Previous transcriptomic analyses have detected MuSK mRNA in quiescent, activated and regenerating SCs(Petrany et al., 2020; Shcherbina et al., 2020; Yue et al., 2020). We confirmed that MuSK transcripts are expressed in SCs isolated from uninjured and regenerating muscle from both 3- and 5- month old mice using gel-based PCR analysis (Fig. 1a-c; Supp. Fig 1a, b). This analysis also showed that all MuSK detected in WT SCs harbors the Ig3 domain (encoded by exons 6 and 7). For clarity we refer to the form containing the Ig3 domain as Full-Length (FL)-MuSK. We did not detect transcripts lacking the Ig3 domain (ΔIg3-MuSK) in isolated WT SCs (Fig. 1c). To detect MuSK protein in SCs we isolated myofibers from adult muscle and double labeled them with antibodies recognizing Pax7 and MuSK. As shown in Fig. 1d, MuSK protein is expressed in SCs from both WT and ΔIg3-MuSK mice.

As a first step towards assessing the role of the MuSK-BMP pathway in SC dynamics we compared both SC number and myofiber diameter in WT and ΔIg3-MuSK TA from 3-month-old mice. We chose this time point since adult SC number and myonuclear content are established by this age and SCs are in their adult quiescent state(Ancel et al., 2021; Bachman and Chakkalakal, 2022; Cramer et al., 2020). Analysis of Pax7+ cells showed that their density was comparable in both genotypes at 3 months (Fig. 2a, c). TA myofiber diameter was also comparable in both genotypes at this age (Fig. 2a, b), which is in agreement with results in a related study(Jaime et al., 2024). These results indicate that the MuSK-BMP pathway does not play a discernable role in the developmental and early postnatal events that establish myofiber size and SC pool numbers in fast muscle.

To determine whether the MuSK-BMP pathway plays a role in SC quiescence in older mice, we next assessed SC number and myofiber diameter at 5 months of age. Both measures showed striking differences between genotypes at this age. SC density in the 5-month-old TA was reduced by almost half in ΔIg3-MuSK compared to WT mice (19±2.1 and 10.6±1.4mm² in WT vs ΔIg3-MuSK, respectively; n=9-10, p=0.004; Fig. 2a, c). On the other hand, myofiber diameter in ΔIg3-MuSK muscle was increased by ∼10% compared to WT (37 ±0.8µm and 41 ± 1.0µm in WT vs ΔIg3-MuSK, respectively; n=8, p=0.006; Fig. 2a, b).

We next asked whether the increase in myofiber size observed in 5-month-old mice was associated with improved muscle function. We performed grip strength analysis of ΔIg3-MuSK male mice at 3- and 5- months of age. As shown in Fig. 2, grip strength is comparable between ΔIg3-MuSK and WT mice at 3 months. In contrast, at 5 months, male ΔIg3-MuSK mice display increased grip strength performance (Fig. 2d). This increase in grip strength was observed in 3 cohorts of male mice (Supp. Fig. 2). We also tested grip strength in one cohort of female mice. Although the average grip strength was higher in 5 month old ΔIg3-MuSK females compared to controls, this effect did not reach statistical significance (Supp. Fig. 2). Thus, muscle function is improved in 5- month-old male ΔIg3-MuSK animals.

The observation that SC numbers are decreased while myofiber diameter is increased in 5- month-old ΔIg3-MuSK TA muscle suggested that SCs in these adult muscles became activated and fused into the myofiber, thus contributing to its growth. To test this idea directly we counted the number of myonuclei in isolated myofibers from 5-month-old ΔIg3-MuSK and WT EDL muscles, an accessible and readily quantifiable model for such measurements. As shown in Fig. 3a, b, the number of myonuclei/myofiber was increased in ΔIg3-MuSK EDL muscle. In addition, the ΔIg3-MuSK myofibers were also longer (Fig. 3a, c). Importantly, the proportion of myonuclei per myofiber length is equivalent at both ages, suggesting that the size of the myonuclear domain is conserved (Fig. 3d). Taken together, these findings indicate that between 3 and 5 months of age in ΔIg3-MuSK muscle SCs become activated, divide, and fuse into the myofiber - resulting in increased length. Further, the conserved proportion of myonuclei/myofiber length indicates that this growth increase scales with the addition of the myonuclei to the myofiber.

