Fibronectin regulates Wnt7a signaling and satellite cell expansion

SUMMARY

The influence of the extracellular matrix (ECM) within the stem cell niche remains poorly understood. We found that Syndecan-4 (Sdc4) and Frizzled-7 (Fzd7) form a co-receptor complex in satellite cells and that binding of the ECM glycoprotein Fibronectin (FN) to Sdc4 stimulates the ability of Wnt7a to induce the symmetric expansion of satellite stem cells. Newly activated satellite cells dynamically remodel their niche by transient high-level expression of FN. Knockdown of FN in prospectively isolated satellite cells severely impaired their ability to repopulate the satellite cell niche. Conversely, in vivo over-expression of FN with Wnt7a dramatically stimulated the expansion of satellite stem cells in regenerating muscle. Therefore, activating satellite cells remodel their niche through autologous expression of FN that provides feedback to stimulate Wnt7a signaling through the Fzd7/Sdc4 co-receptor complex. Thus, FN and Wnt7a together regulate the homeostatic levels of satellite stem cells and satellite myogenic cells during regenerative myogenesis.

INTRODUCTION

Satellite cells are the primary contributor and are indispensible for skeletal muscle regeneration (Wang and Rudnicki, 2011). All satellite cells express the paired-box transcription factor Pax7 (Seale et al., 2000). As determined by lineage tracing, about 10% of these satellite cells have never expressed the myogenic regulatory factor (MRF) Myf5 (Kuang et al., 2007). Using Myf5-Cre and R26R-YFP reporter alleles, we observed that satellite stem cells, which have never expressed Myf5-Cre (Pax7⁺/YFP⁻), extensively contribute to the satellite cell pool after transplantation into muscle. By contrast, satellite myogenic cells, which have expressed Myf5-Cre (Pax7⁺/YFP⁺), are committed to undergo differentiation and do not contribute to the satellite cell pool. Upon activation, satellite stem cells can either undergo a symmetric planar cell division, or alternatively undergo an asymmetric apical-basal cell division to give rise to a satellite myogenic cell (Kuang et al., 2007). Therefore, satellite cells are a heterogeneous population composed of a small fraction of satellite stem cells and a large number of committed satellite myogenic cells (Kuang et al., 2008).

The spatiotemporal regulation of satellite cells during muscle regeneration is remarkably fine-tuned and highly dependent on a variety of extrinsic signals (Bentzinger et al., 2010; Kuang et al., 2008). For example, we recently demonstrated that Wnt7a/Fzd7 signaling through the planar-cell-polarity (PCP) pathway drives the symmetric expansion of satellite stem cells resulting in accelerated and augmented repair of muscle (Le Grand et al., 2009). Other factors that act on satellite cells include Notch ligands, brain-derived neurotrophic factor (BDNF), mechano-growth factor (MGF), hepatocyte growth factor (HGF) and fibroblast growth factor (FGF) (Ates et al., 2007; Brack et al., 2008; DiMario et al., 1989; Kuang et al., 2007; Miller et al., 2000; Mousavi and Jasmin, 2006). Lineage progression and terminal commitment in more advanced stages of muscle regeneration appear to be modulated by a transition towards Insulin-like growth factor 1 (IGF-1) and canonical Wnt signaling (Adi et al., 2002; Allen and Boxhorn, 1989; Brack et al., 2008; Doumit et al., 1996).

Apart from classic signaling molecules, mechanical and structural properties of the niche play an important role for satellite cell function (Cosgrove et al., 2009). Satellite cells cannot be removed from niche and maintained in vitro without a loss of stem cell characteristics (Cosgrove et al., 2009; Wilson and Trumpp, 2006). However, it has recently been demonstrated that isolated satellite cells cultured for short terms on elastic surfaces mimicking the softness of adult skeletal muscle better retain stem cell properties than cells grown on rigid surfaces (Gilbert et al., 2010). This study suggests that a better understanding of the muscle stem cell niche will eventually help us to develop techniques for the ex vivo cultivation of satellite cells perhaps allowing genetic correction and stem cell therapy of diseased muscle.

Structural properties of the satellite cell niche are largely determined by the fiber sarcolemma and the complex extracellular matrix (ECM) components in the basement membrane that surrounds muscle fibers. The basement membrane is primarily composed of collagens, laminins and non-collagenous glycoproteins (Sanes, 2003). Transcriptional profiling of regenerating muscle suggests that the extracellular space is dynamically remodeled during muscle regeneration (Goetsch et al., 2003). Satellite cells express high levels of the Laminin receptors α7β1 Integrin (Itg) and dystroglycan (Burkin and Kaufman, 1999; Cohn et al., 2002). Mice deficient for the Laminin-α2 subunit suffer from muscular dystrophy with severely impaired regeneration which can be rescued by transgenic restoration of a functional basement membrane-dystroglycan linkage (Bentzinger et al., 2005). Moreover, muscles with satellite cells lacking dystroglycan display a blunted regenerative response to injury (Cohn et al., 2002).

Recently, muscle-resident fibroblasts were demonstrated to be required for fully efficient muscle regeneration (Murphy et al., 2011). Fibroblasts secrete a wide variety of ECM molecules and may well influence satellite cells by altering the composition of their extracellular milieu (Serrano and Munoz-Canoves, 2010). Nevertheless, little is known about the causes and consequences of ECM modulation during muscle regeneration. In addition, the molecular mechanisms underlying crosstalk of satellite cells with their structural microenvironment remain largely speculative.

In this study, we report that satellite cells transiently remodel their niche during muscle regeneration with the ECM glycoprotein Fibronectin (FN). We demonstrate that upon muscle injury, FN expressed from satellite cells autologously modulates their expansion within their niche by potentiating Wnt7a signaling. Conversely, loss of FN from the niche impairs the maintenance of the satellite cell pool during muscle regeneration.

