The actin regulator N-WASp is required for muscle-cell fusion in mice

Abstract

A fundamental aspect of skeletal myogenesis involves extensive rounds of cell fusion, in which individual myoblasts are incorporated into growing muscle fibers. Here we demonstrate that N-WASp, a ubiquitous nucleation-promoting factor of branched microfilament arrays, is an essential contributor to skeletal muscle-cell fusion in developing mouse embryos. Analysis both in vivo and in primary satellite-cell cultures, shows that disruption of N-WASp function does not interfere with the program of skeletal myogenic differentiation, and does not affect myoblast motility, morphogenesis and attachment capacity. N-WASp–deficient myoblasts, however, fail to fuse. Furthermore, our analysis suggests that myoblast fusion requires N-WASp activity in both partners of a fusing myoblast pair. These findings reveal a specific role for N-WASp during mammalian myogenesis. WASp-family elements appear therefore to act as universal mediators of the myogenic cell-cell fusion mechanism underlying formation of functional muscle fibers, in both vertebrate and invertebrate species.

Myoblast fusion provides a universal mechanism for formation and growth of multinucleated muscle fibers (1, 2). However, although the genetic regulatory networks governing skeletal mammalian myogenesis are well characterized (3, 4), many of the cellular mechanisms underlying execution of myogenic differentiation, including muscle-cell fusion, are poorly understood.

Studies of myoblast fusion during Drosophila myogenesis have put forward a molecular genetic framework, which ascribes major significance to the contribution of the actin-based cytoskeleton, with a particularly prominent role assigned to the branched actin polymerization machinery centered on the Arp2/3 complex (5–7). Nucleation of branched actin polymerization by Arp2/3 is commonly stimulated by nucleation promoting factors (NPFs) belonging to the WASp-protein family. Therefore, the conserved nature of the Arp2/3 complex and its associated NPFs raises the possibility that elements of this machinery are universal mediators of myoblast fusion. To address this issue, we examined the consequences of disrupting the function of N-WASp, the primary mammalian homolog of WASp-family proteins, during embryonic myogenesis in mice. Here, we report that myogenesis in mouse embryos is severely impaired following disruption of N-WASp function in myogenic tissue, throughout the skeletal muscle field. Although the size and distribution of the progenitor myoblast population is not affected, these cells give rise to thin, mononucleated muscle fibers. Using primary cell cultures, we show that N-WASp–deficient myoblasts are motile, differentiate properly, and assume the morphology of mature myogenic cells, yet fail to fuse. These observations identify a myogenic setting for N-WASp function, and suggest an essential, universal involvement for branched actin nucleation, mediated by WASp-family elements, during the process of myoblast fusion.

Results

Conditional Disruption of Murine N-WASp Results in Abnormal Skeletal Myogenesis.

Disruption of the N-WASp gene results in embryonic lethality at embryonic day E11, characterized by small body size and prominent neural tube and cardiac defects (8). To circumvent these phenotypes, which bar proper study of myogenesis in the absence of N-WASp function, we made use of a conditional, loxP-based allele (referred to as N-WASp^fl), which enables tissue-specific disruption of N-WASp (9). Two Cre driver lines, Myf5^Cre (10) and MyoD^Cre (11), were used for this purpose. Both drivers mediate Cre-based recombination at early phases of the myogenic program, in all skeletal muscle progenitor cells, and are considered highly effective tools for studying the consequences of single-gene disruption on skeletal myogenesis (12). Mice in which N-WASp was disrupted using N-WASp^fl and either of the two myogenic Cre lines completed embryogenesis, but died immediately after birth. Although the general morphology of these conditional knockout animals (referred to herein as N-WASp^cKO) was normal, external examination, as well as tissue dissection, suggested that skeletal muscle mass was greatly reduced (Fig. S1). Furthermore, these animals were incapable of inflating their lungs, a common consequence of impairment to muscle development in mouse embryos, and the likely cause of postnatal lethality. Indeed, immunostaining of histological sections revealed that the musculature of Myf5^Cre/N-WASp^fl/- embryos at E16.5–E18, visualized with antibodies to the heavy chain of muscle myosin (MHC), is poorly developed (Fig. 1 A–D). The effect is extensive, as practically all skeletal muscle groups (body-wall, limb, respiratory, facial, etc.) are affected in this manner.

