Met and Cxcr4 cooperate to protect skeletal muscle stem cells against inflammation-induced damage during regeneration

Acute skeletal muscle injury is followed by an inflammatory response, removal of damaged tissue, and the generation of new muscle fibers by resident muscle stem cells, a process well characterized in murine injury models. Inflammatory cells are needed to remove the debris at the site of injury and provide signals that are beneficial for repair. However, they also release chemokines, reactive oxygen species, as well as enzymes for clearance of damaged cells and fibers, which muscle stem cells have to withstand in order to regenerate the muscle. We show here that MET and CXCR4 cooperate to protect muscle stem cells against the adverse environment encountered during muscle repair. This powerful cyto-protective role was revealed by the genetic ablation of Met and Cxcr4 in muscle stem cells of mice, which resulted in severe apoptosis during early stages of regeneration. TNFα neutralizing antibodies rescued the apoptosis, indicating that TNFα provides crucial cell-death signals during muscle repair that are counteracted by MET and CXCR4. We conclude that muscle stem cells require MET and CXCR4 to protect them against the harsh inflammatory environment encountered in an acute muscle injury.


Introduction
Muscle injury through trauma is common and can be repaired by muscle regeneration (Järvinen et al., 2005;Tidball, 2005;Tidball, 2017). Stem cells reside in the muscle tissue and provide the cellular source for the regeneration process (Chargé and Rudnicki, 2004;Relaix and Zammit, 2012). Muscle stem cells are characterized by the expression of PAX7 and their location in the stem cell niche between the basal lamina and plasma membrane of the muscle fiber (Mauro, 1961;Seale et al., 2000). Muscle stem cells are quiescent in the adult, but can be re-activated upon injury. On one hand, activated muscle stem cells proliferate and generate differentiating cells to repair the muscle, and on the other they can self-renew to repopulate the stem cell niche (Chargé and Rudnicki, 2004;Relaix and Zammit, 2012). A complex interplay between muscle stem cells and their environment occurs during muscle repair. Inflammatory cells and the cytokines they produce provide important cues for muscle stem cells and regulate their activation, proliferation, and differentiation. Therefore, communication between muscle stem cells and the immune system needs to be tightly regulated. Failure of