RESULTS

Sdc4 is a Co-receptor for Fzd7 in Satellite Stem Cells

Fzd7 and its ligand Wnt7a play an important role in regulating the symmetric expansion of Pax7⁺/YFP⁻ satellite stem cells to maintain the homeostatic levels of satellite cells through regeneration (Le Grand et al., 2009). Fzd7 is highly expressed in quiescent Pax7⁺/YFP⁻ satellite stem cells. During Xenopus laevis gastrulation, Sdc4 functions as a Fzd7 co-receptor and both are required for elongation-extension movements induced by planar cell polarity (PCP) signaling (Munoz et al., 2006). Sdc4 is highly expressed in satellite cells and required for normal function (Cornelison et al., 2001; Cornelison et al., 2004)

To investigate whether Fzd7 and Sdc4 are co-receptors in mammalian myogenic cells, we performed co-immunoprecipitation (Co-IP) experiments. Immunoprecipitation of transfected Flag-tagged Fzd7 from primary myoblasts co-precipitated transfected YFP-tagged Sdc4 (Figure 1A). These data support the notion that Fzd7 and Sdc4 are co-receptors.

Figure 1. The FN receptor Sdc4 forms a functional complex with Fzd7.

(A) Co-IP of Sdc4 with the Wnt7a receptor Fzd7 from satellite cell derived primary myoblasts overexpressing (OE) Fzd7-Flag and Sdc4-YFP. Co-IP was performed with an anti-YFP antibody or with an IgG control.

(B) Proximity ligation assay (PLA) of Sdc4 and Fzd7 in activated satellite cells after 42 hours of fiber culture. No interaction is observed in siSdc4 treated cells. Scale bar = 5 μm.

(C) Proximity ligation assay (PLA) of Sdc4 and FN in activated satellite cells after 42 hours of fiber culture. No interaction is observed in siSdc4 treated cells. Scale bar = 5 μm.

(D) Co-IP of Fzd7 with FN from satellite cell derived primary myoblasts overexpressing (OE) Fzd7-Flag and FN. Co-IP was performed with an anti-YFP antibody. siRNA knockdown of endogenous Sdc4 (siSdc4) prevents Co-IP of FN with Fzd7 when compared to siSCR.

(E) Co-IP of Sdc4 with Wnt7a from satellite cell derived primary myoblasts that overexpress (OE) Sdc4-YFP and Wnt7a-HA. Co-IP was performed with an anti-flag antibody. siRNA knockdown of endogenous Fzd7 prevents Co-IP of Sdc4 with Wnt7a.

(F) Rac1 activation assay. Total Rac1 is shown as a loading control. Densitometric quantification represents average grey values ± SEM after subtraction of the background and normalization to total Rac1. The average grey value obtained for empty vector (EV) was set to 100%. n=3. p values are **p < 0.01; *p < 0.05.

We next examined whether endogenous Fzd7 and Sdc4 form a receptor complex in satellite cells using an in-situ proximity ligation assay (PLA) (Fredriksson et al., 2002; Pisconti et al., 2010). Notably, PLA detection of endogenous Fzd7 and Sdc4 with antibodies resulted in a strong signal in activated satellite cells on cultured myofibers (Figure 1B). This signal was completely abolished by knocking down Sdc4 with siRNA (siSdc4). Therefore, we conclude that Fzd7 and Sdc4 form a co-receptor complex in activated satellite cells.

Sdc4 is a high affinity receptor for fibronectin (FN) (Lyon et al., 2000; Woods et al., 2000). We used PLA to assess the binding of FN to Sdc4 in satellite cells, and similarly detected a strong signal in satellite cells on cultured myofibers (Figure 1C). Again, PLA reactivity of Sdc4 and FN antibodies on satellite cells was completely abrogated by siSdc4 treatment. Therefore, we conclude that Sdc4 binds FN on activated satellite cells.

Our previous work has demonstrated that ligation of Wnt7a to Fzd7 activates the planar cell polarity (PCP) pathway (Le Grand et al., 2009). Hence, we investigated whether binding of Wnt7a to Fzd7 in primary myoblasts was dependent on the presence of Sdc4 as a co-receptor. Immunoprecipitation of Flag-tagged Fzd7 from primary myoblasts co-precipitated FN and this interaction was lost following siRNA knockdown of endogenous Sdc4 (Figure 1D). Moreover, immunoprecipitation of YFP-tagged Sdc4 co-precipitated overexpressed Wnt7a-HA, and this interaction was lost following siRNA knockdown of endogenous Fzd7 (Figure 1E). Therefore, we conclude that the Fzd7/Sdc4 co-receptor complex binds both Wnt7a and FN.

Rac1 is associated with Sdc4 and is activated by FN binding (Bass et al., 2007). Rac1 is also a known effector of the PCP pathway (Seifert and Mlodzik, 2007). Therefore, to examine the role played by Sdc4 in PCP signaling, we investigated whether FN stimulation of Sdc4 facilitates Fzd7 dependent Rac1 activation. We observed that over-expression of Fzd7, or stimulation with FN resulted in increased levels of active Rac1 in primary myoblasts (Figure 1F). Notably, FN stimulation of cells over-expressing Fzd7 resulted in markedly increased levels of Rac1 activation. From these experiments, we conclude that the Fzd7/Sdc4-Rac1 co-receptor complex integrates Wnt7a and FN signals to activate PCP signaling.

FN and Wnt7a Signal through Fzd7/Sdc4 to Stimulate Satellite Stem Cell Symmetric Divisions

Our finding that the Fzd7/Sdc4 co-receptor complex integrates Wnt7a and FN signals suggested that FN plays a role in PCP signaling. Therefore, we investigated whether co-activation of the Fzd7/Sdc4 receptor complex with FN and Wnt7a influences the symmetric division of Pax7⁺/YFP⁻ satellite stem cells on single muscle fibers derived from Myf5-Cre/R26R-YFP mice as previously described (Kuang et al., 2007; Le Grand et al., 2009).