Fig. 1.

Skeletal muscle mass of N-WASp^cKO embryos is severely reduced, whereas myogenic differentiation is unaffected. (A–D) Histological sections of wild-type (WT) embryos (A and C) and Myf5^Cre/N-WASp^fl/- embryos (B and D). Muscle fibers are visualized with anti-MHC (red) and nuclei with DAPI (blue). (A and B) Matching sections revealing body wall muscles (BM) and limb muscles (LM) at E16.5. (C and D) Matching sections revealing facial elements at E16.5. EOM, extra ocular muscles. (E and F) RNA in situ hybridization of a myogenin DNA probe to E10.5 WT (E) and Myf5^Cre/N-WASp^fl/- (F) embryos reveals an identical pattern of somitic expression. (G and H) Immuno-localization of MHC in E11.5 WT (G) and Myf5^Cre/N-WASp^fl/- (H) embryos reveals similar patterns of differentiated muscle groups. Somitic (S), forelimb (FL), hindlimb (HL), and temporalis (T) muscles are indicated. (Scale bars: 500 μm in A, 100 μm in C, and 2 mm in F and H.)

Myogenic Program Is Properly Initiated in N-WASp^cKO Mice.

To elucidate the basis for the dramatic effect of disrupting N-WASp on muscle fiber formation, we examined several key features associated with the onset of skeletal myogenesis in N-WASp^cKO embryos. The complex set of genetic programs that govern fate determination during skeletal myogenesis commonly lead to expression of the key differentiation factor myogenin in all skeletal muscle progenitors (3, 4). Using RNA in situ hybridization, we determined that the normal expression pattern of myogenin at E10.5 remains unaltered in N-WASp^cKO embryos (Fig. 1 E and F and Fig. S1). This observation implies both proper initiation of the program underlying skeletal muscle differentiation, as well as proper myogenic patterning within the somites, which will give rise to body-wall skeletal muscles. Myf5^Cre/N-WASp^fl/- embryos further display robust expression of MHC, a second marker of advanced myoblast differentiation, in E11.5 wholemount preparations (Fig. 1 G and H). MHC expression at this stage is displayed by a variety of myoblast populations, consistent with proper specification and differentiation of skeletal muscle progenitors in different anatomical locations.

Given the well established contribution of Arp2/3-based branched actin polymerization to cell motility (13), we next ascertained whether lack of N-WASp function interferes with the motile capacity of skeletal muscle progenitors. To address this issue, we generated N-WASp^cKO embryos using the N-WASp^fl allele, together with the Pax3^Cre driver (14). In such embryos, N-WASp is disrupted in dermomyotome-derived muscle precursors before their differentiation, allowing to assess their capacity to migrate considerable distances away from their somitic origin (15). Pax3^Cre/N-WASp^fl/- embryos do not develop past E12, probably due to expression of the driver within the neural crest and heart progenitors, but survive long enough to allow for analysis of early myogenesis in the absence of N-WASp function. Importantly, MHC-expressing cells in the limb are properly patterned within these embryos, demonstrating that the progenitors migrate away from the somites and populate limb buds and other sites of differentiation and muscle fiber formation (Fig. S1). Furthermore, the expression pattern of Pax7, an early myoblast fate marker, revealed that Pax3^Cre/N-WASp^fl/- embryos possess a normally sized field of limb bud myoblasts (Fig. S1).

Taken together, these observations imply that disruption of murine N-WASp does not interfere with the specification, migration capacity, and differentiation program of skeletal myoblasts during embryogenesis.

Skeletal Muscle Fibers of N-WASp^cKO Mice Are Mononucleated.

