation ( Figures 4 G and 4H). These experiments suggest that EGFR signaling in satellite cells is crucial during regeneration and that supplementation with recombinant EGF can enhance the pool of myogenic progenitors during muscle regeneration.

We observed that nTdTsatellite cells gave rise to roughly 3-fold higher engraftment as Pax7-expressing satellite cells compared with nGFPsatellite cells ( Figures 4 B and 4C ), consistent with our previous report (). By contrast, nGFPsatellite cell-derived myoblasts from the same donors lost all self-renewal capacity and failed to engraft as Pax7-expressing satellite cells despite injecting a 10-fold higher number of cells ( Figure 4 D). Strikingly, treatment of nTdTsatellite cells with EGF for 3 h following isolation increased nGFPprogeny and facilitated engraftment as nGFPsatellite cells ( Figure 4 B). This enhanced differentiation as myonuclei by roughly 3-fold while maintaining stem cell self-renewal. Furthermore, consistent with our findings in ex vivo myofiber and primary myoblast culture, EGF had no effect on transplanted nGFPsatellite cells or nGFPmyoblasts ( Figures 4 C and 4D). This evidence supports that the role of EGF is specific to Myf5satellite stem cells, where it promotes generation of Myf5progenitors that amplify and differentiate to muscle fibers.

In (B)–(D) and (F)–(H), error bars represent means ± SEM; ∗ p < 0.05. In (B) and (D), n = 3 donors; in (C), n = 4 donors; in (F), n = 4 mice; in (G) and (H), n = 3 mice.

(G and H) Quantification of (G) Pax7-expressing and (H) Myog-expressing cells on sections from regenerating EGFR cKO or Pax7-CreERT2 TA muscles 10 days after CTX-induced injury treated with control (saline) or EGF protein.

(F) Quantification of Pax7-expressing cells on sections from non-injured and regenerating EGFR cKO or Pax7-CreERT2 TA muscles 10 days after injury.

(B–D) Representative images of (B) 10,000 nTdT POS transplanted satellite stem cells, (C) 10,000 transplanted nGFP POS satellite cells, or (D) 100,000 transplanted nGFP POS myoblast stained with DAPI (blue), GFP (green), TdTomato (red), and Pax7 (gray). Arrows indicate donor-derived Pax7 + satellite cells.

To explore whether EGFR signaling affects Myf5satellite stem cells, Myf5committed satellite cells, or myoblasts in vivo, we crossed the Myf5-Cre allele with a ROSA26R-nTnG allele () consisting of a CMV/β-actin promoter and a loxP flanked nuclear TdTomato (nTdT) and nuclear GFP (nGFP) cassette to generate a Myf5-Cre nTnG transgenic reporter model where all Myf5cells express nTdT and Myf5cells express nGFP. Isolation of nTdTsatellite cells (Myf5) and nGFPsatellite cells (Myf5) allows us to determine the outcome of asymmetric division following transplantation, where nTdT satellite cells undergoing asymmetric division give rise to one nGFPmyogenic progenitor and one nTdTsatellite stem cell ( Figures S4 A–S4E).

Prigge, J.R., Wiley, J.A., Talago, E.A., Young, E.M., Johns, L.L., Kundert, J.A., Sonsteng, K.M., Halford, W.P., Capecchi, M.R., and Schmidt, E.E. (2013). Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei. Mamm. Genome Published online September 11, 2013. https://doi.org/10.1007/s00335-013-9469-8 .

To test whether EGF acts independently of cell polarity by activating Myf5 expression or enforcing myogenic commitment, we studied the effect of EGF on satellite stem cell-derived myoblasts in culture. Real-time qPCR showed no change in Myf5 (eYFP) activation in cultured myoblasts derived from eYFPor eYFPsatellite cells from Myf5-Cre/R26R-eYFP mice ( Figures S3 H and S3I). Importantly, EGF did not promote eYFP protein expression in eYFPmyoblasts ( Figure S3 J). These data support our hypothesis that polarized EGFR in satellite cells promotes apicobasal division within the myofiber niche.

