A change in cis-regulatory logic underlying obligate versus facultative muscle multinucleation in chordates.

Vertebrates and tunicates are sister groups that share a common fusogenic factor, Myomaker (Mymk), that drives myoblast fusion and muscle multinucleation. Yet they are divergent in when and where they express Mymk. In vertebrates, all developing skeletal muscles express Mymk and are obligately multinucleated. In tunicates, Mymk is expressed only in post-metamorphic multinucleated muscles, but is absent from mononucleated larval muscles. In this study, we demonstrate that cis-regulatory sequence differences in the promoter region of Mymk underlie the different spatiotemporal patterns of its transcriptional activation in tunicates and vertebrates. Although in vertebrates myogenic regulatory factors (MRFs) such as MyoD1 alone are required and sufficient for Mymk transcription in all skeletal muscles, we show that transcription of Mymk in post-metamorphic muscles of the tunicate Ciona requires the combinatorial activity of MRF, MyoD and Early B-cell Factor (Ebf). This macroevolutionary difference appears to be encoded in cis, likely due to the presence of a putative Ebf-binding site adjacent to predicted MRF binding sites in the Ciona Mymk promoter. We further discuss how Mymk and myoblast fusion might have been regulated in the last common ancestor of tunicates and vertebrates, for which we propose two models.

Drosophila Myoblast Fusion: Invasion and Resistance for the Ultimate Union.

Cell-cell fusion is indispensable for creating life and building syncytial tissues and organs. Ever since the discovery of cell-cell fusion, how cells join together to form zygotes and multinucleated syncytia has remained a fundamental question in cell and developmental biology. In the past two decades, Drosophila myoblast fusion has been used as a powerful genetic model to unravel mechanisms underlying cell-cell fusion in vivo. Many evolutionarily conserved fusion-promoting factors have been identified and so has a surprising and conserved cellular mechanism. In this review, we revisit key findings in Drosophila myoblast fusion and highlight the critical roles of cellular invasion and resistance in driving cell membrane fusion.

MTM1-mediated production of phosphatidylinositol 5-phosphate fuels the formation of podosome-like protrusions regulating myoblast fusion.

Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major insights into the mechanisms underlying fusion seem to be conserved from insects to mammals and include the formation of podosome-like protrusions (PLPs) that exert a driving force toward the founder cell. However, the machinery that governs this process remains poorly understood. In this study, we demonstrate that MTM1 is the main enzyme responsible for the production of phosphatidylinositol 5-phosphate, which in turn fuels PI5P 4-kinase α to produce a minor and functional pool of phosphatidylinositol 4,5-bisphosphate that concentrates in PLPs containing the scaffolding protein Tks5, Dynamin-2, and the fusogenic protein Myomaker. Collectively, our data reveal a functional crosstalk between a PI-phosphatase and a PI-kinase in the regulation of PLP formation.

The IRE1α/XBP1 signaling axis drives myoblast fusion in adult skeletal muscle.

Skeletal muscle regeneration involves a signaling network that regulates the proliferation, differentiation, and fusion of muscle precursor cells to injured myofibers. IRE1α, one of the arms of the unfolded protein response, regulates cellular proteostasis in response to ER stress. Here, we demonstrate that inducible deletion of IRE1α in satellite cells of mice impairs skeletal muscle regeneration through inhibiting myoblast fusion. Knockdown of IRE1α or its downstream target, X-box protein 1 (XBP1), also inhibits myoblast fusion during myogenesis. Transcriptome analysis revealed that knockdown of IRE1α or XBP1 dysregulates the gene expression of molecules involved in myoblast fusion. The IRE1α-XBP1 axis mediates the gene expression of multiple profusion molecules, including myomaker (Mymk). Spliced XBP1 (sXBP1) transcription factor binds to the promoter of Mymk gene during myogenesis. Overexpression of myomaker in IRE1α-knockdown cultures rescues fusion defects. Inducible deletion of IRE1α in satellite cells also inhibits myoblast fusion and myofiber hypertrophy in response to functional overload. Collectively, our study demonstrates that IRE1α promotes myoblast fusion through sXBP1-mediated up-regulation of the gene expression of multiple profusion molecules, including myomaker.