Met is required for normal muscle regeneration
To identify factors that directly regulate muscle stem cell behavior in vivo, we systematically assessed chemokine transcripts in regenerating muscle using published data sources (Hirata et al., 2003;Xiao et al., 2011;Bobadilla et al., 2014) and verified their expression using qPCR. A multitude of chemokines are rapidly and strongly induced after injury. In murine tibialis anterior muscle tissue, Tnf and Hgf transcripts were induced 10-500-fold with a time course that peaked 2-3 days after injury ( Figure 1A and B, Figure 1-figure supplement 1, and Supplementary file 1). TNFα is known to orchestrate the inflammatory response and to participate in the communication between immune cells (Saclier et al., 2013a;Turner et al., 2014), and HGF is a proliferation and motility factor that can act as protective factor in tissue injury (Birchmeier et al., 2003;Nakamura and Mizuno, 2010). Hgf transcripts were produced at low levels by quiescent and activated muscle stem cells, demonstrating that other cell types but muscle stem cells produce Hgf in the regenerating muscle ( Figure 1C and Supplementary file 1). This is in accord with previous data on Hgf expression obtained by microarray analysis Latroche et al., 2017; see also Figure 1-figure supplement 1). The HGF receptor MET is expressed in adult muscle stem cells (Cornelison and Wold, 1997), and, in contrast to quiescent muscle stem cells, Met transcripts were upregulated when the cells were activated ( Figure 1D).
To identify the role of HGF/MET during muscle repair, we introduced a loss-of-function mutation in Met in muscle stem cells using a constitutive Pax7 iresCre allele (Pax7 iresCre ;Met flox/flox mice, named hereafter coMet; the genotype of the corresponding control mice used was Pax7 iresCre ;Met +/+ ). Met is known to control migration of myogenic progenitors during development (Bladt et al., 1995). The conditional mutation did not affect muscle progenitor migration because Pax7 (and hence Pax7 iresCre ) starts only to be expressed in progenitors that have already reached their targets (Relaix et al., 2004). Therefore, the Met mutation in myogenic progenitors is introduced after migration is completed, from there on persisting throughout fetal and postnatal development. In the undamaged muscle, neither fiber diameter nor muscle stem cell numbers were changed in coMet mutant compared to control mice (Figures 2 and 3).
Upon injury of the tibialis anterior muscle using cardiotoxin, coMet mutant muscle stem cells were able to regenerate muscle fibers. However, at 7 days post injury (dpi) the diameter of the newly regenerated fibers was smaller in coMet mutants than in control animals, but diameters largely equalized between control and coMet mutants and were no longer significantly different at 20 dpi ( Figure 2A -D, quantified in E). Moreover, the number of PAX7+ stem cells in the regenerated muscle of coMet mice was reduced by 68% at 7 dpi compared to control mice, and also this difference became less pronounced at 20 dpi (47% reduction in coMet mice; Figure 3A-F). Similar deficits were observed when Met was mutated in adult muscle stem cells using the tamoxifen-inducible Pax7 iresCreERT2 allele (Pax7 iresCreERT2Gaka/+ ;Met flox/flox mice treated with tamoxifen, named hereafter Tx Gaka Met as controls, Pax7 iresCreERT2Gaka/+ ;Met +/+ mice treated with tamoxifen were used). Thus, the diameter of new fibers was smaller at 7 dpi in Tx Gaka Met compared to control animals at 7 dpi, but at 20 dpi the difference in fiber diameters was no longer significant ( Figure 2F-J). Moreover, the number of PAX7+ stem cells in the regenerated muscle of Tx Gaka Met mice was reduced by 73% at 7 dpi compared to control, and also   Latroche et al., 2017). and 20 dpi) muscle of control and coMet mutants using antibodies against laminin (red) and sarcomeric myosin (green). DAPI was used as a counterstain. (E) Distribution of Feret fiber diameters in uninjured and regenerating muscle (7 dpi and 20 dpi) of control mice and coMet mutants. (F-I) Immunohistological analysis of regenerating muscle of control and Tx Gaka Met mice using antibodies against laminin (red) and sarcomeric myosin (green). DAPI was used  this difference was less pronounced at 20 dpi (44% reduction in Tx Gaka Met mice) ( Figure 3G-L). In summary, our data indicate that loss of Met in muscle stem cells results in a mild regeneration deficit. This is accompanied by a reduction of muscle stem cell numbers during early stages of regeneration, which is partly compensated for during late stages. Increased proliferation of the remaining stem cell pool might account for this (see below for a more detailed description of the mechanisms). A previous report had indicated that ablation of Met using a distinct tamoxifen-inducible Pax7 CreERT2 allele (Pax7 CreERT2Fan ) resulted in a much more severe muscle regeneration deficit (Webster and Fan, 2013). We used this Cre allele to mutate Met (Pax7 CreERT2Fan/+ ;Met flox/flox mice treated with tamoxifen, named hereafter Tx Fan Met animals; as controls, Pax7 CreERT2Fan/+ ;Met +/+ mice treated with tamoxifen were used), and also detected a very severe muscle regeneration deficit at 7 dpi and 20 dpi compared to control animals at these stages of regeneration ( Figure 2K-O). In particular, extracellular matrix remnants from injured skeletal muscle fibers (i.e., ghost fibers) were abundant at 7 dpi and 20 dpi. Notably, even in the uninjured muscle a 50 % reduction in the number of PAX7+ cells was observed in the Tx Fan Met animals compared to controls. This became more pronounced after injury when a 94 and 65% reduction in stem cell numbers was present at 7 dpi and 20 dpi, respectively, compared to the control animals at these stages of regeneration ( Figure 3M-R). Different recombination efficacies did not account for these differences in phenotypes observed in coMet and Tx Gaka Met animals on one side, and Tx Fan Met animals on the other side ( Figure 3-figure supplement 1A-D). We conclude that the muscle stem cell and regeneration deficits present in Tx Fan Met mutants are apparently not only due to the Met ablation. It should be noted that in the Pax7 CreERT2Fan ; allele, the Pax7 coding sequence is disrupted by Cre, whereas the Pax7 iresCreERT2Gaka and Pax7 iresCre alleles do not interfere with the Pax7 coding sequence (Keller et al., 2004;Lepper et al., 2009;Murphy et al., 2011; see also Figure 3figure supplement 1E for a cartoon of the different Cre alleles used). PAX7 levels are known to affect muscle stem cell behavior and their ability to regenerate the muscle (von Maltzahn et al., 2013;Mademtzoglou et al., 2018). Thus, the absence of one functional Pax7 allele might contribute to the exacerbated muscle stem cell and regeneration phenotypes observed in Tx Fan Met animals.

