Defects In Myoblast Fusion Research Articles

Inter-organ steroid hormone signaling promotes myoblast fusion via direct transcriptional regulation of a single key effector gene

Steroid hormones regulate tissue development and physiology by modulating the transcription of a broad spectrum of genes. In insects, the principal steroid hormones, ecdysteroids, trigger the expression of thousands of genes through a cascade of transcription factors (TFs) to coordinate developmental transitions such as larval molting and metamorphosis. However, whether ecdysteroid signaling can bypass transcriptional hierarchies to exert its function in individual developmental processes is unclear. Here, we report that a singlenon-TF effector gene mediates the transcriptional output of ecdysteroid signaling in Drosophila myoblast fusion, a critical step in muscle development and differentiation. Specifically, we show that the 20-hydroxyecdysone (commonly referred to as "ecdysone") secreted from an extraembryonic tissue, amnioserosa, acts on embryonic muscle cells to directly activate the expression of antisocial (ants), which encodes an essential scaffold protein enriched at the fusogenic synapse. Not only is ants transcription directly regulated by the heterodimeric ecdysone receptor complex composed of ecdysone receptor (EcR) and ultraspiracle (USP) via ecdysone-response elements but also more strikingly, expression of ants alone is sufficient to rescue the myoblast fusion defect in ecdysone signaling-deficient mutants. We further show that EcR/USP and a muscle-specific TF Twist synergistically activate ants expression invitro and invivo. Taken together, our study provides the first example of a steroid hormone directly activating the expression of a single key non-TF effector gene to regulate a developmental process via inter-organ signaling and provides a new paradigm for understanding steroid hormone signaling in other developmental and physiological processes.

The Abl-interactor Abi suppresses the function of the BRAG2 GEF family member Schizo.

Guanine nucleotide exchange factors (GEF) of the BRAG subfamily activate small Arf GTPases, which are pivotal regulators of intracellular membrane traffic and actin dynamics. Consequently, BRAG proteins have been implicated to regulate the surface levels of adhesive and signaling receptors. However, not much is known about the mechanism leading to the regulation of these surface proteins. In this study, we found that the Drosophila BRAG GEF Schizo interacts physically with the Abl-interactor (Abi). schizo mutants display severe defects in myoblast fusion during syncytial muscle formation and show increased amounts of the cell adhesion protein N-cadherin. We demonstrate that the schizo myoblast fusion phenotype can be rescued by the expression of the Schizo GEF (Sec7) and membrane-binding (pleckstrin homology) domain. Furthermore, the expression of the Sec7-PH domain in a wild-type background decreases the amounts of N-cadherin and impairs myoblast fusion. These findings support the notion that the Sec7-PH domain serves as a constitutive-active form of Schizo. Using a yeast-two hybrid assay, we show that the SH3 domain of Abi interacts with the N-terminal region of Schizo. This region is also able to bind to the cytodomain of the cell adhesion molecule N-cadherin. To shed light on the function of Schizo and Abi in N-cadherin removal, we employed epistasis experiments in different developmental contexts of Drosophila. These studies point towards a new model for the regulation of Schizo. We propose that the binding of Abi to the N-terminal part of Schizo antagonizes Schizo function to inhibit N-cadherin removal.

Biasing the conformation of ELMO2 reveals that myoblast fusion can be exploited to improve muscle regeneration

Myoblast fusion is fundamental for the development of multinucleated myofibers. Evolutionarily conserved proteins required for myoblast fusion include RAC1 and its activator DOCK1. In the current study we analyzed the contribution of the DOCK1-interacting ELMO scaffold proteins to myoblast fusion. When Elmo1−/− mice underwent muscle-specific Elmo2 genetic ablation, they exhibited severe myoblast fusion defects. A mutation in the Elmo2 gene that reduced signaling resulted in a decrease in myoblast fusion. Conversely, a mutation in Elmo2 coding for a protein with an open conformation increased myoblast fusion during development and in muscle regeneration. Finally, we showed that the dystrophic features of the Dysferlin-null mice, a model of limb-girdle muscular dystrophy type 2B, were reversed when expressing ELMO2 in an open conformation. These data provide direct evidence that the myoblast fusion process could be exploited for regenerative purposes and improve the outcome of muscle diseases.