Both plasma and cellular FN contain the Hep II domain for binding to Sdc4 (Singh et al., 2010; Woods et al., 2000). Standard fiber medium contains 20% Fetal bovine serum (FBS) resulting in a plasma FN concentration in the range of 5–15 μg/ml (Hayman and Ruoslahti, 1979; Sochorova et al., 1983). To investigate the role of FN on Wnt7a signaling on satellite cells in cultured myofibers, we supplemented the medium with an additional 25 μg/ml soluble plasma FN. As a control, we similarly increased the concentration of the unrelated ECM component Collagen (COL).

Addition of FN alone had no significant effect on the proportion of symmetric satellite stem cell divisions after 42h of culture when compared to COL (Figure 2A). As previously described, Wnt7a drives the symmetric expansion of satellite stem cells by stimulating planar cell divisions parallel to the basal lamina (Le Grand et al., 2009). Accordingly, after 42h of culture, addition of Wnt7a with COL (COL&Wnt7a) resulted in a 73% increase in the number of Pax7⁺/YFP⁻ satellite stem cells (Figure 2A), and a 108% increase in the proportion of symmetric cell divisions when compared to COL alone (Figure 2B). By contrast, application of FN together with Wnt7a (FN&Wnt7a) resulted in a 147% increase in the number of satellite stem cells (Figure 2A), and a 163% increase in the proportion of symmetric cell divisions (Figure 2B). By 72h of culture, FN&Wnt7a treatment resulted in 156% increase in numbers of satellite stem cells relative to COL&Wnt7a treatment (Figure 2C). Strikingly, FN&Wnt7a treatment resulted in the formation of large homogeneous clusters of Pax7⁺/YFP⁻ satellite stem cells after 72h of myofiber culture (Figure 2D). Again, neither FN nor COL alone had a significant effect. Numbers of Pax7⁺/YFP⁺ cells were unchanged under all conditions at 42 hours and slightly increased by ~38% at 72 hours in the FN&Wnt7a condition (Figure S1A and S1B).

Figure 2. The Fzd7/Sdc4 co-receptor complex drives the symmetric expansion of satellite stem cells.

(A) Myofibers were isolated and cultured for 42h in the presence of Collagen (COL), FN, COL and Wnt7a (COL&Wnt7a) or FN and Wnt7a (FN&Wnt7a). FN potentiates the function of Wnt7a driving the expansion of satellite stem cells (Pax7⁺/YFP⁻). Bars represent means ± SEM. n=4. p values are ***p < 0.001; *p < 0.05.

(B) Quantification of satellite stem cell symmetric divisions after 42h of myofiber culture in the presence of COL, FN, COL&Wnt7a or FN&Wnt7a. Bars represent means ± SEM. n=4. p values are **p < 0.01; *p < 0.05.

(C) Quantification of satellite cell populations after 72h of myofiber culture in the presence of COL, FN, COL&Wnt7a or FN&Wnt7a. Bars represent means ± SEM. n=3. p values are *p < 0.05.

(D) FN&Wnt7a treatment results in the formation of clusters of satellite stem cells rather than mixed clusters by 72h of myofiber culture. Arrows indicate Pax7⁺/YFP⁻ satellite stem cells. Scale bar = 25 μm.

(E and F) Antibodies to Sdc4 block the ability of Wnt7a to stimulate satellite stem cells (αSdc4&Wnt7a) when compared to IgG (IgG&Wnt7a) after 42h of myofiber culture. Inhibition of Sdc4 also decreased numbers of Pax7⁺/YFP⁺ cells. Bars represent means ± SEM. n=3. p value is **p < 0.01.

(G) Quantification of satellite cell populations after 42h of myofiber culture in the presence of PBS vehicle, Tenascin-C (TEN), PBS&Wnt7a or TEN&Wnt7a. TEN inhibition of FN binding to Sdc4 antagonizes the effect of Wnt7a on satellite stem cells. Bars represent means ± SEM. n=3. p values is *p < 0.05.

To assess whether a functional Fzd7/Sdc4 receptor complex is required for FN&Wnt7a mediated expansion of the Pax7⁺/YFP⁻ satellite stem cells, we blocked Sdc4 using a neutralizing antibody (Cornelison et al., 2004). When compared to an unspecific IgG in combination with Wnt7a (IgG&Wnt7a), Sdc4 antibody and Wnt7a (αSdc4&Wnt7a) led to a 30% decrease in the numbers of Pax7⁺/YFP⁺ satellite myogenic cells, and a 64% decrease in Pax7⁺/YFP⁻ satellite stem cells after 42h of myofiber culture (Figure 2E and 2F). Moreover, blocking of FN binding to Sdc4 with Tenascin-C (TEN) (Huang et al., 2001) selectively impaired the ability of Wnt7a to stimulate the expansion of the Pax7⁺/YFP⁻ satellite stem cell pool on myofibers cultured for 42h (Figure 2G) without any effect on Pax7⁺/YFP⁺ satellite myogenic cells (Figure S1C). Taken together, these data confirm that Fzd7 and Sdc4 are co-receptors, and that Wnt7a signaling through Fzd7 requires ligation of FN to its receptor Sdc4.

Activated Satellite Cells Remodel their Niche with FN

Numerous cell types have been noted to express FN including fibroblasts, chondrocytes, endothelial cells, macrophages, as well as certain epithelial cells (Hynes and Yamada, 1982). Low levels of FN expression have been described in the interstitium and in capillaries of adult skeletal muscle (Peters et al., 1996). Satellite cells reside closely juxtaposed to muscle fibers in a niche between the sarcolemma and the basal lamina (Charge and Rudnicki, 2004). Immunostaining with antibodies directed to Pax7 and FN revealed that the satellite cell niche does not contain detectable levels of FN in resting tibialis anterior (TA) muscle, which was, however, present in ring-like structures reminiscent of capillaries (Figure 3A). By contrast, five days after acute cardiotoxin (CTX) muscle injury, satellite cells were embedded in a extracellular milieu containing high levels of FN.

Figure 3. Muscle regeneration is accompanied by a transient FN fibrosis.