TMEM8C-mediated fusion is regionalized and regulated by NOTCH signalling during foetal myogenesis.

The location and regulation of fusion events within skeletal muscles during development remain unknown. Using the fusion marker myomaker (Mymk), named TMEM8C in chicken, as a readout of fusion, we identified a co-segregation of TMEM8C-positive cells and MYOG-positive cells in single-cell RNA-sequencing datasets of limbs from chicken embryos. We found that TMEM8C transcripts, MYOG transcripts and the fusion-competent MYOG-positive cells were preferentially regionalized in central regions of foetal muscles. We also identified a similar regionalization for the gene encoding the NOTCH ligand JAG2 along with an absence of NOTCH activity in TMEM8C+ fusion-competent myocytes. NOTCH function in myoblast fusion had not been addressed so far. We analysed the consequences of NOTCH inhibition for TMEM8C expression and myoblast fusion during foetal myogenesis in chicken embryos. NOTCH inhibition increased myoblast fusion and TMEM8C expression and released the transcriptional repressor HEYL from the TMEM8C regulatory regions. These results identify a regionalization of TMEM8C-dependent fusion and a molecular mechanism underlying the fusion-inhibiting effect of NOTCH in foetal myogenesis. The modulation of NOTCH activity in the fusion zone could regulate the flux of fusion events.

Machine learning inference of continuous single-cell state transitions during myoblast differentiation and fusion.

Cells modify their internal organization during continuous state transitions, supporting functions from cell division to differentiation. However, tools to measure dynamic physiological states of individual transitioning cells are lacking. We combined live-cell imaging and machine learning to monitor ERK1/2-inhibited primary murine skeletal muscle precursor cells, that transition rapidly and robustly from proliferating myoblasts to post-mitotic myocytes and then fuse, forming multinucleated myotubes. Our models, trained using motility or actin intensity features from single-cell tracking data, effectively tracked real-time continuous differentiation, revealing that differentiation occurs 7.5-14.5 h post induction, followed by fusion ~3 h later. Co-inhibition of ERK1/2 and p38 led to differentiation without fusion. Our model inferred co-inhibition leads to terminal differentiation, indicating that p38 is specifically required for transitioning from terminal differentiation to fusion. Our model also predicted that co-inhibition leads to changes in actin dynamics. Mass spectrometry supported these in silico predictions and suggested novel fusion and maturation regulators downstream of differentiation. Collectively, this approach can be adapted to various biological processes to uncover novel links between dynamic single-cell states and their functional outcomes.

Phosphatidylserine orchestrates Myomerger membrane insertions to drive myoblast fusion.

Muscle cell fusion is a multistep process where the final step of the reaction drives progression beyond early hemifusion events to complete fusion. This step requires activity of the muscle-specific fusogen Myomerger, a single-pass transmembrane protein containing 84 amino acids with an ectodomain that includes two α-helices. Previous studies have demonstrated that Myomerger acts by destabilizing membranes through generation of elastic stresses in the outer leaflet of the plasma membrane. An obvious question is how such destabilizing activity might be regulated to avoid membrane and cellular damage, and how the two juxtaposed helices cooperate in fusion. Using cellular fusion assays and in vitro liposome assays, we report that the two helices possess unique characteristics, both of which are needed for full activity of the protein. We demonstrate that externalized phosphatidylserine (PS), a lipid previously implicated in myoblast fusion, has a determinant role in the regulation of Myomerger activity. The membrane-proximal, amphipathic Helix-1 is normally disordered and its α-helical structure is induced by PS, making membrane interactions more efficacious. The distal, more hydrophobic Helix-2 is intrinsically ordered, possesses an ability to insert into membranes, and augments the membrane-stressing effects of Helix-1. These data reveal that Myomerger fusogenic activity is an exquisitely orchestrated event involving its two ectodomain helices, which are controlled by membrane lipid composition, providing an explanation as to how its membrane-stressing activity is spatially and temporally regulated during the final step of myoblast fusion.

The serine/threonine kinase Back seat driver prevents cell fusion to maintain cell identity.