PIP3 depletion rescues myoblast fusion defects in human rhabdomyosarcoma cells.

Myoblast fusion is required for myotube formation during myogenesis, and defects in myoblast differentiation and fusion have been implicated in a number of diseases, including human rhabdomyosarcoma. Although transcriptional regulation of the myogenic program has been studied extensively, the mechanisms controlling myoblast fusion remain largely unknown. This study identified and characterized the dynamics of a distinct class of blebs, termed bubbling blebs, which are smaller than those that participate in migration. The formation of these bubbling blebs occurred during differentiation and decreased alongside a decline in phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) at the plasma membrane before myoblast fusion. In a human rhabdomyosarcoma-derived (RD) cell line that exhibits strong blebbing dynamics and myoblast fusion defects, PIP3 was constitutively abundant on the membrane during myogenesis. Targeting phosphatase and tensin homolog (PTEN) to the plasma membrane reduced PIP3 levels, inhibited bubbling blebs and rescued myoblast fusion defects in RD cells. These findings highlight the differential distribution and crucial role of PIP3 during myoblast fusion and reveal a novel mechanism underlying myogenesis defects in human rhabdomyosarcoma.

MiR-7 Restores Phenotypes in Myotonic Dystrophy Muscle Cells by Repressing Hyperactivated Autophagy

Unstable CTG expansions in the 3’ UTR of the DMPK gene are responsible for myotonic dystrophy type 1 (DM1) condition. Muscle dysfunction is one of the main contributors to DM1 mortality and morbidity. Pathways by which mutant DMPK trigger muscle defects, however, are not fully understood. We previously reported that miR-7 was downregulated in a DM1 Drosophila model and in biopsies from patients. Here, using DM1 and normal muscle cells, we investigated whether miR-7 contributes to the muscle phenotype by studying the consequences of replenishing or blocking miR-7, respectively. Restoration of miR-7 with agomiR-7 was sufficient to rescue DM1 myoblast fusion defects and myotube growth. Conversely, oligonucleotide-mediated blocking of miR-7 in normal myoblasts led to fusion and myotube growth defects. miR-7 was found to regulate autophagy and the ubiquitin-proteasome system in human muscle cells. Thus, low levels of miR-7 promoted both processes, and high levels of miR-7 repressed them. Furthermore, we uncovered that the mechanism by which miR-7 improves atrophy-related phenotypes is independent of MBNL1, thus suggesting that miR-7 acts downstream or in parallel to MBNL1. Collectively, these results highlight an unknown function for miR-7 in muscle dysfunction through autophagy- and atrophy-related pathways and support that restoration of miR-7 levels is a candidate therapeutic target for counteracting muscle dysfunction in DM1.

WNT/β-catenin signaling plays a crucial role in myoblast fusion through regulation of nephrin expression during development.

Skeletal muscle development is controlled by a series of multiple orchestrated regulatory pathways. WNT/β-catenin is one of the most important pathways for myogenesis; however, it remains unclear how this signaling pathway regulates myogenesis in a temporal- and spatial-specific manner. Here, we show that WNT/β-catenin signaling is crucial for myoblast fusion through regulation of the nephrin (Nphs1) gene in the Myog-Cre-expressing myoblast population. Mice deficient for the β-catenin gene in Myog-Cre-expressing myoblasts (Ctnnb1F/F;Myog-Cre mice) displayed myoblast fusion defects, but not migration or cell proliferation defects. The promoter region of Nphs1 contains the conserved β-catenin-binding element, and Nphs1 expression was induced by the activation of WNT/β-catenin signaling. The induction of Nphs1 in cultured myoblasts from Ctnnb1F/F;Myog-Cre mice restored the myoblast fusion defect, indicating that nephrin is functionally relevant in WNT/β-catenin-dependent myoblast fusion. Taken together, our results indicate that WNT/β-catenin signaling is crucial for myoblast fusion through the regulation of the Nphs1 gene.