The demonstration that the initial phases of skeletal myogenesis do not require N-WASp led us to examine a “timeline” of histological sections of maturing N-WASp^cKO embryos, stained for informative markers, to identify the stage at which phenotypic defects first arise (Fig. 2). Mixed fields of Pax7-positive precursors and MHC-expressing cells at E10.5, demonstrate proper and timely onset of the early stages of myogenesis in the somites and limbs of N-WASp^cKO embryos (Fig. 2 A, B, E, F, I, and J). As muscle development progresses, mild phenotypic abnormalities can be detected by E14.5, when muscle fibers in N-WASp^cKO embryos appear shorter and thinner than those in age-matched wild-type embryos (Fig. 2 C, G, and K). These phenotypes become highly pronounced as the embryos mature further (Fig. 2 D, H, and L). Significantly, N-WASp^cKO embryonic muscle fibers remain mononucleated, suggesting that their underdeveloped nature results from a failure to incorporate myoblasts via fusion into maturing fibers.

Fig. 2.

Muscle fibers of N-WASp^cKO embryos are properly initiated, but remain mononucleated. (A-D) Myogenesis in WT embryos. (A–D) Somitic (A) and limb (B–D) myogenic cell fields at E10.5 (A and B), E14.5 (C), and E16.5 (D). Pax7 (green) marks myoblast nuclei, MHC (red) marks fibers and all nuclei are visualized by DAPI (blue). (E–L) Corresponding panels showing the progress of myogenesis in Myf5^Cre/N-WASp^fl/- embryos (E–H) and in MyoD^Cre/N-WASp^fl/- embryos (I–L) of similar ages. Arrows point to nuclei within representative multinucleated wild-type fibers in C and D, and to single nuclei within mutant fibers in G, H, K, and L. Representative mononucleated fibers in E16.5 N-WASp^cKO embryos are outlined in H and L. (Scale bar: 50 μm in A.)

Fusion arrest in N-WASp^cKO embryos is associated with enhanced activation of Caspase-3 (Fig. S2 A–C), implying increased apoptosis within the myogenic field. The myogenic differentiation program is not perturbed, however, evidenced by the unaltered ratio between myoblasts expressing Pax7 and those expressing the more advanced differentiation marker MyoD (Fig. S2 D–J).

N-WASp–Depleted Primary Cultures Differentiate Properly but Fail to Fuse.

We next turned to primary cell cultures, to analyze the myogenic requirement for N-WASp at cellular resolution. Toward this end, satellite cell cultures were prepared from isolated muscle fibers of N-WASp^fl/fl mice (16). Disruption of N-WASp in these cultures was achieved following infection with the adenovirus vector Ad-Cre-eGFP, containing both the Cre recombinase and nuclear eGFP. Depletion of N-WASp protein was verified by Western blot analysis (Fig. S3). Differentiation of the adenovirus-infected cultures was induced via serum starvation. N-WASp^fl/fl–derived cultures, separately infected with a vector harboring eGFP alone (Ad-eGFP), harbor an intact N-WASp locus while expressing cytoplasmic eGFP, and therefore served as our primary control.

Monitoring culture differentiation by visualization of cell morphologies and expression of informative markers, revealed a striking difference between the Ad-eGFP– and Ad-Cre-eGFP–infected cultures of N-WASp^fl/fl satellite cells (Fig. 3). Although the control cells readily formed dense arrays of multinucleated myofibers by this protocol (Fig. 3 A–C and E–G), the N-WASp–depleted cultures were primarily composed of individual, mononucleated cells (Fig. 3 D and H). Quantification demonstrated that 67% of control myotubes harbored three or more nuclei, whereas 73% of N-WASp–depleted cells contained only a single nucleus, with nearly the entire remainder composed of binucleated cells (Fig. 3I).

Fig. 3.

Disruption of N-WASp inhibits myoblast fusion in satellite cell cultures. (A–H) Primary cultures derived from satellite cells, isolated from EDL muscles of 4- to 6-wk-old mice, following infection with various adenovirus vectors and 48 h of serum-starvation-induced differentiation. Muscle fibers are visualized with anti-MHC (red) and nuclei with DAPI (blue). GFP (green) is an indicator of viral infection. (A and B) Cultures derived from WT (ICR) mice and infected with Ad-eGFP (A) or Ad-Cre-eGFP (B) viruses. (C and D) Cultures derived from N-WASp^fl/fl mice and infected with Ad-eGFP (C) or Ad-Cre-eGFP (D) viruses. (E–H) Magnified views of cultures matching those shown in A–D. Control cultures (A–C and E–G) formed multinucleated fibers, whereas the fibers in cultures in which N-WASp was disrupted (D and H) remained mononucleated. (I) Graphic presentation of the fusion index assayed following 48 h in differentiation medium (n = number of nuclei within individual fibers; ∼800 nuclei were counted and classified for each of the four infection protocols). (Scale bars: 100 μm in A and 50 μm in E.)