Cell fate specification is essential for every major event of embryogenesis, and subsequent cell maturation ensures individual cell types acquire specialized functions. The mechanisms that regulate cell fate specification have been studied exhaustively, and each technological advance in developmental biology ushers in a new era of studies aimed at uncovering the most fundamental processes by which cells acquire unique identities. What is less appreciated is that mechanisms are in place to ensure cell identity is maintained throughout the life of the organism. The body wall musculature in the Drosophila embryo is a well-established model to study cell fate specification, as each hemisegment in the embryo generates and maintains thirty muscles with distinct identities. Once specified, the thirty body wall muscles fuse with mononucleate muscle precursors that lack a specific identity to form multinucleate striated muscles. Multinucleate body wall muscles do not fuse with each other, which maintains a diversification of muscle cell identities. Here we show the serine/threonine kinase Back seat driver (Bsd) prevents inappropriate muscle fusion to maintain cell identity. Thus, the regulation of cell fusion is one mechanism that maintains cell identity.

Netrin-4 synthesized in satellite cell-derived myoblasts stimulates autonomous fusion.

Satellite cells are indispensable for skeletal muscle regeneration and hypertrophy by forming nascent myofibers (myotubes). They synthesize multi-potent modulator netrins (secreted subtypes: netrin-1, -3, and -4), originally found as classical neural axon guidance molecules. While netrin-1 and -3 have key roles in myogenic differentiation, the physiological significance of netrin-4 is still unclear. This study examined whether netrin-4 regulates myofiber type commitment and myotube formation. Initially, the expression profiles indicated that satellite cells isolated from the extensor digitorum longus muscle (EDL muscle: fast-twitch myofiber-abundant) expressed slightly more netrin-4 than the soleus muscle (slow-type abundant) cells. As netrin-4 knockdown inhibited both slow- and fast-type myotube formation, netrin-4 may not directly regulate myofiber type commitment. However, netrin-4 knockdown in satellite cell-derived myoblasts reduced the myotube fusion index, while exogenous netrin-4 promoted myotube formation, even though netrin-4 expression level was maximum during the initiation stage of myogenic differentiation. Furthermore, netrin-4 knockdown also inhibited MyoD (a master transcriptional factor of myogenesis) and Myomixer (a myoblast fusogenic molecule) expression. These data suggest that satellite cells synthesize netrin-4 during myogenic differentiation initiation to promote their own fusion, stimulating the MyoD-Myomixer signaling axis.

Regulation of the myoblast fusion reaction for muscle development, regeneration, and adaptations.

Fusion of plasma membranes is essential for skeletal muscle development, regeneration, exercise-induced adaptations, and results in a cell that contains hundreds to thousands of nuclei within a shared cytoplasm. The differentiation process in myocytes culminates in their fusion to form a new myofiber or fusion to an existing myofiber thereby contributing more synthetic material to the syncytium. The choice for two cells to fuse and become one could be a dangerous event if the two cells are not committed to an allied function. Thus, fusion events are highly regulated with positive and negative factors to fine-tune the process, and requires muscle-specific fusogens (Myomaker and Myomerger) as well as general cellular machinery to achieve the union of membranes. While a unified vertebrate myoblast fusion pathway is not yet established, recent discoveries should make this pursuit attainable. Not only does myocyte fusion impact the normal biology of skeletal muscle, but new evidence indicates dysregulation of the process impacts pathologies of skeletal muscle. Here, I will highlight the molecular players and biochemical mechanisms that drive fusion events in muscle, and discuss how this key myogenic process impacts skeletal muscle diseases.

Syndecan-4 affects myogenesis via Rac1-mediated actin remodeling and exhibits copy-number amplification and increased expression in human rhabdomyosarcoma tumors.

Skeletal muscle demonstrates a high degree of regenerative capacity repeating the embryonic myogenic program under strict control. Rhabdomyosarcoma is the most common sarcoma in childhood and is characterized by impaired muscle differentiation. In this study, we observed that silencing the expression of syndecan-4, the ubiquitously expressed transmembrane heparan sulfate proteoglycan, significantly enhanced myoblast differentiation, and fusion. During muscle differentiation, the gradually decreasing expression of syndecan-4 allows the activation of Rac1, thereby mediating myoblast fusion. Single-molecule localized superresolution direct stochastic optical reconstruction microscopy (dSTORM) imaging revealed nanoscale changes in actin cytoskeletal architecture, and atomic force microscopy showed reduced elasticity of syndecan-4-knockdown cells during fusion. Syndecan-4 copy-number amplification was observed in 28% of human fusion-negative rhabdomyosarcoma tumors and was accompanied by increased syndecan-4 expression based on RNA sequencing data. Our study suggests that syndecan-4 can serve as a tumor driver gene in promoting rabdomyosarcoma tumor development. Our results contribute to the understanding of the role of syndecan-4 in skeletal muscle development, regeneration, and tumorigenesis.