Novel nesprin-1 mutations associated with dilated cardiomyopathy cause nuclear envelope disruption and defects in myogenesis.

Nesprins-1 and -2 are highly expressed in skeletal and cardiac muscle and together with SUN (Sad1p/UNC84)-domain containing proteins and lamin A/C form the LInker of Nucleoskeleton-and-Cytoskeleton (LINC) bridging complex at the nuclear envelope (NE). Mutations in nesprin-1/2 have previously been found in patients with autosomal dominant Emery–Dreifuss muscular dystrophy (EDMD) as well as dilated cardiomyopathy (DCM). In this study, three novel rare variants (R8272Q, S8381C and N8406K) in the C-terminus of the SYNE1 gene (nesprin-1) were identified in seven DCM patients by mutation screening. Expression of these mutants caused nuclear morphology defects and reduced lamin A/C and SUN2 staining at the NE. GST pull-down indicated that nesprin-1/lamin/SUN interactions were disrupted. Nesprin-1 mutations were also associated with augmented activation of the ERK pathway in vitro and in hearts in vivo. During C2C12 muscle cell differentiation, nesprin-1 levels are increased concomitantly with kinesin light chain (KLC-1/2) and immunoprecipitation and GST pull-down showed that these proteins interacted via a recently identified LEWD domain in the C-terminus of nesprin-1. Expression of nesprin-1 mutants in C2C12 cells caused defects in myoblast differentiation and fusion associated with dysregulation of myogenic transcription factors and disruption of the nesprin-1 and KLC-1/2 interaction at the outer nuclear membrane. Expression of nesprin-1α2 WT and mutants in zebrafish embryos caused heart developmental defects that varied in severity. These findings support a role for nesprin-1 in myogenesis and muscle disease, and uncover a novel mechanism whereby disruption of the LINC complex may contribute to the pathogenesis of DCM.

Drosophila Kette coordinates myoblast junction dissolution and the ratio of Scar-to-WASp during myoblast fusion.

ABSTRACTThe fusion of founder cells and fusion-competent myoblasts (FCMs) is crucial for muscle formation in Drosophila. Characteristic events of myoblast fusion include the recognition and adhesion of myoblasts, and the formation of branched F-actin by the Arp2/3 complex at the site of cell–cell contact. At the ultrastructural level, these events are reflected by the appearance of finger-like protrusions and electron-dense plaques that appear prior to fusion. Severe defects in myoblast fusion are caused by the loss of Kette (a homolog of Nap1 and Hem-2, also known as NCKAP1 and NCKAP1L, respectively), a member of the regulatory complex formed by Scar or WAVE proteins (represented by the single protein, Scar, in flies). kette mutants form finger-like protrusions, but the electron-dense plaques are extended. Here, we show that the electron-dense plaques in wild-type and kette mutant myoblasts resemble other electron-dense structures that are known to function as cellular junctions. Furthermore, analysis of double mutants and attempts to rescue the kette mutant phenotype with N-cadherin, wasp and genes of members of the regulatory Scar complex revealed that Kette has two functions during myoblast fusion. First, Kette controls the dissolution of electron-dense plaques. Second, Kette controls the ratio of the Arp2/3 activators Scar and WASp in FCMs.

FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1).

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.

WHAMY is a novel actin polymerase promoting myoblast fusion, macrophage cell motility and sensory organ development in Drosophila.

Wiskott-Aldrich syndrome proteins (WASPs) are nucleation-promoting factors (NPF) that differentially control the Arp2/3 complex. In Drosophila, three different family members, SCAR (also known as WAVE), WASP and WASH (also known as CG13176), have been analyzed so far. Here, we characterized WHAMY, the fourth Drosophila WASP family member. whamy originated from a wasp gene duplication and underwent a sub-neofunctionalization. Unlike WASP, we found that WHAMY specifically interacted with activated Rac1 through its two CRIB domains, which were sufficient for targeting WHAMY to lamellipodial and filopodial tips. Biochemical analyses showed that WHAMY promoted exceptionally fast actin filament elongation, although it did not activate the Arp2/3 complex. Loss- and gain-of-function studies revealed an important function of WHAMY in membrane protrusions and cell migration in macrophages. Genetic data further implied synergistic functions between WHAMY and WASP during morphogenesis. Double mutants were late-embryonic lethal and showed severe defects in myoblast fusion. Trans-heterozygous mutant animals showed strongly increased defects in sensory cell fate specification. Thus, WHAMY is a novel actin polymerase with an initial partitioning of ancestral WASP functions in development and subsequent acquisition of a new function in cell motility during evolution.