(A) In homeostatic muscle tissue, satellite cells are found in close proximity to FN rich areas resembling capillaries. Upon injury the muscle is highly saturated with FN and the satellite cells are deeply embedded within it. Arrows denote Pax7 expressing satellite cells. Scale bar = 50μm.

(B) Regeneration time course after CTX injury of the TA muscle. FN levels increase at day 5 after CTX compared to the ECM component LM. Scale bar = 50 μm.

(C) qPCR from whole muscle cDNA at the given time points after CTX injury. The expression of FN correlates with Pax7. Data points represent mean ± SEM. n=3. “no injury” was set to 100% for both genes.

(D) FN expression in freshly FACS isolated cells from injured and uninjured muscle. Quiescent satellite cells (QSC) and activated satellite cells (ASC) are compared to non-satellite cells from uninjured (nSC-U) and injured (nSC-I) muscle. Bars represent means ± SEM. n=3. p value is *p < 0.05.

(E) Microarray heat map representing ECM genes from quiescent satellite cells (Quie.), proliferating myoblasts (Prol.) and 2 or 5 day differentiated (2d diff./5d diff.) myofibers. The probe for FN (Fn1) shows the highest signal in proliferating myogenic cells and is substantially lower in Quie. and diff. (Asterisk). Signal intensities represent the average of n=3 microarrays per condition for Prol. and diff. and n=1 microarray for Quie.

To study FN expression dynamics during regeneration, the TA muscle was injured by injection of CTX and analyzed at several time-points after injury. In contrast to the unrelated ECM component Laminin (LM), FN in muscle cross sections peaked at 5-days post injury and declined to baseline thereafter (Figure 3B). Quantitative real-time PCR (qPCR) using whole muscle lysates confirmed that maximal FN expression after injury correlated with Pax7 expression and therefore with tissue satellite cell content (Figure 3C).

To examine the expression of FN in satellite cells, we FACS isolated activated satellite cells (ASC) from four day CTX injured muscles of Pax7-zsGreen reporter mice (Bosnakovski et al., 2008) based on zsGreen fluorescence, and compared them to quiescent satellite cells (QSC) from injured muscle by qPCR (Figure 3D, S2A, S2B and S2C). For comparison we also collected an equal number of Pax7-zsGreen negative non-satellite cells from injured (nSC-I) and uninjured (nSC-U) muscles. qPCR confirmed that nSC-I and nSC-U did not express Pax7 (Figure S2A). The activation marker MyoD was expressed at significantly higher levels in ASC when compared to QSC (Figure S2B), while the expression of Spry1, a quiescence marker (Shea et al., 2010), tended to be higher in QSC (Figure S2C). ASC expressed 55 fold more FN than QSC (Figure 4B). Moreover, ASC expressed 40% of FN levels expressed in the nSC-I population, which largely consists of cells secreting high levels of ECM molecules such as fibroblasts, immune and vessel associated cells. qPCR over the EIIIA and EIIIB splice sites of FN revealed that myogenic cells express mostly cellular FN (cFN) that, in contrast to plasma FN (pFN), is locally bound in the cellular niches and does not diffuse (To and Midwood, 2011) (Figure S2D).

Figure 4. Activated satellite cells express FN to remodel their niche.

(A) Quiescent satellite cells which were directly fixed after fiber isolation, only express marginal amounts of FN, whereas proliferating activated satellite cells after 42h of fiber culture express high levels of FN. Scale bar = 5 μm.

(B) After 72h of fiberculture the majority of Pax7 positive satellite cells stain strongly for FN. Scale bar = 50 μm.

(C) 42h activated satellite cells that were stained with FN antibody before permeabilization (non perm.). Scale bar = 10 μm.

(D) Activated satellite cells on fibers that were directly fixed after isolation from regenerating muscle five days after CTX injury express high levels of FN underneath the intact basal-lamina. Scale bar = 5 μm.

(E) In dividing asymmetric satellite cell doublets on fibers after 42 hours of culture, satellite stem cells (Pax7⁺/YFP⁻) contain lower levels of FN that the apical satellite myogenic cell (Pax7⁺/YFP⁺). Scale bar = 5 μm.

(F) Background corrected, pooled average grey values of FN staining from >10 asymmetric divisions (as illustrated in Figure S3A). The area that was densitometrically analyzed for each cell in an individual division was kept constant. The YFP+ cell was set to 100% for each individual division.

These results demonstrate that satellite cells are a source of FN during muscle regeneration. To further investigate the expression pattern of FN and other ECM components by myogenic cells, we performed microarray gene expression analysis on prospectively isolated quiescent satellite cells (Quie.), versus established proliferating satellite cell-derived myoblasts (Prol.), and differentiated myotubes (2d and 5d diff.) (Figure 3E, Table S1). Several probes, including Vitronectin (Vtn), Biglycan (Bgn), Decorin (Dcn), Perlecan (HSPG2), Laminin subunits (Lama2 and Lamc1) and Nidogen (Nid1) showed a high hybridization signal in quiescent satellite cells when compared to primary myoblasts or differentiated myotubes. Notably, in proliferating primary myoblasts the probe for FN (Fn1) showed an intensive hybridization signal, which was 60–70% lower in quiescence or differentiation. When compared to FN, none of the other ECM components were as strongly upregulated in proliferation with respect to the other conditions. These data confirm that the expression of FN by myogenic cells is dynamically regulated and depends on the activation and differentiation state.

To investigate the expression of FN protein in activated satellite cells, we stained quiescent satellite cells on freshly isolated single myofibers (0h) or activated satellite cells on cultured fibers for 42h with anti-FN antibody (Figure 4A). FN was barely detectable in quiescent satellite cells, but was strongly upregulated in activated satellite cells at 42h in culture. After 72h of culture the majority of satellite cells on single fibers were readily identified by high-level expression of FN (Figure 4B).