We next queried the role of ΔIg3-MuSK in muscle regeneration, a chief function of adult SCs. TA muscles from 5-month-old WT and ΔIg3-MuSK TA were injured on day 0 by BaCl₂ injection and muscles were harvested at 3-, 5-, 7-, 14-, 22-, and 29-days post-injury (dpi; Fig. 4). We assessed muscle morphology, myofiber diameter, and SC density at each timepoint. The ΔIg3-MuSK muscle regenerated efficiently, with full restoration of muscle structure achieved by 22 dpi, which was comparable to WT (Fig. 4b, c, e). The regeneration rate was increased as judged by histoarchitecture (Fig. 4b, c), increased Pax7+ density at 5 dpi (Fig. 4d) and, in the male animals, increased myofiber diameter at 7 dpi (Fig. 4e). The increased Pax7+ cells at 5 dpi and the increased myofiber size at 7dpi indicate that the early stages of muscle regeneration are accelerated in ΔIg3-MuSK mice.

Notably, the myofiber diameters at the completion of regeneration (22 dpi) were comparable in both genotypes (Fig. 4e). Moreover, the density of SCs in the regenerated muscle (22 and 29 dpi) was indistinguishable in WT and ΔIg3-MuSK (Fig. 4d). This comparable restoration of myofiber size and SC density indicates that the intrinsic regulatory mechanisms governing SC dynamics during regeneration, including stemness and regenerative capacity, are maintained in ΔIg3-MuSK muscle.

The studies presented above utilized mice with constitutive (germline) knock-in of the ΔIg3-MuSK allele. However, MuSK is also highly expressed by neuromuscular junction (NMJ) myonuclei, and at lower levels in subsets of myonuclei and interstitial cells(Jaime et al., 2024; Petrany et al., 2020; Zeng et al., 2022; Zhang et al., 2021). Thus, there were two key questions outstanding: 1) Is the function of the MuSK-BMP pathway in SC dynamics cell autonomous; and 2) are there developmental events regulated by this pathway that might contribute to its role in adult SCs? To address both these points we utilized tamoxifen-inducible, conditional Pax7^CreERT2;R26R^{LSL- tdTomato};MuSK-Ig3^loxP/loxP mice. These mice license the expression of ΔIg3-MuSK only in cells expressing Pax7 and at the age determined by the tamoxifen administration. In addition, it marks them with the TdTomato reporter. As a control, we used Pax7^CreERT2;R26R^LSL-tdTomato, in which tamoxifen injection induces TdTomato expression in Pax7+ cells, but the MuSK allele is unaffected. We induced Cre-recombination using tamoxifen administered at 3 months of age and then harvested the TA muscles at 5 months of age (Fig. 5a). PCR analysis of RNA isolated from pre-fixed, FACS-sorted SCs confirmed that the Cre-recombination of the floxed Ig3 domain of MuSK was effective (Supp. Fig. 3a, b). As shown in Fig. 5, Cre-mediated expression of ΔIg3-MuSK in SCs at 3 months resulted in an ∼9% increase in both muscle weight and myofiber diameter when assessed at 5 months of age (0.035±0.0007 and 0.038±0.0007 g, n=5-8 mice/group, p=0.007; and 36.0±0.78 and 39.3±1.2 µ, n=5-7 mice/group, p=0.037 for weights and diameters, respectively; Fig. 5). Finally, we also counted the number of myonuclei per myofiber in EDL muscles from the same animals (see Methods). Fig. 5e shows that there was an ∼22% increase in the myonuclear number in the Pax7^CreERT2;R26R^LSL-tdTomato;MuSK-Ig3^loxP/loxP compared to controls (Pax7^CreERT2;R26R^LSL-tdTomato; 234.2±9.8 and 286. ±16.2, respectively, t-test, p=0.008; n=3 per genotype). The increased TA weight, myofiber size and myonuclear number observed in these experiments indicates that MuSK acts in a SC-autonomous manner and that the expression of MuSK in the adult SC is sufficient for this role.

The results presented above indicate that the MuSK-BMP pathway is important for maintaining SC quiescence in resting adult muscle, and that perturbation of this pathway leads to the activation of these stem cells. To provide direct evidence for such activation and to gain insight to the mechanistic basis of this state change we interrogated the gene expression of FACs-sorted SCs from uninjured 5-month-old WT and ΔIg3-MuSK muscle (n= 6 animals/genotype). To preclude artefactual activation during cell isolation, animals were perfusion-fixed prior to harvest. We then used the NanoString nCounter Stem Cell Characterization Panel to assess gene expression in WT and ΔIg3-MuSK SCs. This panel includes 770 genes involved in multiple stem cell types including stem-cell regulatory signaling pathways, epigenetics, metabolism, differentiation signaling, and lineage specification. Hierarchical clustering revealed significant changes (p < 0.05) in the expression of 21 of the genes in this panel. Nineteen of the differentially expressed genes (DEGs) were upregulated including TBLX1, HDAC2, PPP2R2A, SMAD4, CAP2, CREBBP, MDH1, NCOR1, RRAGC and CUL1; while two genes, XIST and CACYBP, were downregulated (Fig. 6a, b).