Phospholipase D1 facilitates second-phase myoblast fusion and skeletal muscle regeneration.

Myoblast differentiation and fusion is a well-orchestrated multistep process that is essential for skeletal muscle development and regeneration. Phospholipase D1 (PLD1) has been implicated in the initiation of myoblast differentiation in vitro. However, whether PLD1 plays additional roles in myoblast fusion and exerts a function in myogenesis in vivo remains unknown. Here we show that PLD1 expression is up-regulated in myogenic cells during muscle regeneration after cardiotoxin injury and that genetic ablation of PLD1 results in delayed myofiber regeneration. Myoblasts derived from PLD1-null mice or treated with PLD1-specific inhibitor are unable to form mature myotubes, indicating defects in second-phase myoblast fusion. Concomitantly, the PLD1 product phosphatidic acid is transiently detected on the plasma membrane of differentiating myocytes, and its production is inhibited by PLD1 knockdown. Exogenous lysophosphatidylcholine, a key membrane lipid for fusion pore formation, partially rescues fusion defect resulting from PLD1 inhibition. Thus these studies demonstrate a role for PLD1 in myoblast fusion during myogenesis in which PLD1 facilitates the fusion of mononuclear myocytes with nascent myotubes.

Novel Drosophila model of myotonic dystrophy type 1: phenotypic characterization and genome-wide view of altered gene expression.

Myotonic dystrophy type 1 (DM1) is a multisystemic RNA-dominant disorder characterized by myotonia and muscle degeneration. In DM1 patients, the mutant DMPK transcripts containing expanded CUG repeats form nuclear foci and sequester the Muscleblind-like 1 splicing factor, resulting in mis-splicing of its targets. However, several pathological defects observed in DM1 and their link with disease progression remain poorly understood. In an attempt to fill this gap, we generated inducible transgenic Drosophila lines with increasing number of CTG repeats. Targeting the expression of these repeats to the larval muscles recapitulated in a repeat-size-dependent manner the major DM1 symptoms such as muscle hypercontraction, splitting of muscle fibers, reduced fiber size or myoblast fusion defects. Comparative transcriptional profiling performed on the generated DM1 lines and on the muscleblind (mbl)-RNAi line revealed that nuclear accumulation of toxic CUG repeats can affect gene expression independently of splicing or Mbl sequestration. Also, in mblRNAi contexts, the largest portion of deregulated genes corresponded to single-transcript genes, revealing an unexpected impact of the indirect influence of mbl on gene expression. Among the single-transcript Mbl targets is Muscle protein 20 involved in myoblast fusion and causing the reduced number of nuclei in muscles of mblRNAi larvae. Finally, by combining in silico prediction of Mbl targets with mblRNAi microarray data, we found the calcium pump dSERCA as a Mbl splice target and show that the membrane dSERCA isoform is sufficient to rescue a DM1-induced hypercontraction phenotype in a Drosophila model.

Asymmetric Mbc, active Rac1 and F-actin foci in the fusion-competent myoblasts during myoblast fusion in Drosophila