MET and CXCR4 signaling cooperates during muscle regeneration
The CXCR4 receptor is expressed in developing and adult muscle stem cells and mediates CXCL12 signals that stimulate their proliferation and migration (Vasyutina et al., 2005;Odemis et al., 2007;Griffin et al., 2010). Cxcl12 is expressed by various cell types of the immune system. qPCR demonstrated that muscle tissue and PAX7+ cells expressed Cxcl12 transcripts in both uninjured and regenerating muscle, and confirmed that Cxcr4 transcripts were present in PAX7+ cells ( Figure 4A-C, Figure 4-figure supplement 1, and Supplementary file 1). Cxcr4 and Met are known to cooperate during muscle development (Vasyutina et al., 2005). We therefore tested whether this cooperativity was also observed in adult muscle stem cells and whether it would have an impact on muscle repair using Cxcr4 and Met double mutant mice (Pax7 iresCreERT2Gaka/+ ;Cx-cr4 flox/flox ;Met flox/flox mice treated with tamoxifen, hereafter called Tx Gaka Cxcr4;Met animals; Pax7 ire-sCreERT2Gaka/+ ;Cxcr4 +/+ ;Met +/+ treated with tamoxifen served as controls). Mutations of Cxcr4 and Met in muscle stem cells did not obviously affect muscle formation or muscle stem cell numbers ( Figure 5, Figure 5-figure supplement 1). However, Tx Gaka Cxcr4;Met double mutant mice at 7 dpi displayed a very severe regeneration deficit compared to control mice at 7 dpi. In particular, formation of myofibers was strongly impaired ( Figure 5A-E). Further, the number of muscle stem cells detected at 7 dpi was decreased by 93 % as compared to control mice at 7 dpi ( Figure 5F-J).
The severe regeneration deficit was accompanied by widespread fibrosis, persisting macrophages, as a counterstain. (J) Distribution of Feret fiber diameters in uninjured and regenerating muscle (7 dpi and 20 dpi) of control and Tx Gaka Met mice.
(K-N) Immunohistological analysis of regenerating (7 dpi and 20 dpi) muscle of control and Tx Fan Met mice using antibodies against laminin (red) and sarcomeric myosin (green). DAPI was used as a counterstain. (O) Distribution of Feret fiber diameters in uninjured and regenerating (7 dpi and 20 dpi) muscle of control and Tx Fan Met mice. Scale bars, 100 µm. In (

Muscle stem cells deficient for Met and Cxcr4 are susceptible to apoptosis
We next assessed the mechanisms by which the Cxcr4 and Met mutations affect muscle stem cell maintenance in the injured muscle. We observed a pronounced increase in apoptosis of PAX7+ cells at 4 dpi in the double mutants and a severe decrease in the number of PAX7+ muscle stem cells ( Figure 6). A less pronounced enhancement of apoptosis of muscle stem cells was observed in Tx Gaka Met single mutants, whereas the Tx Gaka Cxcr4 single mutation did not significantly impair survival as compared to control animals ( Figure 6). Thus, the signals provided by CXCR4 and MET protect muscle stem cells from apoptosis in the acutely injured muscle.
CXCR4 and MET signals stimulate muscle stem cell proliferation in vitro (Allen et al., 1995;Cornelison, 2008). However, in the regenerating muscle in vivo, ablation of Cxcr4 and Met in muscle stem cells did not impair their proliferation. On the contrary, EdU incorporation showed that proliferation of muscle stem cells increased in the Tx Gaka Cxcr4;Met double and Tx Gaka Met single mutants (Figure 6figure supplement 1), possibly due to compensatory mechanisms. Moreover, in Tx Gaka Cxcr4;Met double and Tx Gaka Met single mutants, the ratio of MyoG+/Pax7+ cells was slightly increased, indicating that differentiation was mildly enhanced (Figure 6-figure supplement 2). We conclude that CXCR4 and MET signals cooperate to convey powerful cyto-protective functions.   Latroche et al., 2017).

