MuSK is expressed in SCs from WT and ΔIg3-MuSK mice.

(a) MuSK protein domain schematic. Full-length (FL)-MuSK contains the Ig3 domain, while it is lacking in ΔIg3-MuSK. (b) Schematic of MuSK exon organization including approximate locations of forward (F) and reverse (R) PCR primers used for detection of either FL-MuSK, containing exons 6 & 7 (which encode the Ig3 domain) or ΔIg3-MuSK, lacking these exons. Note that the presence of two small (30nt) alternatively-spliced exons, ‘5a’ and ‘5b’ result in multiple bands (See also Supplemental Fig. 1). (c) PCR analysis of FACs-sorted SCs: WT SCs express FL-MuSK; endogenous ΔIg3-MuSK is not detected. SCs from ΔIg3-MuSK mice express only the form lacking exons 6 and 7. (d) IHC of isolated WT and ΔIg3-MuSK adult EDL myofibers cultured for 1d and stained for nuclei (DAPI), SCs (Pax7) and MuSK (see Methods). Note that MuSK is expressed in SCs of both genotypes. White dotted line denotes myofiber perimeter.

Changes in TA myofiber size, SC number, and grip strength emerge between 3 and 5 months of age in ΔIg3-MuSK mice.

(a) Representative IHC images of 3- and 5-month-old WT and ΔIg3-MuSK TA used for Pax7+ quantification and myofiber size analysis. Pax7 (red) and laminin (green) staining; arrows indicate SCs. (b) At 3 months, myofiber minimum Feret diameter is equivalent in WT and ΔIg3-MuSK muscle (n=3 male mice, unpaired t-test, 3 mo. p=0.6). At 5 months, myofiber minimum Feret diameter is increased by about 10% (37 ±0.80µm and 41 ± 1.0µm in WT vs ΔIg3-MuSK, respectively; n=8 male mice, unpaired t-test p=0.006). (c) At 3 months of age, the number of Pax7+ cells per area is equivalent between WT and ΔIg3-MuSK muscle (n=10-11 male & female mice, unpaired t-test, p=0.6). At 5 months, the number of Pax7+ cells per area is reduced by almost half in ΔIg3-MuSK muscle (19±2.1 and 10.6±1.4mm2 in WT vs ΔIg3-MuSK, respectively; n=9-10 male & female mice, unpaired t-test, p=0.004). (d) At 3 months, grip strength is comparable between the two genotypes (n=10 males, unpaired t-test, p=0.64). In contrast, at 5 months, ΔIg3-MuSK mice have increased grip strength (n=6-10 males, unpaired t-test, p=0.029; see also Supplemental Fig. 2).

Increased myofiber myonuclei number and length in 5-month-old ΔIg3-MuSK EDL.

(a) Representative DAPI-stained images of isolated WT and ΔIg3-MuSK EDL myofibers from 5 mo. old mice. (b) Note that myonuclear number/myofiber is increased ∼13% in ΔIg3-MuSK compared to WT (245 ±9.0 and 282±9.0 in WT and ΔIg3-MuSK myofibers, respectively; n=5 male mice, 7-10 myofibers per mouse; p=0.005; unpaired t-test). (c) 5-month-old ΔIg3-MuSK myofibers length is increased ∼10% compared to WT (3714±72 and 4089±79 in WT and ΔIg3-MuSK myofibers, respectively; n=5 male mice, 7-10 myofibers per mouse; t- test, p=0.0007). (d)The numbers of nuclei per myofiber length was comparable in ΔIg3-MuSK and WT myofibers (n=5 male mice, 7-10 myofibers per mouse; unpaired t-test, p=0.99)

Efficient, accelerated regeneration in 5-month-old ΔIg3-MuSK muscle.

(a) Experimental design. WT and ΔIg3-MuSK TA muscles were injected with BaCl2 at day 0 and muscle harvested at the indicated time points. (b) Hematoxylin and eosin stain of uninjured and regenerating ΔIg3-MuSK and WT male TA muscle. (c.) IHC images of uninjured and regenerating ΔIg3-MuSK and WT male TA muscle used to quantify Pax7+ cells and myofiber size; laminin (green), Pax7 (red), DAPI (blue). (d) The number of Pax7+ cells is reduced in resting control ΔIg3-MuSK muscle at 5 months (see also Fig. 2a, c). However, during regeneration the number of Pax7+ cells in ΔIg3-MuSK muscle is increased relative to WT at 5 dpi (n=8 mice (4 male, 4 female; 5dpi t-test p= 0.023). (e) Analysis of myofiber size in females (left) and males (right) reveals increase in minimum Feret diameter of ΔIg3-MuSK myofibers in uninjured mice (see also fig. 2b). Following injury, an increase in myofiber size was observed at 7 dpi in ΔIg3-MuSK males (n=4 mice/sex, t- test, Male 7dpi p= 0.038). No significant differences in Feret diameter were observed in regenerating female WT compared to ΔIg3-MuSK muscle.

Expression of ΔIg3-MuSK in SCs from 3 months of age is sufficient to mediate increased TA muscle weight and myofiber size at 5 months.