There was an error published in Development138, 1551-1562.We have been unable to reproduce the data in Figure 1H, reporting that the myoblast fusion defect in mbc mutant embryos is rescued by expression of constitutively activated Rac1. Although we are unable to confirm the source of the error, all stocks have been re-confirmed and all other results in the original publication independently confirmed by two of the authors.Revised data pertaining to Figure 1 of the original paper are provided in Fig. 1 below. All are lateral views of stage 16 embryos stained as in the original publication.Results shown in Fig. 1A,B,E are as originally published (original Figure 1 panels A, B and I, respectively). Of note, the rescued embryo in Fig. 1B exhibits dorsal closure defects characteristic of mbc mutants, confirming that rescue is only occurring in the musculature as expected. It is apparent in Fig. 1C,D, however, that constitutively active Rac1 does not rescue fusion in an mbc mutant embryo (as originally shown in Figure 1H of the original publication). We do note that FCMs expressing activated Rac1 exhibit an increased tendency to cluster around founder cells, suggesting that migration is increased in these FCMs. These cells often also adopt a more elongated appearance with more pronounced filopodia (Fig. 1D). Additionally, the data in Fig. 1F confirm that pan-mesodermal expression of activated Rac does not rescue the myoblast fusion defect and, moreover, causes severe perturbations owing to higher and earlier expression of activated Rac1.To confirm that the bipartite Mbc-Elmo guanine nucleotide exchange factor does function in the Rac1 pathway, we examined the consequences of their overexpression in the musculature (Fig. 2, below).As anticipated, this overexpression caused defects in myoblast fusion reminiscent of those described for overexpression of activated Rac1 itself (Luo et al., 1994). Notably, this myoblast fusion defect is suppressed by loss of one copy of Rac1 and Rac2 (Fig. 2B), consistent with their activation by Mbc and Elmo.In summary, the primary conclusions of the original publication remain intact. However, our current results do not support the additional conclusion that the only role of mbc is to activate Rac1. Although Mbc and Elmo appear to be capable of activating this small GTPase, the inability of constitutively activated Rac1 to rescue fusion in mbc mutant embryos mutants suggest that Mbc is required for more than the simple activation of Rac1, perhaps controlling its localization or activating another small GTPase.The authors apologize to readers and anyone who experimentally followed up on these results for this mistake.

T.P.35 FHL1 is beneficial in reducing muscle wasting in dystrophic FRG1 mice

Abstract Four and a half LIM 1 (FHL1) is a protein highly expressed in skeletal muscle, and we have previously reported that FHL1 can promote myoblast fusion in vitro and skeletal muscle hypertrophy in vivo by activating the calcineurin/NFATc1 pathway. Therefore FHL1 has potential as a therapeutic target for muscle diseases that display a myoblast fusion defect and/or progressive muscle wasting. The focus of this study was to determine if FHL1 can reduce muscle wasting in the dystrophic FRG1 mouse. FRG1 is one of several candidate genes for Facioscapulohumeral muscular dystrophy and FRG1 transgenic mice show resemblance to this disease clinically and pathologically. The dystrophy in FRG1 mice is characterised by progressive muscle wasting and is accompanied by spinal kyphosis caused by weakness of the trapezius muscle. We generated myoblast cell lines overexpressing FRG1 that exhibited a myoblast fusion defect, and as FHL1 enhances myoblast fusion we generated FRG1 mice over-expressing FHL1 by crossing our FHL1 skeletal muscle-specific transgenic mice with the dystrophic FRG1 mouse model. X-ray images of 6-week old mice revealed that FHL1 reduced the kyphosis of FRG1 mice. Analysis of muscle weights in mice revealed an increased muscle mass in FRG1/FHL1 mice relative to FRG1 mice, indicating FHL1 reduces muscle wasting. Histological analysis of muscle further revealed an increase in the average myofibre cross-sectional area and increase in the proportion of larger myofibres in FRG1/FHL1 mice compared to FRG1 mice. This beneficial effect of FHL1 was present in 6-week old FRG1/FHL1 mice and sustained into adulthood (12-weeks). FRG1/FHL1 mice also exhibited a reduction in fibrosis and fat accumulation in muscle further supporting the sustained benefit of FHL1 in reducing the dystrophic pathology of FRG1 mice. This study confirms that FHL1 is sufficient to reduce the dystrophy in FRG1 mice and is a potential future therapeutic target in treating muscle diseases.