To interrogate the dynamics of FN expression in satellite cells, we fixed single fibers as early as 8 hours after isolation and activation, and stained for FN. To exclude endocytosis of FN from the culture medium we depleted it from pFN using collagen-sepharose (Figure S2E). 8 hour activated satellite cells were readily identified by high-level expression of FN (Figure S4F). Most FN immunoreactivity was intracellular, suggesting its presence in the secretory pathway. Incubation of 42 hour activated satellite cells with FN antibody before permeabilization revealed that after prolonged activation a large fraction of FN protein was extracellularly localized (Figure 4C). To confirm that FN is locally bound in the niche of ASC in vivo, we isolated single muscle fibers from mice that had been injured for five days with CTX. Notably, FN expression was detectable in discrete domains around activated satellite cells within their niche beneath the intact basal-lamina (Figure 4D).

To assess the expression of FN in Pax7⁺/YFP⁻ satellite stem cells versus Pax7⁺/YFP⁺ satellite myogenic cells, we analyzed asymmetric satellite cell divisions found on cultured individual myofibers at 42h after isolation. This experiment revealed that FN expression was markedly up regulated in Pax7⁺/YFP⁺ satellite myogenic cells relative to Pax7⁺/YFP⁻ satellite stem cells (Figure 4E). Stringent washing conditions during the staining procedure were used to enrich for intracellular FN in the secretory pathway allowing for the quantification of protein levels in doublets resulting from asymmetric cell divisions (Figure 4F and S3A). Quantitative analysis of immunostaining grey values from >10 randomly selected asymmetric doublets using non-saturating concentrations of FN antibody revealed that Pax7⁺/YFP⁻ cells contain about 60% of the FN levels found in Pax7⁺/YFP⁺ cells (Figure 4F).

We next compared FACS-purified Pax7⁺/YFP⁻ primary cells in passages <3 to Pax7⁺/YFP⁺ cells. Cultured Pax7⁺/YFP⁻ cells expressed 40% of Myf5 and 66% of FN mRNA when compared to Pax7⁺/YFP⁺ cells (Figure S3B and S3C). Moreover, compared to Pax7⁺/YFP⁺ cells, Pax7⁺/YFP⁻ cells contained lower levels of FN protein (Figure S3D). The observation that Pax7⁺/YFP⁺ satellite myogenic cells express elevated levels of FN relative to Pax7⁺/YFP⁻ satellite stem cells intriguingly suggests that Wnt7a signaling is primed in satellite stem cells by FN originating from satellite myogenic cells following an asymmetric division.

FN Knockdown Impairs Satellite Cell Function

Our experiments indicate that Fzd7 and Sdc4 are co-receptors, and that Wnt7a signaling through Fzd7 requires ligation of FN to its receptor Sdc4. Therefore, the observation that activated satellite cells upregulate FN to remodel their niche suggests that FN expression provides feedback to modulate Wnt7a-induced PCP signaling in satellite cells.

To investigate the cell-autonomous role of satellite cell-derived FN, we treated single myofibers isolated from the TA muscle of Myf5-Cre/R26R-YFP mice with a validated duplexed silencer select siRNA for FN (siFN) or with a scrambled siRNA (siSCR) (Daley et al., 2009; Daley et al., 2011) in pFN free culture medium. Removal of pFN from the culture medium decreased the number of both Pax7⁺/YFP⁺ and Pax7⁺/YFP⁻ per fiber after 42h of culture (siSCR in Figure 5A and 5C) when compared to normal serum (COL in Figure 2A and S1A or PBS in Figure 2G and S1C). Transfection of siFN further reduced the number of Pax7⁺/YFP⁺ cells by 51% from 4.7 per fiber in the siSCR control to 2.3 (Figure 5A) and the number of Pax7⁺/YFP⁺ divisions by 56% from 0.9 per fiber to 0.4 (Figure 5B). Strikingly, siFN reduced the number of Pax7⁺/YFP⁻ cells by 80% from 0.4 per fiber in the siSCR condition to 0.08 (Figure 5C). Moreover, the symmetric proliferation of Pax7⁺/YFP⁻ cells was completely abolished by siFN (Figure 5D).

Figure 5. Knock down of FN impairs satellite cell function.

(A) FN was knocked down in satellite cells on isolated Myofibers in pFN free culture medium for 42h. siFN reduces the number of Pax7⁺/YFP⁺ cells per fiber when compared to the siSCR control. Bars represent means ± SEM. n=3. p values are is *p < 0.05.

(B) siFN reduces the number of symmetric Pax7⁺/YFP⁺ divisions. Bars represent means ± SEM. n=3. p value is *p < 0.05.

(C) Knockdown of FN severely reduces the number of Pax7⁺/YFP⁻ cells per fiber. Bars represent means ± SEM. n=3. p value is *p < 0.05.

(D) No symmetric Pax7⁺/YFP⁻ division could be detected (n.d.= none detected) in the siFN condition when compared to siSCR. Bars represent means ± SEM. n=3.

This data demonstrates a cell-autonomous requirement of satellite cells for FN that is pronounced in the Pax7⁺/YFP⁻ population. Moreover, the cell-autonomous loss FN expression appears to phenocopy the effect of knockout of Sdc4 from satellite cells (Cornelison et al., 2004) or antibody inhibition of Sdc4 (Figure 2E and 2F). Together, these data support the notion that cell-autonomous FN expression is not only required for Wnt7a signaling through the Fzd7/Sdc4 co-receptor complex in satellite stem cells, but is also essential for Sdc4 function in all satellite cells.

Cell-autonomous FN is Essential for the Maintenance of the Satellite Cell Pool

To investigate the satellite cell phenotype resulting from FN knockdown in muscle tissue we injected a self-delivering FN siRNA into the TA at 3 days after CTX injury. This treatment resulted in a 59% reduction in satellite cell numbers relative to siSCR injected muscles when examined 10 days after injury (Figure S4A and S4B). siRNA knockdown of FN reduced expression levels by 58% after 5 days in whole muscle tissue (Figure S4C).