(a.) Experimental design. At 3 months of age tamoxifen was administered for 5 consecutive days to Pax7CreERT2;R26RLSL-tdTomato;MuSK-Ig3loxP/loxP(test group, purple) and Pax7CreERT2;R26RLSL-tdTomato (control group, dark teal) mice, which were harvested 65-70 days later at ∼5 months of age. (b.) TA muscle weights were increased ∼9% in Pax7CreERT2;R26RLSL-tdTomato;MuSK-Ig3loxP/loxPmice (n=5-8 mice/group; 2 TAs per mouse), (0.035±0.0007 and 0.038±0.0007 g; t-test, p=0.007). (c.) Myofiber cross-sectional minimum Feret diameter was also increased (∼9%) in Pax7CreERT2;R26RLSL- tdTomato;MuSK-Ig3loxP/loxP mice (n=5-7 mice/group; 36.0±0.78 and 39.3±1.2; t-test, p=0.037). (d.) Representative images of Pax7CreERT2;R26RLSL-tdTomato (control, left) and Pax7CreERT2;R26RLSL-tdTomato;MuSK-Ig3loxP/loxP(test, right) representative IHC images of anti-laminin (green) used to measure myofiber size. (e.) Nuclei number per EDL myofiber was increased (∼22%) in test compared to controls (234.2±9.8 and 286. ±16.2, respectively, t-test, p=0.008).

SCs are activated in uninjured ΔIg3-MuSK mice.

(a) Heatmap of 21 differentially expressed genes between the two genotypes detected by nCounter analysis using the Stem Cell Characterization Panel. (b) Comparison of the DEGs in WT and ΔIg3-MuSK SCs detected by nCounter analysis with the DEGs detected in SCs from either ‘young’ or ‘aged’ animals by RNAseq analysis during early damage-induced activation (uninjured vs. 1 dpi; GSE121589). “*” indicates whether each gene was significantly upregulated or downregulated in the respective data sets.

A model for the function of the MuSK-BMP pathway in adult SCs.

MuSK is expressed in both WT and ΔIg3-MuSK SCs (red) where it functions in a cell autonomous manner. Top. In wildtype the MuSK Ig3 domain mediates high affinity BMP binding and MuSK-BMP signaling, maintaining SC quiescence and myofiber size. Bottom. In ΔIg3-MuSK, BMP binding and signaling are reduced, disrupting SC quiescence. Between 3- and 5- months of age SCs expressing ΔIg3-MuSK are activated in uninjured adult muscle and divide and fuse into the myofiber, resulting in increased myofiber size, myonuclear number and grip strength. SCs in 5-month-old ΔIg3-MuSK muscle maintain stemness and can support regeneration and reestablishment of the SC pool following injury.

MuSK expression in SCs from uninjured and regenerating WT and ΔIg3-MuSK muscle

(a) Exon organization of MuSK. The two alternatively-spliced 30nt exons (5a and 5b) are indicated by asterisks. The PCR primers used for panels (b) and (c) are denoted F1 and F2, respectively. (b.) SCs from 3- and 5- month-old uninjured WT muscle express FL-MuSK; ΔIg3-MuSK is not detected. SCs from resting muscle from constitutive ΔIg3-MuSK mice express only ΔIg3-MuSK, as predicted. (c.) SCs from regenerating 5-month-old WT muscle 5 dpi express FL-MUSK, but no detectable ΔIg3-MuSK. SCs from regenerating muscle in ΔIg3-MuSK mice 5 dpi express ΔIg3-MuSK transcripts. Note that MuSK has two alternatively-spliced 30 nucleotide exons (5a and 5b) between exons 5 and 6 (Fig. 1a). Alternative splicing of the 5b exon is the basis for the multiple bands detected(Jaime et al., 2024).

Grip strength in ΔIg3-MuSK and WT mice at 3 and 5 months of age in multiple cohorts.

(a) Three different cohorts of male mice were used to assess 3 mo. grip strength (t-test, p=0.64, p=0.02, p=0.18). (b) One cohort of female mice was used to assess 3 mo. grip strength (t-test, p=0.38) (c) Three separate cohorts of male mice were used to assess 5 mo. grip strength (t-test, p=0.029,p=0.036, p=0.021). (d) One cohort of female mice was used to assess 5 mo. grip strength (t-test, p=0.16). (Note that left panels in a. and c. are the same data presented in the Fig. 2d and are shown here for comparison.)

Tamoxifen treatment at 3 months induces Cre-mediated recombination of the MuSK-Ig3 domain in SCs from Pax7CreERT2;R26RLSL-tdTomato;MuSK-Ig3loxP/loxPmice.

(a.) Experimental design. Pax7CreERT2;R26RLSL-tdTomato;MuSK-Ig3loxP/loxP and Pax7CreERT2;R26RLSL-tdTomato mice were injected with 5 daily doses of tamoxifen at 3 months of age and the muscles harvested 2 months later. Prior to muscle harvest mice were lightly perfusion fixed with 0.5% PFA to capture SCs in their in vivo state. RNA was isolated from FACS-purified SCs. (b.) Gel-based PCR analysis of FL-MuSK and ΔIg3-MuSK expression using the F1 and R primers (see Supp. Fig 1a.). Only FL-MuSK is detected in SCs from Pax7CreERT2;R26RLSL- tdTomato mice and only ΔIg3-MuSK (617 and 587bp) is detected in control Pax7CreERT2;R26RLSL-tdTomato;MuSK-Ig3loxP/loxP mice. See also Supp. Fig. 1 for details on primer location and band identification.

Nanostring nCounter unsupervised results.

Heatmap showing the unsupervised hierarchical clustering of WT SCs (orange, n=6) and ΔIg3-MuSK SCs (blue, n=6) generated via analysis of NanoString Stem Cell Characterization panel on Rosalind. (b) Ranking of gene set functions according to the Global Significance Scores quantified by Nanostring software, based on unsupervised clustering.

Female ΔIg3-MuSK TA myofibers are larger than WT at 3 and at 5 months.

At 3 months and 5 months, the minimum Feret diameter is about 10% larger in ΔIg3-MuSK mouse muscle compared to WT (n=3-4 female mice, unpaired t-test, 3 mo. p=0.03; 5 mo. p=0.01)