The Arf-GEF Schizo/Loner regulates N-cadherin to induce fusion competence of Drosophila myoblasts

Myoblast fusion is a key process in multinucleated muscle formation. Prior to fusion, myoblasts recognize and adhere to each other with the aid of cell-adhesion proteins integrated into the membrane. Their intracellular domains participate in signal transduction by binding to cytoplasmic proteins. Here we identified the calcium-dependent cell-adhesion protein N-cadherin as the binding partner of the guanine-nucleotide exchange factor Schizo/Loner in Drosophila melanogaster. N-cadherin was expressed in founder cells and fusion-competent myoblasts of Drosophila during the first fusion phase. Our genetic analyses demonstrated that the myoblast fusion defect of schizo/loner mutants is rescued in part by the loss-of-function mutation of N-cadherin, which suggests that Schizo/Loner is a negative regulator of N-cadherin. Based on our findings, we propose a model where N-cadherin must be removed from the myoblast membrane to induce a protein-free zone at the cell–cell contact point to permit fusion.

A TGFβ-Smad4-Fgf6 signaling cascade controls myogenic differentiation and myoblast fusion during tongue development

The tongue is a muscular organ and plays a crucial role in speech, deglutition and taste. Despite the important physiological functions of the tongue, little is known about the regulatory mechanisms of tongue muscle development. TGFβ family members play important roles in regulating myogenesis, but the functional significance of Smad-dependent TGFβ signaling in regulating tongue skeletal muscle development remains unclear. In this study, we have investigated Smad4-mediated TGFβ signaling in the development of occipital somite-derived myogenic progenitors during tongue morphogenesis through tissue-specific inactivation of Smad4 (using Myf5-Cre;Smad4(flox/flox) mice). During the initiation of tongue development, cranial neural crest (CNC) cells occupy the tongue buds before myogenic progenitors migrate into the tongue primordium, suggesting that CNC cells play an instructive role in guiding tongue muscle development. Moreover, ablation of Smad4 results in defects in myogenic terminal differentiation and myoblast fusion. Despite compromised muscle differentiation, tendon formation appears unaffected in the tongue of Myf5-Cre;Smad4(flox/flox) mice, suggesting that the differentiation and maintenance of CNC-derived tendon cells are independent of Smad4-mediated signaling in myogenic cells in the tongue. Furthermore, loss of Smad4 results in a significant reduction in expression of several members of the FGF family, including Fgf6 and Fgfr4. Exogenous Fgf6 partially rescues the tongue myoblast fusion defect of Myf5-Cre;Smad4(flox/flox) mice. Taken together, our study demonstrates that a TGFβ-Smad4-Fgf6 signaling cascade plays a crucial role in myogenic cell fate determination and lineage progression during tongue myogenesis.

Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts

The formation of the larval body wall musculature of Drosophila depends on the asymmetric fusion of two myoblast types, founder cells (FCs) and fusion-competent myoblasts (FCMs). Recent studies have established an essential function of Arp2/3-based actin polymerization during myoblast fusion, formation of a dense actin focus at the site of fusion in FCMs, and a thin sheath of actin in FCs and/or growing muscles. The formation of these actin structures depends on recognition and adhesion of myoblasts that is mediated by cell surface receptors of the immunoglobulin superfamily. However, the connection of the cell surface receptors with Arp2/3-based actin polymerization is poorly understood. To date only the SH2-SH3 adaptor protein Crk has been suggested to link cell adhesion with Arp2/3-based actin polymerization in FCMs. Here, we propose that the SH2-SH3 adaptor protein Dock, like Crk, links cell adhesion with actin polymerization. We show that Dock is expressed in FCs and FCMs and colocalizes with the cell adhesion proteins Sns and Duf at cell-cell contact points. Biochemical data in this study indicate that different domains of Dock are involved in binding the cell adhesion molecules Duf, Rst, Sns and Hbs. We emphasize the importance of these interactions by quantifying the enhanced myoblast fusion defects in duf dock, sns dock and hbs dock double mutants. Additionally, we show that Dock interacts biochemically and genetically with Drosophila Scar, Vrp1 and WASp. Based on these data, we propose that Dock links cell adhesion in FCs and FCMs with either Scar- or Vrp1-WASp-dependent Arp2/3 activation.