We next asked whether the set of DEG SC genes detected in uninjured ΔIg3-MuSK were characteristic of an activated state. We compared the hits in the ΔIg3-MuSK nCounter set to a published RNAseq dataset of SC DEGs from resting and injured (1 dpi) WT muscle from young (2-3 months) and aged (20-22 months) muscle (GSE121589; Shcherbina et al., 2020). Our analysis revealed striking similarities in the sets of SC DEGs from uninjured ΔIg3-MuSK and damaged WT muscles. As shown in Fig. 6a, b, 17 of the 19 upregulated genes in uninjured ΔIg3-MuSK SCs are also upregulated in SCs from old and/or young activated WT SCs. XIST, one of the two genes downregulated in ΔIg3-MuSK SCs, was also reduced in both young and aged SCs from damaged WT muscle. This concordance of gene expression patterns indicates that ΔIg3-MuSK SCs in 5-month-old uninjured muscle are in a state of early activation.

Mice were bred and housed at the Brown University Animal Care Facility (ACF). Animals were group-housed in cages of up to five animals. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Brown University and done in accordance with institutional guidelines for animal care.

Germ line MuSK^ΔIg3/ΔIg3 mice, referred to throughout this paper as ‘ΔIg3-MuSK’ mice, were generated by Crispr-mediated removal of exons 6 and 7 of MuSK, which encode the Ig3 domain and were on the C57Bl/6 background. Details of the production and characterization of this model have been reported(Jaime et al., 2024).

For conditional mutations we generated a MuSK-Ig3^loxP/loxP mouse using CRISPR/Cas9, in which the exons encoding the Ig3 domain of MuSK (exons 6 and 7) are flanked with loxP sites. Tamoxifen-inducible Pax7^CreERT2;R26R^LSL-tdTomatomice were provided by Bradley Olwin(Pawlikowski et al., 2015). These lines were crossed to generate Pax7^CreERT2;R26R^LSL-tdTomato;MuSK-Ig3^loxP/loxPmice. Recombination was induced with five daily intraperitoneal (IP) injections of tamoxifen (Sigma-Aldrich) in corn oil dosed at 0.1 mg tamoxifen/ g mouse weight. Mice were treated with tamoxifen at 3 months of age to induce Cre-recombination (Supp. Fig. 2).

For Pax7 antibody staining muscles were harvested from perfusion-fixed animals. Briefly, animals were anesthetized using 2,2,2-Tribromoethanol (250 mg/kg, Sigma Aldrich) followed by perfusion with 20mL of 4% PFA in PBS prior to TA harvest. Muscles were dehydrated in 30% sucrose in PBS overnight, frozen in OCT using liquid nitrogen, and stored at −80°C. In other experiments mice were euthanized with CO₂ followed by cervical dislocation. TA muscles were then flash frozen using freezing isopentane and stored at −80°C.

Frozen sections (10µm) were obtained on a Leica Cryostat. Sections from flash frozen muscle were post-fixed in 4% paraformaldehyde (PFA) for 10 minutes at room temperature. Tissue sections and isolated myofibers were permeabilized with 0.25% Triton-X100 (Sigma-Aldrich) in PBS followed by blocking with 2% bovine serum albumin (Sigma-Aldrich) and 5% goat serum (Thermo Fisher) in PBS. Sections and myofibers that were stained with anti-Pax7 antibody (DSHB) also included a block with 1:50 Mouse on Mouse Ig Blocking Reagent (MKB-2213, Vector Labs). Incubation with primary antibody was at 4°C overnight followed by incubation with secondary antibody at room temperature for 1 hr. To visualize EdU incorporation, the Click-it Plus EdU Cell Proliferation kin, Alexa flour-488 kit (C10637, Thermo-Fisher) was used following the manufacturer’s instructions. Primary antibodies were 1:20 anti-Pax7 (Developmental Studies Hybridoma Bank), 1:1000 rabbit anti-laminin (L9393, Sigma-Aldrich), MuSK myasthenia gravis (MG) patient IgG4 fraction was generously provided by M. Huijbers(Huijbers et al., 2013). Secondary antibodies against IgG1 or IgG of the appropriate species were conjugated to Alexa-488, Alexa-555, or Alexa-647 (Invitrogen) and used at 1:1000. Myofibers were incubated with 1µg/mL DAPI (D1306, Invitrogen) for 5 minutes at room temperature and then mounted in Mowiol supplemented with DABCO (Sigma-Aldrich) as an anti-fade agent. Sections were mounted with Vectashield+DAPI as an anti-fade agent (Vector Laboratories).