Depletion of N-WASp from the satellite cell cultures was associated with enhanced apoptosis, matching our reported observations in N-WASp^cKO embryos. To ascertain that the block in myoblast fusion was not a consequence of the reduced cell density resulting from cell death, we reassessed the fusion capacity of Ad-eGFP-Cre–infected cultures of N-WASp^fl/fl satellite cells that were plated at a 2.5-fold higher confluency than the standard assay. Myoblast fusion upon serum starvation is arrested to a similar extent under these conditions (74% mononucleated cells). In a corresponding experiment, control (Ad-eGFP–infected) cultures were plated at a fourfold lower confluency than the standard assay. Myoblast fusion upon serum starvation declines somewhat, but remains robust under these conditions (43% of myotubes harbor three or more nuclei).

Although strongly deficient in their capacity to generate multinucleated myotubes, N-WASp–depleted cells displayed a variety of features associated with proper myogenic differentiation before onset of fusion. Thus, these cells adopted the elongated, spindle-like morphology characteristic of differentiated myocytes (Fig. 4 A and B), and expressed robust, normal levels of differentiation markers such as MyoD, Myogenin, and MHC (Fig. S3). Importantly, interfaces between neighboring pairs of N-WASp–depleted cells displayed pronounced recruitment of β-catenin, a marker and component of productive myogenic cell attachments (17, 18), suggesting that cell–cell contact was properly initiated (Fig. 4 C and D).

Fig. 4.

Cultured N-WASp–depleted myoblasts cannot fuse, but retain major features of differentiation. (A–D) Differentiating satellite cell cultures derived from N-WASp^fl/fl mice and infected with either Ad-eGFP (A and C), leaving N-WASp intact, or with Ad-Cre-eGFP (B and D), leading to disruption of N-WASp. (A and B) Differentiated myoblasts and muscle fibers are visualized with anti-MHC (red), and nuclei with DAPI (blue). (C and D) Localization pattern of β-catenin (red), including strong accumulation at adhering myoblast interfaces (arrowheads), is maintained in N-WASp–depleted cells. A strong β-catenin signal is displayed along contact surfaces of 4.2 ± 1.6% of control cell pairs and 4.8 ± 1.5% of N-WASp–depleted cell pairs (n ∼ 900, P = 0.31). (E-H) Initial (E and G) and final (F and H) frames of movies documenting progress of differentiation in satellite cell culture derived from N-WASp^fl/fl mice, and infected either with Ad-eGFP (E and F) or with Ad-Cre-eGFP (G and H). The N-WASp–depleted cells are highly motile, and establish persistent contacts with neighboring cells, but fail to fuse. Filming of the myoblast cultures was initiated (t = 0) 48 h after the onset of differentiation via serum starvation. Thus, although similar numbers of cells were exposed to either the Ad-eGFP or Ad-Cre-eGFP viruses, enhanced cell death in the nonfusing culture eliminates a sizable proportion of the cells, resulting in a sparse appearance. (Scale bars: 100 μm in A and 25 μm in C and G.)

Time-lapse imaging of live satellite cell cultures was used to monitor their dynamic behavior following adenovirus infection and serum starvation (Fig. 4 E–H and Movies S1 and S2). As can be readily ascertained from this analysis, N-WASp–depleted cultures share many features with the Ad-eGFP–infected controls, but fail to generate myotubes. Thus, the N-WASp–depleted cells are highly motile, moving at an average speed of 1.67 ± 0.23 μm/min, somewhat faster than the controls (1.14 ± 0.35 μm/min, n = 14). Furthermore, the N-WASp–depleted cells undergo a variety of cell-shape changes, and are clearly capable of prolonged cell–cell contact (up to 45 min). However, these associations consistently fail to result in fusion and myotube formation. Taken together, the satellite-cell culture data strongly imply that the failure of N-WASp–depleted cells to form multinucleated myotubes results from a specific arrest in cell fusion, whereas all other aspects of myogenesis are unaffected.