To confirm the specific effect of EGFR signaling, siRNA against EGFR (siEGFR) was transfect into satellite cells on single EDL myofibers from Myf5-Cre/R26R-eYFP mice. Similar to inhibition with lapatinib, siEGFR reduced the rate of asymmetric division by 65% compared with scrambled siRNA (siSCR) ( Figure 3 J). Transfection of siEGFR also increased the rate of symmetric division and increased satellite stem cell numbers by 76% ( Figures 3 K and 3L). This suggests that EGF can change mitotic orientation by recruitment of centrosomes along polarized p-EGFR in satellite cells.

EGFR signaling can orient the mitotic axis of polarized epithelial MDCK (Madin-Darby canine kidney) cells along the apicobasal axis (). Moreover, asymmetric satellite cell divisions occur in an apicobasal orientation (). Therefore, single EDL myofibers were cultured for 36 h, and satellite cell centrosomes were detected by immunostaining for phosphorylated Aurk (p-Aurk). Importantly, EGF increased the proportion of total mitotic satellite cells along the apicobasal axis by 50% ( Figure 3 H). We did not observe changes to the proliferation rate of satellite cells at 36 h with EGF, around 5% of cells undergoing mitosis were marked by p-Aurk staining ( Figures S3 C and S3D). Moreover, there was no change in the rate of S phase entry in eYFPor eYFPsatellite cell populations following EGF treatment, as determined by EdU (5-ethynyl-2’-deoxyuridine) incorporation over the first 20h of culture ( Figure 3 I).

To investigate whether EGFR activation is a driver of asymmetric division, EGF was added to cultures of single EDL myofibers isolated from Myf5-Cre/R26R-eYFP mice. Strikingly, EGF treatment increased the asymmetric satellite stem cell division rate 2.5-fold ( Figure 3 G).

In sublaminar satellite cells, p-EGFR was polarized in 63% of satellite cells ( Figure 3 C) localized to the basal surface on the opposite cortex to the myofiber ( Figure S3 B). Surprisingly, p-EGFR was restricted to a constrained streak-like domain ( Figure 3 B), and this polarized localization was maintained as satellite cells enter M phase ( Figure 3 D). p-EGFR is basolaterally localized in satellite cells on myofibers isolated and immediately fixed following injury-induced activation in vivo ( Figure 3 E; Video S1 ). In injured muscle, p-EGFR and Aurka follow the expression pattern of Pax7, peaking between 3–7 days of regeneration ( Figure 3 F). Early activation of EGFR and Aurka correlates with expansion of the proliferating satellite cell population (), and expression of Myog in differentiating myogenic progenitors ( Figure 3 F). Together, this suggests that basally localized EGFR primes satellite stem cells for asymmetric divisions.

To gain insight into the function of EGFR in satellite stem cell asymmetric division, we examined EGFR localization and activation in vitro and in vivo. Immunofluorescence on muscle sections revealed that EGFR protein is polarized to the basal surface of quiescent satellite cells ( Figure 3 A). To examine the signaling status of EGFR, we immunostained satellite cells on myofibers cultured in serum-free medium with and without EGF stimulation. In quiescent satellite cells, EGFR is inactive by immunostaining activated EGFR phosphorylated on Tyr1068 (p-EGFR) ( Figure 3 B). Following 1 h stimulation with EGF, activated p-EGFR was detected in most satellite cells ( Figures 3 B and 3C).

In (H), (G), and (J)–(L), error bars represent means ± SD; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005. In (C), n = 3 mice; in (E), n = 43 and n = 46 cells, respectively; in (F), n = 2 mice per time point; in (G), n = 6 mice; in (H), n = 5 mice; in (I), n = 3 mice; in (J)–(L), n = 4 mice.