Previous work has shown that within the satellite cell population, Pax7⁺/YFP⁻ satellite stem cells are the cell type than can repopulate the stem cell niche in transplantation paradigms (Kuang et al., 2007). Because our data suggested that FN is of essential importance for the function of Pax7⁺/YFP⁻ cells we decided to test for the consequences of loss of cell-autonomous FN from transplanted satellite cells. To address this question we isolated satellite cells, performed an ex vivo siRNA knockdown of FN, then transplanted the cells back into muscle, and enumerated repopulation of the satellite cell niche (Figure 6A). Briefly, quiescent satellite cells were FACS purified from Pax7-zsGreen reporter mice based on zsGreen expression and transfected with siFN or siSCR for three hours on ice. After extensive washing, 15,000 transfected satellite cells were either injected into the TA of immunosupressed mice that had received a CTX injury two days previously, or cultured for three days for qPCR validation of knockdown efficiency. Three weeks after transplantation, mice were sacrificed and the engraftment of Pax7 and zsGreen double-positive (Pax7⁺/zsGreen⁺) cells was assessed by immunostaining of muscle sections (Figure 6B). Strikingly, ex vivo siRNA knockdown of FN in prospectively isolated satellite cells resulted in a 65% reduction of their engraftment three weeks following injection (Figure 6C). When siRNA treated freshly isolated satellite cells were cultured instead of transplanted, siFN reduced FN mRNA by 50% after three days (Figure S5). Importantly, resident satellite cells in the injected TA muscle displayed no significant change in their numbers (Figure 6D) indicating that siFN transfection remained limited to ex vivo. Taken together, these results demonstrate that cell-autonomous expression of FN by activated satellite cells within their niche is indispensable for the homeostatic regulation of the satellite cell pool size during regenerative myogenesis.

Figure 6. Cell-autonomous FN is essential for the maintenance of satellite cells in their niche.

(A) Scheme of the siRNA knockdown strategy that was used to test the function of cell-autonomous FN for satellite cells in vivo.

(B) Three weeks after transplantation, donor derived cells are observed as zsGreen⁺/Pax7⁺ cells (yellow arrowheads) in host tissue. Scale bar = 50 μm.

(C) Knockdown of FN in transplanted satellite cells resulted in a 65% reduction in their number. Only Pax7⁺/zsGreen⁺ donor cells were included in the quantification. Bars represent means ± SEM. n=3. p value is *p < 0.05.

(D) The number of resident Pax7⁺/zsGreen⁻ satellite cells is not significantly changed by transplantation of siFN or siSCR treated satellite cells. Bars represent means ± SEM. n=3.

FN and Wnt7a Cooperate In vivo

To elucidate whether increased FN levels are capable of modulating the satellite cell response to Wnt7a stimulation in vivo, we electroporated Wnt7a-HA and/or FN expression plasmids into the TA muscle and quantified the number of satellite cells after seven days. Electroporation of the CMV-FN expression plasmid resulted in a 51% decrease in the numbers of Pax7⁺ satellite cells relative to electroporation of empty vector (EV) while CMV-Wnt7a lead to a 49% increase (Figure S6). To determine the number of satellite stem cells after electroporation we used Myf5-nLacZ mice (Tajbakhsh et al., 1996) which facilitate the ready detection of Pax7⁺/β-Gal⁻ cells by immunostaining on histological sections (Kuang et al., 2007). β-Gal antibody-staining revealed that CMV-FN plasmid alone did not significantly change numbers of Pax7⁺/β-Gal⁻ satellite cells (Figure 7A). However, CMV-Wnt7a increased numbers Pax7⁺/β-Gal⁻ satellite cells by 289%. Importantly, the combination CMV-FN and CMV-Wnt7a plasmid lead to a 654% increase in the number of Pax7⁺/β-Gal⁻ satellite cells. Pax7⁺/β-Gal⁻ satellite cells were evenly distributed throughout the muscle cross-sections and we did not observe focal accumulation around Wn7a-HA expressing fibers (Figure 7B). Co-electroporation of CMV-FN and CMV-Wnt7a plasmids did not significantly change total satellite cell numbers. This result strongly supports the assertion that Wnt7a and FN stimulate PCP signaling to drive the symmetric expansion of satellite stem cells during regenerative myogenesis.

Figure 7. Wnt7a and FN stimulate the expansion of satellite stem cells in muscle tissue.

(A) Plasmid vectors expressing FN and Wnt7a were electroporated into TA muscles of Myf5-LacZ mice. After 7d the electro-damage induced regeneration is accompanied by an increase in the amount of Pax7⁺/β-gal⁻ satellite stem cells for Wnt7a and for Wnt7a&FN when compared to empty vector (EV). A significant increase in Pax7⁺/β-gal⁻ satellite stem cell numbers can be observed for FN&Wnt7a when compared to Wnt7a alone. Bars represent means ± SEM. n=3. p values are ***p < 0.001; **p < 0.01; *p < 0.05.

(B) At seven days following electroporation, the effect of FN and Wnt7a is readily apparent. Arrows indicate Pax7⁺/β-gal⁻ satellite stem cells. Scale bar = 50μm.

DISCUSSION

Wnt7a/Fzd7 signaling stimulates symmetric stem cell divisions to regulate the overall numbers and proportion of satellite stem cells versus committed satellite myogenic cells (Le Grand et al., 2009). Our study revealed that during the initial proliferative response to injury, committed satellite myogenic cells release elevated quantities of FN into their microenvironment. FN ligation to the Fzd7/Sdc4 receptor complex during this stage of muscle regeneration is required for Wnt7a to induce the expansion of the satellite stem cell pool. Interference with this mechanism by knockdown of FN leads to a dramatic reduction of the overall satellite cell pool after injury and therefore impairs the regenerative potential of muscle.

Our results demonstrate that satellite cells dynamically auto-regulate the FN content in their niche and a loss of this ability due to siRNA knockdown leads to an impaired maintenance of satellite cells. Intriguingly, satellite stem cells produce markedly lower amounts of FN than satellite myogenic cells. This suggests that during an asymmetric cell division, the committed daughter cell remodels the niche to instruct the remaining satellite stem cell to become more responsive to Wnt7a/Fzd7 signaling. We speculate that the presentation of FN to satellite stem cells from satellite myogenic cells provides a feedback mechanism to modulate the overall size of the satellite cell pool and control the ratio between satellite stem cells and their committed myogenic daughter cells.