Immunofluorescence imaging was performed using a Nikon E800, a Nikon Ti2-E Widefield, or a Zeiss LSM 800 Confocal Microscope. Data acquisition was performed using the Zen software (blue edition, Zeiss). Images used for Pax7 counting and myofiber size analysis were collected and scored blinded.

ΔIg3-MuSK mouse muscles used for myofiber cross sectional size analysis were from perfusion fixed animals and Pax7^CreERT2;R26R^LSL-tdTomato;MuSK-Ig3^loxP/loxP muscles used for myofiber cross sectional size analysis were from flash frozen muscle. Images of TA muscle sections immunostained with anti-laminin antibody (L9393, Sigma-Aldrich) and captured using a Nikon E800 fluorescent microscope or with a Zeiss LSM 800 Confocal Microscope with a 20X objective (images within the same experiment were captured using the same microscope). Images were processed using MyoVision software to determine minimum Feret diameter measurements for each myofiber(Briguet et al., 2004; Wen et al., 2018). Output images were manually examined, and any misidentified fibers were excluded, 650-1800 fibers examined per mouse. Representative images were acquired with a Zeiss LSM 800 Confocal Laser Scanning Microscope with a 20x objective. The average myofiber min Feret diameter for each mouse was used for statistical analysis using an unpaired t-test in GraphPad Prism 9. Analysis was done blinded.

Extensor digitorum longus (EDL) muscle was dissected and enzymatically digested in 400-U/mL collagenase II (Worthington) at 37°C for 1.5 hours. Collagenase was inactivated by the addition of DMEM supplemented with 15% horse serum. Flame-polished Pasteur pipettes were used to agitate and separate individual myofibers, which were then immediately fixed in 4% paraformaldehyde for 10 minutes, followed by 3 washes of PBS prior to any immunostaining.

After immunostaining myofibers (see “Immunohistochemistry” methods), z-stacked tiled images were taken of whole myofibers with a Nikon Ti2-E Widefield Fluorescence Microscope using Zen software (blue edition, Zeiss). The automated measurement function was used to quantify all DAPI stained nuclei as well as total myofiber length.

Grip strength analysis was done to assess front-limb muscle strength of WT and ΔIg3-MuSK male and female mice at 3 and 5.5 months of age. Mice were lifted by their tail and guided to use both front paws to grasp the pull-bar assembly connected to the grip strength meter (Columbus Instruments). The mouse was pulled away from the sensor until their grip on the bar was broken and the peak amount of force in kilograms per unit force (KGF) was recorded. Five trials were performed sequentially per animal and then averaged together and normalized to mouse weight. Analysis was done blinded to genotype.

Briefly, 5-month-old mice were anesthetized with isoflurane (induced with 4% isoflurane, maintained at ∼1.5% isoflurane and 400 mL O2 per min) and placed on a temperature-controlled platform to maintain core body temperature between 35°C and 37°C. Injury to the left TA muscle was induced by first piercing the muscle with a 28-gauge needle ∼15 times followed by injection of 50μL of 1.2% BaCl2 in PBS. To relieve pain, mice were treated with 0.5mg/mL of Buprenorphine Sustained-Release (Bup-SR) at the time of injury. For EdU labeling, mice received 50mg/kg 5- ethynyl-2’-deoxyuridine (EdU, Thermo Fisher) in PBS by intraperitoneal injection 24 hr before harvest. Injured muscle was harvested at 3-, 5-, 7-, 14-, 22-, and 29-days post injury (dpi). Control muscle from uninjured mice was also collected.