(J–L) Number of (J) asymmetric satellite stem cell divisions, (K) symmetric satellite stem cell divisions, and (L) eYFP Neg satellite stem cells per myofiber at 42 h of culture after transfection with siRNA against EGFR (siEGFR) normalized to scrambled siRNA (siSCR).

(I) Quantification of EdU-labeled satellite cells on myofibers from Myf5-Cre/R26R-eYFP mice cultured in vehicle or EGF-containing medium supplemented with EdU for 20 h and with a 20-h chase prior to fixation.

(H) Number of apicobasally oriented mitotic satellite cell divisions at 36 h of culture in EGF-containing medium normalized to vehicle stained with p-Aurk (green). The host myofiber is outlined with a dashed line. DAPI is shown in blue.

(G) Number of asymmetric satellite stem cell divisions per myofiber at 42 h of culture in EGF-containing medium normalized to vehicle.

(F) Immunoblot analysis of p-EGFR, EGFR, Aurka, Pax7, and Myog expression in uninjured TA muscles or 3, 7, 14, and 21 days after saline or cardiotoxin (CTX) injection.

(E) Analysis of EGFR and p-EGFR localization from injured EDL muscle fixed 2 days after injury and manually dissociated and then stained with DAPI (blue), EGFR or p-EGFR (green), and Pax7 (red).

(D) Polarized p-EGFR (green) staining in mitotic p-H3-expressing (white) and Pax7-expressing (red) satellite cells on EDL myofibers at 36 h. DAPI is shown in blue.

(C) Quantification of p-EGFR staining in satellite cells on EDL myofibers at 1 h of culture in vehicle or EGF-containing medium.

(B) Signaling status of p-EGFR (green) in Pax7-expressing (red) and DAPI-positive (blue) cells on EDL myofibers at 1 h of culture in vehicle or EGF-containing medium.

(A) Localization of EGFR (green) in Pax7-expressing (red) satellite cells on an immunostained muscle section. The basal surface of the satellite cell is attached to a basal lamina that surrounds both the cell and its host fiber. Dashed lines are based on autofluorescence of the myofiber sarcolemma.

To investigate whether inhibition of EGFR or Aurka prevents myogenic differentiation generally, we treated differentiating primary myoblasts with lapatinib, TC-A2317, or vehicle controls. Interestingly, unlike satellite cells on myofibers, inhibition of EGFR signaling in differentiating myoblasts led to a slight increase in activation of the differentiation marker Myog ( Figure S2 B), consistent with reports regarding siRNA knockdown of EGFR in mouse and human myoblasts (). Likewise, Aurka inhibition by TC-A2317 modestly increased the percentage of Myog-expressing cells ( Figure S2 B). These results suggest that EGFR and Aurka have different roles in satellite cell self-renewal and progenitor differentiation.

Similarly, we tested Aurka inhibition using TC-A2317 on single EDL myofibers isolated from Myf5-Cre/R26R-eYFP mice. Comparable with EGFR inhibition, TC-A2317 decreased the rate of asymmetric divisions by 74% ( Figures 2 E and 2F). This shift to symmetric divisions increased the number of satellite stem cells by 77% ( Figure 2 G). Consistently, inhibition of Aurka with TC-A2317 did not affect cell proliferation ( Figure 2 H). These results suggest that EGFR and Aurka signaling regulate the mode of satellite stem cell division.

To confirm our lead compound activity in satellite cell asymmetric division and validate that our findings are not specific to FDB muscle satellite stem cells, we cultured satellite cells on myofibers isolated from extensor digitorum longus (EDL) muscles from Myf5-Cre/R26R-eYFP mice and monitored changes in cell proliferation and symmetric or asymmetric cell division. Inhibition of EGFR signaling resulted in a shift toward satellite stem cell symmetric division, as evidenced by an 83% decrease in the number of asymmetric divisions observed ( Figures 2 A, 2B, and S2 A). This change in satellite stem cell division mode resulted in a 71% increase in the number of satellite stem cells ( Figure 2 C). Importantly, EGFR inhibition did not change the total numbers of satellite cells ( Figure 2 D), indicating that EGFR signaling does not affect cell proliferation.