Sdc4 is thought to play a role in cell adhesion through association with α5β1 integrin at focal adhesions. Following Sdc4 binding to FN in the extracellular matrix, the Sdc4 cytoplasmic domain binds phosphatidylinositol 4,5-bisphosphate, which stimulates PKCα activation, leading to the activation of small GTPases and assembly of focal adhesions (Oh et al., 1997; Saoncella et al., 1999; Couchman, 2003; Lim et al., 2003; Dovas et al., 2006). Sdc4 was also found to function as a Fzd7 co-receptor during Xenopus Laevis gastrulation. Sdc4 and Fzd7 are both required for PCP-regulated convergent extension movements during gastrulation (Davidson et al., 2006; Munoz et al., 2006). Here we demonstrate the Fzd7/Sdc4 co-receptor complex exists in mammalian cells. Our experiments further revealed that FN binding the Fzd7/Sdc4 receptor complex potentiates the ability of Wnt7a to activate the PCP mediated symmetric expansion of satellite stem cells. Sdc4 has also been suggested to be the co-receptor for CXCR4 (Hamon et al., 2004). It is interesting to speculate that remodelling of the satellite cell microenvironment with FN also potentiates cytokine signaling.

Sdc4 knockout in the mouse leads to an impaired activation and proliferation of satellite cells (Cornelison et al., 2004). This phenotype strikingly resembles the FN loss-of-function phenotype and suggests that cell-autonomous production of FN ligand in the satellite cell niche is required for Sdc4 function. Since Syndecans have been implicated in cell migration and survival, loss of cell-autonomous FN might also influence these factors (Beauvais and Rapraeger, 2004). Interestingly, Sdc4 knockout mice also display disorganized muscle fibers that do not align with the axis of the former myofibers after muscle injury. These findings suggest an additional role of Sdc4 in defining cell-polarity during differentiation. Wnt7a mediated PCP signalling does not influence differentiation (von Maltzahn et al., 2011). Therefore, polarity determination of differentiating cells by Sdc4 might involve a variation of PCP signaling that is independent of Wnt7a.

FN has been previously demonstrated to inhibit myogenic differentiation (Podleski et al., 1979). Our data indicate that satellite cell derived myogenic precursor cells (mpc) entering the differentiation program downregulate FN expression. Interestingly, mutant mice lacking Membrane-type 1 matrix metalloproteinase (MT1-MMP), which cleaves FN, display pronounced defects in differentiation and fusion of myogenic precursors (Ohtake et al., 2006). This suggests that downregulation of FN in the microenvironment of mpc’s is important for their differentiation.

Degenerative muscle diseases are often accompanied by fibrotic scarring due the persistent excess deposition of ECM components such as FN and certain types of COL (Wynn, 2008). Fibrosis is generally considered to impede satellite cell function and muscle repair (Mann et al., 2011). Intriguingly, our results revealed that a transient FN-rich fibrosis is required for the maintenance of satellite cells in their niche during muscle regeneration. This suggests that the temporary deposition of FN in the satellite niche is an indispensable physiological response of muscle to injury and it is likely that deregulation of this process due to chronic fibrosis might contribute to the pathology of degenerative muscular diseases by perturbing the spatiotemporal regulation of satellite cell function.

The expression of transforming growth factor-β (TGF-β) is frequently increased in muscular dystrophy (Serrano and Munoz-Canoves, 2010). TGF-β is a factor that triggers the production of COL and FN in fibroblasts, a process that has been linked to muscle fibrosis (Grande et al., 1997; Ignotz and Massague, 1986; LeRoy et al., 1990). Moreover, it has also been demonstrated that TGF-β can drive cultured myoblasts into a fibrotic cell type (Li et al., 2004). However, depending on concentration, this factor appears to have beneficial effects for myogenic cells (Carlson et al., 2009). TGF-β may also be involved in the control of FN expression in satellite cells. Thus, the permissive versus non-permissive effects of TGF-β on myogenesis could reflect the role of TGF-β in regulating FN biogenesis in satellite cells.

Circulating pFN does not contain the alternatively spliced EIIIB and EIIIA modules that are present in cellular FN cFN but both FN isoforms contain the Sdc4 binding Hep II domain (Singh et al., 2010; Woods et al., 2000). pFN is a major component of the fibrin clot in the early wound-healing response (To and Midwood, 2011) and could potentially be involved in the initial regulation of satellite cells at the site of acute muscle injury. Moreover, fibroblasts and macrophages are highly abundant in early phases of skeletal muscle regeneration and have been shown to critically influence satellite cells (Chazaud et al., 2003; Murphy et al., 2011; Zhang et al., 2011). Both cell types express FN (Hynes and Yamada, 1982) and might well contribute to the transient FN-rich pro-myogenic fibrosis during muscle regeneration. Other cell types involved in the regulation of satellite cells such as fibro/adipogenic progenitors (FAPs) or PW1+/Pax7− interstitial cells (PICs) could also be potential sources of FN (Joe et al., 2010; Mitchell et al., 2010).

Importantly, our experiments revealed that the niche of activated satellite cells in-situ consists of a well-defined FN microdomain underneath the basal-lamina in the absence of other cell types. Moreover, myogenic cells mostly produce cFN that is locally bound and does not diffuse. Despite of these findings we cannot exclude that other cell types, that only transiently accompany satellite cells during muscle regeneration, contribute to the dynamic remodeling of their niche with FN. Our results demonstrate that, next to activated satellite cells, non-satellite cells are significant contributors to the FN response after injury. Future studies utilizing cell-type specific Cre recombinase expression and conditional alleles of FN will help to clarify to which degree FN derived from distinct cell types is involved in regulation of the satellite cell pool during myogenesis.