MET and CXCR4 signaling protects muscle cells from TNFα-induced apoptosis
We next aimed to identify the factor that induces apoptosis of Met;Cxcr4 mutant muscle stem cells in the injured muscle. The pro-inflammatory cytokine TNFα is induced at the early stages of muscle regeneration and has pro-as well as anti-apoptotic effects on many cell types (Darnay and Aggarwal, 1999;Malka et al., 2000;Collins and Grounds, 2001;Zador et al., 2001;Warren et al., 2002;Aggarwal, 2003). We thus asked whether TNFα production might be responsible for the observed cell death. If freshly isolated muscle stem cells were cultured in media containing 2 % horse serum, TNFα induced apoptosis ( Figure 7A and B). This TNFα-induced cell death of cultured cells was rescued by the addition of HGF and CXCL12, or by the addition of 10 % fetal calf serum. No cooperative effect of HGF and CXCL12 was observed in this cell culture setting ( Figure 7C and D).
Finally, using neutralizing antibodies against TNFα,we tested whether the loss of muscle stem cells in the absence of MET and CXCL12 signaling during regeneration in vivo was caused by TNFα. The efficacy TNFα antibodies was verified in a cell culture experiment (Figure 7-figure supplement  1). We observed a pronounced rescue of PAX7+ cells in the regenerating muscle of Tx Gaka Met and Tx Gaka Cxcr4;Met mutant mice after injection of TNFα neutralizing antibodies ( Figure 7E-P). Taken together, these data demonstrate that MET and CXCR4 signaling cooperate in vivo to protect muscle stem cells from TNFα-induced apoptosis in the inflammatory environment encountered after injury.

Discussion
Muscle injury results in an acute inflammatory response causing the recruitment of macrophages and neutrophils. These cells remove cellular debris at the site of injury and provide signals that are beneficial for muscle repair. In addition, they release a multitude of chemokines, as well as reactive oxygen species and enzymes needed to degrade the debris, thereby creating a hostile environment that muscle stems cells have to withstand in order to regenerate the muscle and self-renew (Tidball, 2005;Chazaud et al., 2009;Saclier et al., 2013b;Londhe and Guttridge, 2015;Tidball, 2017). Our analysis of the in vivo function of MET and CXCR4 demonstrates an important cooperative role in muscle repair that protects stem cells against the adverse environment created by the acute inflammatory response.
Previous studies had shown that HGF can elicit muscle stem cell proliferation in culture and that CXCL12 has mitogenic activity on myogenic C2C12 cells (Allen et al., 1995;Gal-Levi et al., 1998;Odemis et al., 2007). Further, injection of HGF into the intact muscle activates muscle stem cells (Tatsumi et al., 1998), and ablation of Met in muscle stem cells interferes with entry into G alert , a 'alerted' state of quiescence observed in muscle stem cells after injury of the contralateral muscle or of other unrelated organs (Rodgers et al., 2014). HGF/MET signaling also affects additional aspects of muscle stem cell biology. In particular, HGF suppresses differentiation of cultured myogenic cell lines and of primary muscle stem cells (Gal-Levi et al., 1998;Siegel et al., 2009). Thus, HGF had been implicated in multiple aspects of muscle stem cell behaviors, but its role as cyto-protective factor had not been addressed.
Interestingly, cyto-protective functions of HGF/MET were reported in several cell types and injury models, indicating that HGF might be part of a general defensive mechanism in response to tissue damage. In particular, ectopic application of HGF prior to or shortly after an insult protects cells in the liver, kidney, and heart from damage (Ueda et al., 1999;Zhou et al., 2013;Matsumoto et al., 2014;Pang et al., 2018). Moreover, after injury to the liver, kidney, heart, or skeletal muscle, increased HGF expression can be observed in the damaged organs, and plasma levels of HGF rise quickly after injury (Michalopoulos and DeFrances, 1997;Nakamura et al., 2000;Matsumoto and Nakamura, 2001). It was proposed that release from extracellular matrix might account for the fast rise in HGF plasma levels (Shimomura et al., 1995;Tatsumi et al., 1998). In addition, various cytokines, among them interleukin-1 and interleukin-6, activate HGF transcription, which might account for the increased HGF transcripts observed after tissue damage (Birchmeier et al., 2003). We demonstrate here that loss of Met impairs the resistance of muscle stem cells against acute inflammation. Moreover, in vivo the additional loss of Cxcr4 exacerbated the deficits observed after loss of Met. The cooperative effect that we detected here in vivo is reflected by the fact that both receptors, Met and Cxcr4, use in part overlapping downstream signaling cascades but also activate distinct signaling molecules. Tyrosine  (Birchmeier et al., 2003;Gentile et al., 2008). CXCR4 uses G-proteins to transmit signals into the cytoplasm, which involves activation of second messenger-regulated serine/threonine kinases or ion channels. However, CXCR4 also activates RAS/MAPK, PI3-kinase/AKT, and CRK signaling, which is particularly well documented in cancer cells (Teicher and Fricker, 2010). Among these cascades, PI3-kinase/AKT is well known to act anti-apoptotically, and MAPK/ERK signals can counteract the apoptotic activity of TNFα (Tran et al., 2001;Franke et al., 2003).
TNFα is one of many pro-inflammatory cytokines that are rapidly induced upon acute muscle injury, and TNFα is highly expressed by pro-inflammatory macrophages. The primary role of TNFα is to regulate immune cells, but it also affects the proliferation and differentiation of cultured muscle cells (Wallach et al., 1999;Li, 2003;Luo et al., 2005;Palacios et al., 2010). Mice lacking TNFα receptors p55 and p75 show that TNFα does not play an essential role in muscle regeneration, indicating that this cytokine seems to act redundantly with other factors (Collins and Grounds, 2001). However, systemic injection of TNFα neutralizing antibodies protected dystrophic skeletal muscle of mdx mice from necrosis and increased the number of PAX7+ cells (Palacios et al., 2010). This indicates that TNFα exacerbates muscle fiber damage and, in addition, impairs muscle stem cell maintenance in dystrophic muscle. Our analysis indicates that TNFα signals are also damaging for muscle stem cells during acute inflammation after injury, but that endogenous HGF and CXCL12 may counteract this. Effects of TNFα are modulated by other signals, and particularly MAPK/ERK activity can override the apoptotic TNFα signal (Tran et al., 2001;Aggarwal, 2003;Wada and Penninger, 2004;Lu and Xu, 2006;Lau et al., 2011). Acute skeletal muscle injury resulting in inflammation is a common clinical condition caused by trauma, severe contraction, chemicals, myotoxins, and ischemia. Similarly, acute inflammation is observed in muscle diseases like dystrophy (Kharraz et al., 2014;Tidball et al., 2018).
Our genetic experiments indicate that HGF/MET and CXCL12/CXCR4 signaling protects muscle stem cells against the noxious environment generated by the inflammatory response. Exogenous HGF was previously tested in muscle injury and increased the numbers of activated muscle stem cells, but did not enhance fiber growth (Miller et al., 2000). Thus, in healthy muscle, endogenous factors, among them HGF, suffice to ensure appropriate regeneration. Nevertheless, in muscle disease where repair mechanisms fail, enhanced cyto-protection of muscle stem cells appears to be beneficial (Palacios et al., 2010). Whether HGF/MET and CXCL12/CXCR4 signaling protects against TNFα-induced damage in such disease settings will need further investigation.