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.

Caveolin 3 suppresses phosphorylation-dependent activation of sarcolemmal nNOS.

Mutations of the caveolin 3 gene cause autosomal dominant limb-girdle muscular dystrophy (LGMD)1C. In mice, overexpression of mutant caveolin 3 leads to loss of caveolin 3 and results in myofiber hypotrophy in association with activation of neuronal nitric oxide synthase (nNOS) at the sarcolemma. Here, we show that caveolin 3 directly bound to nNOS and suppressed its phosphorylation-dependent activation at a specific residue, Ser1412 in the nicotinamide adenine dinucleotide phosphate (NADPH)-flavin adenine dinucleotide (FAD) module near the C-terminus of the reduction domain, in vitro. Constitutively active nNOS enhanced myoblast fusion, but not myogenesis, in vitro. Phosphorylation-dependent activation of nNOS occurred in muscles from caveolin 3-mutant mice and LGMD1C patients. Mating with nNOS-mutant mice exacerbated myofiber hypotrophy in the caveolin 3-mutant mice. In nNOS-mutant mice, regenerating myofibers after cardiotoxin injury became hypotrophic with reduced myoblast fusion. Administration of NO donor increased myofiber size and the number of myonuclei in the caveolin 3-mutant mice. Exercise also increased myofiber size accompanied by phosphorylation-dependent activation of nNOS in wild-type and caveolin 3-mutant mice. These data indicate that caveolin 3 inhibits phosphorylation-dependent activation of nNOS, which leads to myofiber hypertrophy via enhancing myoblast fusion. Hypertrophic signaling by nNOS phosphorylation could act in a compensatory manner in caveolin 3-deficient muscles.

Insights into the biological functions of Dock family guanine nucleotide exchange factors.

Rho GTPases play key regulatory roles in many aspects of embryonic development, regulating processes such as differentiation, proliferation, morphogenesis, and migration. Two families of guanine nucleotide exchange factors (GEFs) found in metazoans, Dbl and Dock, are responsible for the spatiotemporal activation of Rac and Cdc42 proteins and their downstream signaling pathways. This review focuses on the emerging roles of the mammalian DOCK family in development and disease. We also discuss, when possible, how recent discoveries concerning the biological functions of these GEFs might be exploited for the development of novel therapeutic strategies.

Temporal Expression of Myogenic Regulatory Genes in Different Chicken Breeds during Embryonic Development.

The basic units of skeletal muscle in all vertebrates are multinucleate myofibers, which are formed from the fusion of mononuclear myoblasts during the embryonic period. In order to understand the regulation of embryonic muscle development, we selected four chicken breeds, namely, Cornish (CN), White Plymouth Rock (WPR), White Leghorn (WL), and Beijing-You Chicken (BYC), for evaluation of their temporal expression patterns of known key regulatory genes (Myomaker, MYOD, and MSTN) during pectoral muscle (PM) and thigh muscle (TM) development. The highest expression level of Myomaker occurred from embryonic days E13 to E15 for all breeds, indicating that it was the crucial stage of myoblast fusion. Interestingly, the fast-growing CN showed the highest gene expression level of Myomaker during the crucial stage. The MYOD gene expression at D1 was much higher, implying that MYOD might have an important role after hatching. Histomorphology of PM and TM suggested that the myofibers was largely complete at E17, which was speculated to have occurred because of the expression increase in MSTN and the expression decrease in Myomaker. Our research contributes to lay a foundation for the study of myofiber development during the embryonic period in different chicken breeds.

Puromycin-sensitive aminopeptidase is required for C2C12 myoblast proliferation and differentiation.