N-WASp Is Required in both Fusing Partners.

A recurrent issue in studies of both Drosophila and mammalian myoblast fusion, with broad mechanistic implications, is whether fusion-related elements function in one or both fusing myogenic cells (19–23). We therefore used the N-WASp^fl/fl–derived satellite-cell cultures to assess the requirement for N-WASp in this context. Control (Ad-eGFP–infected) or N-WASp–depleted (Ad-Cre-eGFP–infected) cells derived from N-WASp^fl/fl muscles were separately mixed, at 1:1 ratios, with N-WASp^fl/fl–derived cells infected with an adenovirus-RFP vector (Ad-RFP). Fusion capacity was determined by monitoring the appearance of myotubes containing both GFP and RFP. A significant portion (∼35%) of the myotubes generated from mixtures of Ad-eGFP and Ad-RFP cells displayed the combined GFP-RFP signal (Fig. 5 A and B–B′′), demonstrating efficient fusion between the two myoblast populations. In contrast, when Ad-RFP cells were mixed with N-WASp–depleted (Ad-Cre-eGFP–infected) myoblasts, myotubes with a mixed GFP–RFP content constituted only 5% of those formed (Fig. 5 A and C–C′′). These observations imply that fusion between pairs of myoblasts requires functional N-WASp in both partners.

Fig. 5.

N-WASp is required in both fusing cells during myoblast–myoblast and myotube–myoblast fusion. (A–C′′) Differentiating satellite cell cultures derived from N-WASp^fl/fl mice, infected with Ad-RFP, and mixed with similar cells infected with either Ad-eGFP (B–B′′), leaving N-WASp intact in all cells, or with Ad-Cre-eGFP (C–C′′), leading to disruption of N-WASp only in GFP-expressing cells. GFP (green) and RFP (red) serve as indicators of viral infection and of cell content mixing following fusion. Note that although eGFP and RFP are cytoplasmic proteins, they will, on occasion, stain nuclei as well. Arrowheads in B′′ point to examples of “mixed” fibers, which are rare in N-WASp–depleted cells. (A) Quantification of the percentage of multinucleated fibers displaying a combined RFP and GFP signal, as a result of fusion between the RFP and GFP myoblast populations. ∼300 fibers were examined. In all experiments, the fusion index was assayed following 48 h of coculturing, as described in Materials and Methods. (D–F′′) N-WASp^fl/fl–derived satellite cells infected with either Ad-eGFP (E–E′′), or with Ad-Cre-eGFP (F–F′′), mixed with RFP-expressing myotubes, generated earlier by fusion of Ad-RFP–infected N-WASp^fl/fl satellite cells. Nuclei visualized with DAPI (blue), in addition to the viral GFP and RFP markers. (D) Quantification of the percentage of multinucleated fibers displaying a combined RFP and GFP signal, as a result of fusion between RFP-expressing myotubes and GFP-expressing myoblasts. Depletion of N-WASp from myoblasts compromises their ability to fuse with myotubes expressing N-WASp. ∼600 fibers were examined. (G-I′′) Mixing of Ad-RFP–infected N-WASp^fl/fl satellite cells with myotubes originating from N-WASp^fl/fl–derived satellite cells, and infected after myotube formation with either Ad-eGFP (H–H′′) or with Ad-Cre-eGFP (I–I′′). Markers are as in B–C′′. (G) Quantification of the percentage of multinucleated fibers displaying a combined RFP and GFP signal, as a result of fusion between GFP-expressing myotubes and RFP-expressing myoblasts. Depletion of N-WASp from myotubes compromises their ability to fuse with myoblasts expressing N-WASp. ∼450 fibers were examined. (Scale bars: 50 μm in A, D, and G.)