Combinatorial Binding Leads to Diverse Regulatory Responses: Lmd Is a Tissue-Specific Modulator of Mef2 Activity

Understanding how complex patterns of temporal and spatial expression are regulated is central to deciphering genetic programs that drive development. Gene expression is initiated through the action of transcription factors and their cofactors converging on enhancer elements leading to a defined activity. Specific constellations of combinatorial occupancy are therefore often conceptualized as rigid binding codes that give rise to a common output of spatio-temporal expression. Here, we assessed this assumption using the regulatory input of two essential transcription factors within the Drosophila myogenic network. Mutations in either Myocyte enhancing factor 2 (Mef2) or the zinc-finger transcription factor lame duck (lmd) lead to very similar defects in myoblast fusion, yet the underlying molecular mechanism for this shared phenotype is not understood. Using a combination of ChIP-on-chip analysis and expression profiling of loss-of-function mutants, we obtained a global view of the regulatory input of both factors during development. The majority of Lmd-bound enhancers are co-bound by Mef2, representing a subset of Mef2's transcriptional input during these stages of development. Systematic analyses of the regulatory contribution of both factors demonstrate diverse regulatory roles, despite their co-occupancy of shared enhancer elements. These results indicate that Lmd is a tissue-specific modulator of Mef2 activity, acting as both a transcriptional activator and repressor, which has important implications for myogenesis. More generally, this study demonstrates considerable flexibility in the regulatory output of two factors, leading to additive, cooperative, and repressive modes of co-regulation.

Talin 1 and 2 are required for myoblast fusion, sarcomere assembly and the maintenance of myotendinous junctions

Talin 1 and 2 connect integrins to the actin cytoskeleton and regulate the affinity of integrins for ligands. In skeletal muscle, talin 1 regulates the stability of myotendinous junctions (MTJs), but the function of talin 2 in skeletal muscle is not known. Here we show that MTJ integrity is affected in talin 2-deficient mice. Concomitant ablation of talin 1 and 2 leads to defects in myoblast fusion and sarcomere assembly, resembling defects in muscle lacking beta1 integrins. Talin 1/2-deficient myoblasts express functionally active beta1 integrins, suggesting that defects in muscle development are not primarily caused by defects in ligand binding, but rather by disruptions of the interaction of integrins with the cytoskeleton. Consistent with this finding, assembly of integrin adhesion complexes is perturbed in the remaining muscle fibers of talin 1/2-deficient mice. We conclude that talin 1 and 2 are crucial for skeletal muscle development, where they regulate myoblast fusion, sarcomere assembly and the maintenance of MTJs.

Analysis of the cell adhesion molecule sticks-and-stones reveals multiple redundant functional domains, protein-interaction motifs and phosphorylated tyrosines that direct myoblast fusion in Drosophila melanogaster.

The larval body wall muscles of Drosophila melanogaster arise by fusion of founder myoblasts (FMs) and fusion-competent myoblasts (FCMs). Sticks-and-Stones (SNS) is expressed on the surface of all FCMs and mediates adhesion with FMs and developing syncytia. Intracellular components essential for myoblast fusion are then recruited to these adhesive contacts. In the studies herein, a functional analysis of the SNS cytodomain using the GAL4/UAS system identified sequences that direct myoblast fusion, presumably through recruitment of these intracellular components. An extensive series of deletion and site-directed mutations were evaluated for their ability to rescue the myoblast fusion defects of sns mutant embryos. Deletion studies revealed redundant functional domains within SNS. Surprisingly, highly conserved consensus sites for binding post-synaptic density-95/discs large/zonula occludens-1-domain-containing (PDZ) proteins and serines with a high probability of phosphorylation play no significant role in myoblast fusion. Biochemical studies establish that the SNS cytodomain is phosphorylated at multiple tyrosines and their site-directed mutagenesis compromises the ability of the corresponding transgenes to rescue myoblast fusion. Similar mutagenesis revealed a requirement for conserved proline-rich regions. This complexity and redundancy of multiple critical sequences within the SNS cytodomain suggest that it functions through a complex array of interactions that likely includes both phosphotyrosine-binding and SH3-domain-containing proteins.