(G and H) Number of (G) eYFP Neg and (H) total Pax7-expressing satellite stem cells per myofiber at 42 h of culture in the presence of TC-A2317 normalized to the DMSO control (vehicle).

(E and F) Number of (E) asymmetric and (F) symmetric satellite stem cell divisions per myofiber at 42 h of culture in the presence of TC-A2317 normalized to the DMSO control (vehicle).

(C and D) Number of (C) eYFP Neg and (D) total Pax7-expressing satellite stem cells per myofiber at 42 h of culture in the presence of lapatinib normalized to the DMSO control (vehicle).

(A and B) Number of (A) asymmetric and (B) symmetric satellite stem cell divisions per myofiber at 42 h of culture in the presence of lapatinib normalized to the DMSO control (vehicle).

To identify specific gene targets of the EGFR-Erbb and Aurk inhibitors, we examined gene expression using microarray data () to correlate the expression pattern of the EGFR-Erbb and Aurk family with possible regulatory function in satellite cell self-renewal. Only EGFR from the EGFR-Erbb family was highly expressed in freshly isolated satellite cells, whereas Erbb2, Erbb3, and Erbb4 were expressed at low levels ( Figures 1 D and S1 D). This agrees with published data where EGFR protein is detectable in satellite cells and myoblasts but is lost in differentiation (). Aurka and Aurkb were expressed moderately in satellite cells. However, they were highly expressed in proliferating myoblasts, consistent with their function in regulating the cell cycle ( Figures 1 D and S1 E). Aurkc was only expressed late in differentiation ( Figure 1 D). This information suggested that EGFR, Aurka, and Aurkb are likely targets of the inhibitors identified in our screen.

Several compounds effective in stimulating satellite stem cell expansion were inhibitors of the EGFR-Erbb or aurora kinase (Aurk) pathways ( Figure 1 C). Notably, lapatinib, a Food and Drug Administration (FDA)-approved and clinically experienced EGFR-Erbb2 inhibitor, was identified as a lead compound. Furthermore, the top hit from the screen, ZM 449829, and its prodrug, ZM 39923 hydrochloride, have known inhibitory actions on EGFR (). Of the Aurk inhibitors, ZM 447439 and JNJ-7706621 are extensively studied inhibitors against both Aurka and Aurkb (), whereas TC-A2317 is a VX-680 variant that exhibits higher specificity for Aurka ().

Altogether, 640 well-characterized pharmacological compounds were screened against Wnt7a as a positive control (). We identified 43 candidate compounds as inducers of satellite stem cell expansion ( Table S1 ). Consistent with p38MAPK driving satellite cell commitment (), several inhibitors of the p38MAPK pathway, including SB203580, were confirmed to increase satellite stem cell numbers in the screen ( Figure 1 C), validating that the screening platform reliably identified compounds capable of driving symmetric expansion of satellite stem cells.

Recombination at the R26R-eYFP allele results in individual genotypes for satellite stem cells and committed satellite cells. Therefore, primer combinations designed against the recombination status of the R26R-eYFP allele can be used to quantify numbers of satellite stem cells and committed satellite cells by real-time qPCR ( Figures S1 A and S1B). Consistent with manual enumeration of satellite stem cells by immunofluorescence, real-time qPCR quantification of the recombination state of genomic DNA isolated from myofiber cultures accurately detected a 1.5-fold increase in satellite stem cell numbers after Wnt7a stimulation ( Figure S1 C). Thus, this method enables us to analyze changes in the stem cell population. We adapted this system to a 96-well format using myofibers isolated from flexor digitorum brevis (FDB) muscles, which have a higher satellite cell-to-myonuclei ratio and are amenable for transfer by pipetting (), to screen well-characterized small-molecule inhibitors.