The related question of the requirement for N-WASp during fusion between individual myoblasts and preformed fibers was first addressed via similar assays, in which GFP-expressing myoblasts were mixed with RFP-expressing myotubes. Again, the source material for all myogenic cells was N-WASp^fl/fl–derived satellite-cell cultures infected with different adenoviral vectors. Although ∼25% of RFP-expressing myotubes (generated from Ad-RFP–infected cells) incorporate control (Ad-eGFP–infected) myoblasts, only 5% of such myotubes fuse with Ad-Cre-eGFP–infected cells lacking N-WASp function (Fig. 5 D–F′′). In a complimentary set of experiments, multinucleated myotubes derived from N-WASp^fl/fl satellite cells were infected with either of the GFP-bearing viruses and mixed with Ad-RFP–infected myoblasts. The myoblasts fused efficiently with the control, Ad-eGFP–infected myotubes (∼35% of fibers expressed both RFP and GFP), but fusion with Ad-Cre-eGFP–infected (i.e., N-WASp–depleted) myotubes, was minimal (Fig. 5 G–I′′).

We thus repeatedly observed that fusion is poorly executed in a variety of mixed culture assays, when N-WASp is present only in one of a pair of fusing cells. Hence, muscle cell fusion in the mouse requires a functional contribution from N-WASp in all fusing cells, and this holds true both for myoblast–myoblast and myotube–myoblast fusion events.

Discussion

Our study demonstrates a myogenic role for the nucleation-promoting factor N-WASp as an essential mediator of myoblast fusion in mice. Involvement in myoblast fusion constitutes the primary, if not exclusive, requirement for N-WASp during myogenic differentiation, as many other aspects of the myogenic program appear to proceed normally following disruption of N-WASp, both during embryogenesis and in primary satellite cell cultures. These include timely expression of differentiation markers, morphological changes associated with myoblast maturation, and cell motility.

Our findings extend previous studies that identified an involvement of actin-related signaling elements such as Rac, CDC42, and DOCK1 in murine myoblast fusion (23, 24), to the level of the cellular machinery directly engaged in nucleation of branched microfilament arrays. Noteworthy in this context is the observed requirement for Nap1, a regulator of the WAVE NPF, during fusion of cultured murine myogenic cell lines (25). An essential involvement of WASp/WAVE-family elements in myoblast fusion has been previously established for the different phases of Drosophila myogenesis (21, 26–32).

A variety of fusion-related roles for Drosophila WASp have been proposed, including involvement in establishment of an adhesive structure between myoblasts (30), expansion of nascent fusion pores (21, 26), and contribution to the formation of fusion-associated F-actin “foci” (27, 32). At present, our analysis does not allow for assignment of such specific cellular roles to N-WASp as a mediator of mammalian myoblast fusion. Importantly, a discrete actin-based structure associated with the fusion process in mammalian muscles is still missing. Indications that such structures may exist come from myogenic cell-culture studies (25, 33), but have not been reported in vivo or in primary culture studies, and we were not able to identify them in the course of the current study.

Our demonstration that N-WASp function is required in both partners of a fusing cell pair contrasts with our previous findings in Drosophila. Using cell-type-specific expression methods, we have shown that expression of Drosophila WASp and its associated element WIP in either partner of a fusing cell pair is sufficient for fusion to occur (21, 28). This mechanistic distinction may be reflective of the inherent difference between the invertebrate and mammalian systems, in which the former is based on fusion between unequal “founder cells” and “fusion competent” myoblasts, whereas the latter employs a uniform myoblast pool (34). Thus, although contribution of the WASp-based cellular machinery dedicated to branched microfilament nucleation constitutes a universal feature of the muscle cell-fusion process, aspects of the underlying mechanism may differ according to the specific developmental context.

Materials and Methods

Mouse Genetics.

The following scheme was used to generate N-WASp^cKO mice. Mice bearing constructs expressing Cre recombinase in myogenic tissue were crossed with heterozygotes for an N-WASp null allele (8). Progeny bearing both the Cre driver construct and the null N-WASp allele were then crossed with N-WASp^fl homozygotes (9). Genotypes were determined by PCR of tail tissue. All mice were maintained inside a barrier facility, and experiments were performed in accordance with the Weizmann Institute of Science regulations for animal care and handling.