To prevent artificial activation of quiescent SCs during isolation, mice were perfusion fixed with 0.5% PFA and then muscle was digested with collagenase II (Worthington) as described(Yue et al., 2020; Yue and Cheung, 2020). Pooled hind limb muscles were used for analysis of SCs from resting muscle and pooled TA muscles were used for analysis of SCs from regenerating muscle 5 dpi. The resulting cell suspension was blocked with DMEM + 15% horse serum (Cytiva sh30074) for 10 min at 4°C with rocking. Cells were then incubated for 45 min at 4°C with rocking with 1:250 of each of the following antibodies: Negative selection markers: BV421 Rat anti-mouse anti-CD31 (562939, BD Biosciences), and BV421 Rat anti-mouse CD45 (563890, BD Biosciences), BV421 Rat anti-Mouse Ly-6aA/E (562729, BD Biosciences); positive selection marker: Mouse anti-integrin alpha 7 Alexa fluor 647 (FAB3518R, R&D Systems). Cells were rinsed, resuspended in PBS and sorted using a BD FACSAria IIu cell sorter equipped with 355nm, 407nm, 488nm, 561nm, and 633nm lasers. The machine was optimized for purity and viability. UltaComp eBeads were used for calibration (01-222-41, Thermo Scientific). Total RNA was isolated from FACS-sorted SCs using a miRNeasy FFPE Kit (Qiagen) as described(Yue and Cheung, 2020).

RNA from SCs of 5 - 5.5-month-old resting muscle (n=6/genotype) was isolated as outlined above and samples from all mice were kept separate. Gene expression was analyzed with a Nanostring nCounter Stem Cell Characterization panel (NanoString Technologies). Multiplex gene expression with 770 genes was performed by measurement of the abundance of each mRNA transcript of interest using multiplexed hybridization and digital readouts of fluorescent bar-coded probes that are hybridized to each transcript. Hybridization of samples was performed, and the obtained products were run according to the manufacturer’s instructions. Data were collected on the nCounter Sprint profiler and further analyzed by _ROSALIND® (https://rosalind.bio/), with a HyperScale architecture developed by ROSALIND, Inc. (San Diego, CA). Read Distribution percentages, violin plots, identity heatmaps, and sample MDS plots were generated as part of the QC step.

ROSALIND® follows the nCounter® Advanced Analysis protocol of dividing counts within a lane by the geometric mean of the normalizer probes from the same lane. Housekeeping probes to be used for normalization are selected based on the geNorm algorithm as implemented in the NormqPCR R library(Perkins et al., 2012). Fold changes and p values are calculated using the fast method as described in the nCounter® Advanced Analysis 2.0 User Manual. P-value adjustment is performed using the Benjamini-Hochberg method of estimating false discovery rates (FDR). Clustering of genes for the final heatmap of differentially expressed genes was done using the Partitioning Around Medoids method using the fpc R library(Hennig, n.d.) that takes into consideration the direction and type of all signals on a pathway, and the position, role and type of every gene. Hypergeometric distribution was used to analyze the enrichment of pathways, gene ontology, domain structure, and other ontologies. The topGO R library(Alexa and Rahnenfuhrer, n.d.) was used to determine local similarities and dependencies between GO terms in order to perform Elim pruning correction. Several database sources were referenced for enrichment analysis, including Interpro(Mitchell et al., 2019), NCBI(Geer et al., 2010), MsigDB(Liberzon et al., 2011; Subramanian et al., 2005), REACTOME (Fabregat et al., 2018), WikiPathways(Slenter et al., 2018). Enrichment was calculated relative to a set of background genes relevant for the experiment. Gene expression data was exported and heat maps were generated in GraphPad prism 9.

An RNA-sequencing dataset (GSE121589) from NCBI’s Gene Expression Omnibus database was analyzed(Shcherbina et al., 2020). This data set includes bulk RNA-seq of isolated SCs from young (2-3 mo. old) and aged (22-24 mo. old) muscle from uninjured and from regenerating muscle at 1, 3, 5, and 7 dpi. The raw data files were reprocessed by trimming adapters and low quality reads using TrimGalore! followed by alignment to the Ensembl GRCm38 mouse genome using HiSat2(Kim et al., 2015). StringTie was used to determine the transcripts per million (TPM) of genes in each sample and DESeq2 was used to identify differentially expressed genes (DEGs)(Love et al., 2014; Pertea et al., 2016). Expression of DE genes identified with the nCounter panel were assessed. Highly regulated genes between quiescent SCs (day 0) and early-activated SCs (day 1 and 3 post injury) were indicated by significant (<0.05) change in TPM.

Statistical analysis was performed with GraphPad Prism software using appropriate tests and minimum of 95% confidence interval for significance. Specific details of the statistical tests are given in the figure legends.