In summary, we have discovered a novel physiological mechanism regulating the satellite cell pool during muscle regeneration. We demonstrate that committed satellite cells contribute to a dynamic temporal FN fibrosis. FN in the satellite cell niche is required for the maintenance of the overall satellite cell pool during muscle regeneration. Moreover, FN is necessary to potentiate Wnt7a signaling through the Fzd7/Scd4 receptor complex, which controls the regulation of satellite stem cell numbers. Our identification of a molecular mechanism that integrates growth factor signaling and structural information within the stem cell niche, to direct the expansion of the satellite cell pool during adult myogenesis represents an important advance in our understanding of muscle biology.

EXPERIMENTAL PROCEDURES

Mice and Animal Care

6–8 week old Myf5-Cre/R26R-YFP mice were obtained by crossing Myf5-Cre mice with R26R-YFP reporter mice (Srinivas et al., 2001; Tallquist et al., 2000). Pax7-zsGreen and Myf5-nLacZ mice were generated as previously described (Bosnakovski et al., 2008; Tajbakhsh et al., 1996). Mice were maintained inside a barrier facility, and experiments were performed following the University of Ottawa regulations for animal care and handling.

Myofiber Isolation and Culture

Single myofibers were isolated from the EDL muscles as previously described (Rosenblatt et al., 1995). Isolated myofibers were cultured in suspension in horse serum coated dishes (Kuang et al., 2006). Fiber medium contained 20% FBS (Hyclone) and 1% chick embryo extract (CEE, Accurate Chemicals) and DMEM with 2% L-glutamine, 4.5% glucose, and 110mg/ml sodium pyruvate. For Wnt stimulation, recombinant Wnt7a was added to the fiber medium to a final concentration of 100ng/ml (R&D Systems). To expose the fibers to increased FN or COL levels, human plasma fibronectin (BD biosciences) or rat tail collagen in PBS (VWR) were added to increase the concentration in the medium by 25μg/ml. For inhibition of FN binding to Sdc4 in fibercultures, 5 μg/ml Tenascin-C (R&D Systems) was added to the fiber medium. For inhibition of Sdc4, 20 μg/ml chicken anti-Sdc4 (Cornelison et al., 2004) was added to the fiber medium.

Primary Myoblast Isolation and Culture

Pax7⁺/YFP⁺, Pax7⁺/YFP⁻ and total satellite cells were obtained from hind limb muscles and FACS isolated as previously described (Kuang et al., 2006; Le Grand et al., 2009). For myoblast culture, satellite cells were sorted and plated on COL coated dishes (BD biosciences) in Ham’s F10 medium supplemented with 20% FBS and 5 ng/ml of basic FGF (Millipore).

Western blotting and immunoprecipitation

For Co-IP experiments and Rac1 activation assay satellite cell derived primary myoblasts were transfected with Lipofectamine 2000 according to the manufacturer’s instructions. For Co-IP the cells were treated with Disuccinimidyl suberate crosslinker prior to lysis (Pierce). Cell extracts were obtained by RIPA buffer lysis in the presence of protease inhibitor cocktail (Nacalai). GFP-Trap beads (Allele Biotechnology) or anti-flag M2 beads (Sigma) were used for Co-IP according to the manufacturer’s recommendations. Whole muscle extracts for western blotting and grey value densitometry of western blots were performed as previously described (Bentzinger et al., 2008). Denaturing SDS-PAGE was performed using standard techniques. Rac1 activation assay was performed according to the manufacturer’s instructions (Pierce).

Tissue and satellite cell siRNA knockdown

For FN knockdown in satellite cells and subsequent transplantation, cells were FACS purified from Pax7-zsGreen mice by gating for zsGreen and Hoechst (Bosnakovski et al., 2008). Directly after isolation the cells were lipofected with a validated duplexed silencer select siRNA for FN (Daley et al., 2009; Daley et al., 2011) for three hours on ice. Silencer select FN siRNA was: Sense (5-->3): CCG UUU UCA UCC AAC AAG A (TT) and anti-sense (3-->5): U CUU GUU GGA UGA AAA CGG (GT). After siRNA transfection satellite cells were washed several times with FACS buffer. 15,000 cells for each condition were resuspended in 0.9% NaCl and immediately transplanted into muscles of FK506 immunosupressed mice that had been injured two days before. Scd4 and FN were knocked down in satellite cells in fiberculture as previously described (Le Grand et al., 2009). Silencer select siRNA to FN is described above, Sdc4 siRNA was: Sense (5-->3): GUU ACG ACU UGG GCA AGA A (TT) and anti-sense (3-->5): UUC UUG CCC AAG UCG UAA C (TG). siRNA to Fzd7 has been previously described (Le Grand et al., 2009). In all siRNA knockdown experiments, except for the in vivo knockdown (Figure 5C, 5D and 5E), scrambled siRNA Silencer Select Negative Control No. 1 was used as a control (Ambion). For tissue knockdown the validated FN siRNA sequence was modified to the Accell self-delivering format (Dharmacon). 100μg Accell siRNA was injected into muscles two days after CTX injury. FN Accell siRNA was: Sense (5-->3): CCG UUU UCA UCC AAC AAG A (dGdT) and anti-sense (3-->5): (dTdG) G GCA AAA GUA GGU UGU UCU (5′-P). A similarly modified scrambled sequence was used as a negative control.

Statistical Analysis

Densitometry of grey values from western blots and FN staining in asymmetric doublets was performed with the Image-J software. Compiled data are expressed as mean ± standard error of the mean (SEM). Experiments were done with a minimum of three biological replicates. For statistical comparisons of two conditions, the Student’s t-test was used. The level of significance is indicated as follows: *** p< 0.001, ** p< 0.01, * p< 0.05.

Highlights.

• Fzd7 and Sdc4 form a co-receptor complex in satellite cells

• FN stimulates Wnt7a activity through the Fzd7/Sdc4 co-receptor complex

• Activating satellite cells remodel their niche with FN

• FN regulates the satellite cell pool during muscle regeneration