The satellite cell microenvironment is required to provide necessary signals for asymmetric divisions (). Therefore, we designed a scalable method to quantify satellite stem cell fate decisions without removing them from their native niche. Using Myf5-Cre () and R26R-eYFP () alleles, Cre-mediated recombination at the R26R-eYFP allele and expression of yellow fluorescent protein following Myf5 activation discriminate Myf5satellite stem cells and Myf5committed satellite cells. Culturing single myofibers from Myf5-Cre/ROSA26-eYFP mice for 42 h, where 80% of satellite cells have undergone a single round of cell division, we can quantify symmetric and asymmetric satellite stem cell divisions as well as committed satellite cell divisions through the expression of eYFP ( Figure 1 A).

(D) Microarray heatmap representing genes from the EGFR-Erbb and Aurk family from prospectively isolated satellite cells, cultured myoblasts in vitro, and 2- and 5-day-differentiated myotubes.

(C) Relative changes to satellite stem cell numbers with small-molecule treatment sorted by changes to eYFPsatellite stem cell numbers compared with vehicle (DMSO) controls. Wnt7a was a positive control. Screening hits are listed in Table S1

(A) Symmetric satellite stem cell division, asymmetric satellite stem cell division, and committed satellite cell division on single Myf5-Cre/R26R-eYFP myofibers after 42 h of culture stained with Pax7 (red), eYFP (green), and DAPI (blue).

Discussion

Neg satellite stem cells through an in-niche small-molecule screen ( Extrinsic regulation of tissue-specific stem cell fate is critical for balancing regeneration and stem cell maintenance. Effectors acting on long-term engrafting stem cells are amplified through the proliferation of committed progenitors. Here we identified EGFR-Aurka signaling as a polarity regulator in long-term engrafting Myf5satellite stem cells through an in-niche small-molecule screen ( Figure 1 C).

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Konig S. Epidermal growth factor receptor down-regulation triggers human myoblast differentiation. Olwin and Hauschka, 1988 Olwin B.B.

Hauschka S.D. Cell surface fibroblast growth factor and epidermal growth factor receptors are permanently lost during skeletal muscle terminal differentiation in culture. EGFR signaling transiently increases during skeletal muscle regeneration ( Figure 3 F), localizing to the basal surface of satellite cells 2 days after injury ( Figure 3 E; Video S1 ). Transient EGFR signaling directs satellite cells to self-renew or give rise to committed progeny during regeneration in vivo. Specific deletion of EGFR in satellite cells dysregulates the stem cell pool and results in a reduction in the number of Pax7-expressing cells by 10 days after injury ( Figure 4 F). Exogenous EGF supplementation enhances the numbers of Pax7-expressing cells and Myog-expressing progenitors, where loss of EGFR expression in satellite cells abrogates the effects of EGF ( Figures 4 G and 4H). Despite reduced numbers of Pax7-expressing cells in EGFR cKO mice, we did not observe fewer Myog-expressing cells during regeneration. These data suggest that EGFR cKO satellite stem cells precociously commit to compensate the demand of progenitors necessary for differentiation or directly differentiate, like previous reports of EGFR downregulation in myoblasts (), resulting in exhaustion of the stem cell pool.

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Rudnicki M.A. Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division. Troy et al., 2012 Troy A.

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Olwin B.B. Coordination of satellite cell activation and self-renewal by Par-complex-dependent asymmetric activation of p38α/β MAPK. Ono et al., 2015 Ono Y.