Satellite Cell Cultures.

Primary myoblast cultures were established as previously described (16), following isolation of satellite cells from EDL muscles of N-WASp^fl or ICR (4–6 wk old) mice. Cells were grown at 37 °C, 5% (vol/vol) CO₂ in proliferation medium (BIOAMF-2, Biological Industries), which was replaced on a daily basis. For fusion assays, 2 × 10⁴ cells were seeded and grown for 16 h on a Lab-Tek eight-well chamber slide (Nunc, pretreated with Matrigel (BD Biosciences) for 30 min at 37 °C), exposed to viral infection for 24 h, and induced to differentiate by serum starvation in differentiation medium (16). Viral infection was achieved by overlaying cells with 300 μL of medium containing 6.5 × 10⁷ viral particles per μL of Ad5CMV-eGFP, Ad5CMVCre-eGFP (both from Gene Transfer Vector Core, University of Iowa, MOI = 40), or Adeno-RFP (Capital Biosciences). For fusion index-determination assays, cells were transferred to differentiation medium and cultured for 48 h. For assays monitoring fusion between cocultured cell populations, infected N-WASp^fl myoblasts were first cultured separately in differentiation medium, at low confluency, for 24 h. A total of 2 × 10⁴ cells from each infected culture were then mixed, seeded in an 8-well chamber slide, and set to fuse for a further 48 h. Myoblast–myotube coculturing involved the mixing, for 24 h, of a similarly prepared myoblast population with N-WASp^fl–derived myotubes that had been previously infected with the relevant viral vector.

Live Imaging.

Satellite cell cultures were prepared and treated as for fusion assays, except that seeding was onto Matrigel-treated 35-mm glass-bottom dishes (MatTek). Time-lapse movies were obtained using an Applied Precision DeltaVision imaging system. Z stacks of five images were acquired at regular intervals and compiled to form maximum intensity point projection images. For velocity measurements, the temporal location of cells was tracked manually using a Matlab GUI. Cell velocity was determined from the average distance made by the cell in 1 min.

Immunohistochemistry.

Immunolabeling protocols of mouse tissue paraffin sections and of satellite cell cultures were as described (35, 36). Primary antibodies used included mouse anti-MHC (MH-20, Developmental Studies Hybridoma Bank, 1:5), mouse anti-Pax7 (Developmental Studies Hybridoma Bank, 1:5–1:10), rabbit anti-MyoD (sc-304, Santa Cruz Biotechnology, 1:100), anti-cleaved Caspase 3 (Cell Signaling, 1:50), and mouse anti-β-catenin (BD Biosciences, 1:50). Cy2-, Cy3-, or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch) were used at a dilution of 1:200–1:500. Images were acquired using a Zeiss LSM710 confocal microscopy system. Immuno-detection of MHC on embryo whole mounts was performed as described (37) using alkaline phosphatase-conjugated anti-MHC (clone MY32, Sigma-Aldrich, 1:400) and NBT/BCIP substrate (Roche Applied Science).

In Situ Hybridization.

Whole-mount in situ hybridization was performed using digoxigenin-labeled antisense riboprobes synthesized from cDNA as described (38). Images were obtained using a Leica MZ16FA stereomicroscope attached to a digital camera (DC300F, Leica Microsystems).

Western Blotting.

Protein extracts were obtained following collection and sonication of cultured satellite cells in RIPA buffer (10 mM Tris/150 mM NaCl/5 mM EDTA/1% Triton X-100/0.1% SDS/1% Sodium deoxycholate, and freshly added protease inhibitors). Western blotting protocols were as described (39). Primary antibodies used included mouse anti-MyoD (sc-32758), mouse anti-Myogenin (sc-12732), rabbit anti N-WASp (sc-H-100), and rabbit anti-Emerin (sc-15378), all from Santa Cruz Biotechnology Monoclonal, as well as mouse anti-MHC n2.261 (Developmental Studies Hybridoma Bank). Secondary antibodies used were either anti-rabbit HRP or anti-mouse HRP (Jackson ImmunoResearch). Chemiluminescent detection was performed using EZ-ECL (Biological Industries), according to the manufacturer’s directions.