Materials and methods
counterstain. (G) Quantification of PAX7+ cells in regenerating muscle (4 dpi) of control mice treated with TNFα neutralizing antibodies or control IgG. (H) Quantification of PAX7+ TUNEL+ cells in regenerating muscle (4 dpi) of control mice treated with TNFα neutralizing antibodies or control IgG. (I, J) Immunohistological analysis of muscle stem cells (PAX7+, red) and apoptotic cells (TUNEL staining, green) in injured muscle (4 dpi) of Tx Gaka Met mutants treated with TNFα neutralizing antibodies or control IgG 2 hr before acute injury. DAPI was used as a counterstain. (K) Quantification of PAX7+ cells in regenerating (4 dpi) muscle of Tx Gaka Met mice treated with TNFα neutralizing antibodies or control IgG. (L) Quantification of PAX7+ TUNEL+ cells in regenerating muscle from Tx Gaka Met mice treated with TNFα neutralizing antibodies or control IgG. (M, N) Immunohistological analysis of muscle stem cells (PAX7+, red) and apoptotic cells (TUNEL staining, green) in injured muscle (4 dpi) of Tx Gaka Cxcr4;Met mutants treated with TNFα neutralizing antibodies or control IgG 2 hr before acute injury. DAPI was used as a counterstain.

RNA isolation and qPCR
RNA from the entire muscle and from FACS-isolated muscle stem cells was extracted using TRIzol reagent (15596026, Thermo Fisher Scientific) following the manufacturer's instructions. qPCR was performed using SYBR green master mix (4309155, Thermo Fisher Scientific) as described previously (Bröhl et al., 2012). PCR primers are listed in Key resources table. β-Actin was used for normalization.