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Zammit P.S. Muscle stem cell fate is controlled by the cell-polarity protein Scrib. EGFR is an epithelial polarity regulator () that adds to a body of evidence suggesting that quiescent satellite cells share polarity regulators found in epithelial cell types. Unlike polarized epithelial cells, satellite cells do not have a luminal surface. The basal surface of satellite cells attaches to the basal lamina and the apical surface to the myofiber. M-cadherin, a well-known satellite cell marker, and its partner β-catenin are localized on the apical surface and form cell-cell adherence junctions with the myofiber (). Pard3, a member of the Par complex, is localized along the apical surface of satellite cells (). This suggests that the apical surface of satellite cells resembles the apicolateral domain of epithelial cells. Indeed, the polarity effector Scribble is apically distributed in satellite cells and segregates to the committed daughter cell during asymmetric divisions (). This distinction makes satellite cells a unique system to study polarity effector function in asymmetric stem cell division.

Dumont et al., 2015b Dumont N.A.

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Rudnicki M.A. Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division. Importantly, EGF treatment rescues asymmetric divisions in dystrophin-deficient satellite stem cells ( Figures 6 E and 6F). This suggests that redundancies in overlapping polarity signaling can provide compensation toward establishing asymmetric division. Moreover, EGF stimulated the productive generation of myogenic progenitors required to form new myofibers ( Figures 7 E–7J). Unlike mislocalized Par complex proteins in mdx satellite cells (), EGFR is properly localized and activated in response to EGF stimulation ( Figure 6 A). This suggests that EGFR-mediated polarity can function without dystrophin-dystroglycan signaling.

Sambasivan et al., 2011 Sambasivan R.

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Galy A. Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Uezumi et al., 2010 Uezumi A.

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Tsuchida K. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle. Strikingly, mdx muscles electroporated with an EGF-expressing vector are larger, contain more myogenic progenitors to form new myofibers, and have reduced myofiber branching associated with the mdx phenotype ( Figures 7 B–7J). Nascent myofibers inhibit adipogenic differentiation of fibroadipogenic progenitors in the muscle; therefore, tissue fibrosis is directly affected by the rate of muscle regeneration (). In agreement with this, EGF-electroporated muscles had less fibrosis compared with controls ( Figures 7 C and 7D). Importantly, these changes to the muscle architecture are translated to direct enhancements to muscle function, lasting at least 150 days after treatment ( Figures 7 K and 7L). The increase in specific force generation observed with EGF-treated TA muscles indicates additional contractile muscle fibers or an increased myofiber cross-sectional area from reduced fibrosis and attenuates dystrophic progression.

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Blau H.M. Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx/mTR mice. Duddy et al., 2015 Duddy W.

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Partridge T. Muscular dystrophy in the mdx mouse is a severe myopathy compounded by hypotrophy, hypertrophy and hyperplasia. Yucel et al., 2018 Yucel N.

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Blau H.M. Humanizing the mdx mouse model of DMD: the long and the short of it. Importantly, balancing symmetric expansion and asymmetric commitment in satellite stem cells is critical to maintain muscle regeneration in DMD. Stimulation of satellite cell expansion with compounds such as Wnt7a () can boost regenerative outcomes by promoting a larger pool of satellite cells able to offset deficits in differentiation. Speculatively, prolonged satellite cell expansion may result in proliferative stress and could accelerate telomere shortening, leading to stem cell exhaustion (). Reorienting satellite cell divisions to promote myogenic commitment with EGF tips the balance to promote an increase in the absolute number of asymmetric division similar to a WT context ( Figure 6 F) and improve regenerative outcomes ( Figure 7 ). The effect of EGF stimulation on human satellite cell function is an exciting prospect because the pathological progression of human DMD is more severe than the mdx mouse model ().

Our findings provide proof-of-principle evidence to support functional rescue of mdx satellite cells in vivo by stimulating the EGFR-driven polarity pathway. We envision that stimulation of muscle stem cell function can be combined with dystrophin restoration to restore muscle function in DMD. Future studies will further elucidate the role of EGFR signaling in the regulation of stem cell polarity and the potential for small-molecule activation of EGFR signaling as a therapeutic modality for the treatment of DMD.