Immunohistochemistry
Immunohistochemistry was performed on 12 μm cryo-sections of muscle biopsy samples fixed in Zamboni's fixative for 20 min as described previously (Bröhl et al., 2012). For staining of Pax7, sections were incubated in Antigen Unmasking Solution buffer (H-3300, Vector Laboratories) for 20 min at 80 °C. Primary and secondary antibodies used are listed in Key resources table. Primary antibodies were incubated overnight, and secondary antibodies for 1 hr at 4 °C in blocking solution. DAPI (D9542, Sigma-Aldrich) was used as a counterstain to label nuclei. To detect apoptotic cells, Pax7 immunohistochemistry was combined with TUNEL TMR Red detection kit according to the manufacturer's instruction (12156792910, Roche). To monitor proliferating cells, EdU (50 µg/g body weight) was given i.p. 2 hr before the isolation of the muscle. EdU was detected using Click chemistry (EdU-Click 647, BCK-EdU647, baseclick GmbH) and Biotin picolyl azide (900912, Sigma-Aldrich) as substrate. Detection was performed with fluorophore-coupled streptavidin. Images were acquired using a LSM700 confocal microscope and processed using Adobe Photoshop (Adobe Systems).

Isolation of muscle stem cells and muscle injury
Muscle stem cells were isolated from skeletal muscle using fluorescent-activated cell sorting (FACS) as described (Bröhl et al., 2012). Shortly, muscle tissue was minced, enzymatically digested with 1.5 U/ ml NB4G Collagenase (S1745401, Serva), and 2.4 U/ml Dispase (04942078001, Sigma-Aldrich). Mononucleated cells were isolated and labeled with antibodies against VCAM, Sca1, CD45, CD31 (AF643, rndsystems; BD Bioscience). VCAM+ Sca1 CD31-CD45-cells were isolated using a BD Aria III sorter (BD Bioscience) and dead cells were excluded by propidium iodide staining (P4864, Sigma-Aldrich). Muscle stem cells from regenerating muscles were isolated from animals carrying Pax7 nGFP allele using the digestion procedure described above. Mono-nucleated cells GFP+ cells were isolated by FACS. Cells were collected in TRIzol RNA extraction reagent (15596026, Thermo Fisher Scientific) for RNA isolation or in DMEM/10 % FCS for cultivation. Muscle injury was induced by injecting 30 µl of cardiotoxin (10 µM; C9759, Sigma-Aldrich) into the tibialis anterior muscle of 8-to 12-week-old mice. Muscle injected with phosphate buffered saline (10010056, Thermo Fisher Scientific) was used as a control. Recombination using CreERT2 alleles was induced as described (Murphy et al., 2011), and the injury was induced 10 days after the last tamoxifen administration. Antigen affinity-purified polyclonal goat human/mouse TNFα antibody (AF-410-NA, rndsystems, LOT NQ2519111, NQ2520111, NQ2418041) was dissolved in PBS and 100 µg were injected in a single injection i.p. 2 hr before the cardiotoxin injection. Mice injected with 100 µg goat IgG (AB-108-C, rndsystems) served as control. The animals were analyzed 4 days after injury.

Computational analysis and statistics
Gene expression levels of freshly isolated and cultured muscle stem cells were previously determined using gene expression microarrays Latroche et al., 2017). The *.CEL files of scanned Affymetrix mRNA expression microarrays were downloaded from the GEO repository (accession codes GSE47177 and GSE103684, n = 3 replicates/condition). Normalization and background corrections were performed using the AffySTExpressionCreator v0.14 on the GenePattern Server (Reich et al., 2006) running the Robust Multi-array Average (RMA) algorithm (Irizarry et al., 2003). The relative signal intensities of gene expression of muscle stem cell activation were plotted against the time axis.
Three or more animals were used per genotype and experiment. Microsoft Excel and GraphPad Prism 9 were used for statistical analysis. Data were analyzed using an unpaired, two-tailed t-test. p-values < 0.05 were considered significant. Results are shown as arithmetical mean ± standard error of the mean (SEM) and the dots represent the mean of individual animals. ns: not significant, p>0.05, *p<0.05, **p<0.01, ***p<0.001.

Additional files
Supplementary files • Supplementary file 1. Tnf, Hgf, and Cxcl12 expression levels during muscle regeneration. Expression levels of Tnf, Hgf, and Cxcl12 mRNA after acute injury were determined in the entire muscle by qPCR. Uninjured and 1-7 days post injury (dpi) were assessed, and expression was normalized to the expression in the uninjured muscle. The values are displayed as means ± SEM. p-Values are shown in brackets. β-Actin was used for normalization.
• Transparent reporting form Data availability All data generated or analysed during this study are included in the manuscript and supporting files.