The ubiquitin-proteasome system is a major protein degradation pathway in the cell. Proteasomes produce several peptides that are rapidly degraded to free amino acids by intracellular aminopeptidases. Our previous studies reported that proteolysis via proteasomes and aminopeptidases is required for myoblast proliferation and differentiation. However, the role of intracellular aminopeptidases in myoblast proliferation and differentiation had not been clarified. In this study, we investigated the effects of puromycin-sensitive aminopeptidase (PSA) on C2C12 myoblast proliferation and differentiation by knocking down PSA. Aminopeptidase enzymatic activity was reduced in PSA-knockdown myoblasts. Knockdown of PSA induced impaired cell cycle progression in C2C12 myoblasts and accumulation of cells at the G2/M phase. Additionally, after the induction of myogenic differentiation in PSA-knockdown myoblasts, multinucleated circular-shaped myotubes with impaired cell polarity were frequently identified. Cell division cycle 42 (CDC42) knockdown in myoblasts resulted in a loss of cell polarity and the formation of multinucleated circular-shaped myotubes, which were similar to PSA-knockdown myoblasts. These data suggest that PSA is required for the proliferation of myoblasts in the growth phase and for the determination of cell polarity and elongation of myotubes in the differentiation phase.

The fusogenic synapse at a glance.

Cell-cell fusion is a fundamental process underlying fertilization, development, regeneration and physiology of metazoans. It is a multi-step process involving cell recognition and adhesion, actin cytoskeletal rearrangements, fusogen engagement, lipid mixing and fusion pore formation, ultimately resulting in the integration of two fusion partners. Here, we focus on the asymmetric actin cytoskeletal rearrangements at the site of fusion, known as the fusogenic synapse, which was first discovered during myoblast fusion in Drosophila embryos and later also found in mammalian muscle and non-muscle cells. At the asymmetric fusogenic synapse, actin-propelled invasive membrane protrusions from an attacking fusion partner trigger actomyosin-based mechanosensory responses in the receiving cell. The interplay between the invasive and resisting forces generated by the two fusion partners puts the fusogenic synapse under high mechanical tension and brings the two cell membranes into close proximity, promoting the engagement of fusogens to initiate fusion pore formation. In this Cell Science at a Glance article and the accompanying poster, we highlight the molecular, cellular and biophysical events at the asymmetric fusogenic synapse using Drosophila myoblast fusion as a model.

WNT/ß-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/ß-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/ß-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 ß-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 ß-catenin-binding element, and Nphs1 expression was induced by the activation of WNT/ß-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/ß-catenin-dependent myoblast fusion. Taken together, our results indicate that WNT/ß-catenin signaling is crucial for myoblast fusion through the regulation of the Nphs1 gene.

The regulatory role of Myomaker and Myomixer-Myomerger-Minion in muscle development and regeneration.

Skeletal muscle plays essential roles in motor function, energy, and glucose metabolism. Skeletal muscle formation occurs through a process called myogenesis, in which a crucial step is the fusion of mononucleated myoblasts to form multinucleated myofibers. The myoblast/myocyte fusion is triggered and coordinated in a muscle-specific way that is essential for muscle development and post-natal muscle regeneration. Many molecules and proteins have been found and demonstrated to have the capacity to regulate the fusion of myoblast/myocytes. Interestingly, two newly discovered muscle-specific membrane proteins, Myomaker and Myomixer (also called Myomerger and Minion), have been identified as fusogenic regulators in vertebrates. Both Myomaker and Myomixer-Myomerger-Minion have the capacity to directly control the myogenic fusion process. Here, we review and discuss the latest studies related to these two proteins, including the discovery, structure, expression pattern, functions, and regulation of Myomaker and Myomixer-Myomerger-Minion. We also emphasize and discuss the interaction between Myomaker and Myomixer-Myomerger-Minion, as well as their cooperative regulatory roles in cell-cell fusion. Moreover, we highlight the areas for exploration of Myomaker and Myomixer-Myomerger-Minion in future studies and consider their potential application to control cell fusion for cell-therapy purposes.

APC/CFzr regulates cardiac and myoblast cell numbers, and plays a crucial role during myoblast fusion.

Somatic muscles are formed by the iterative fusion of myoblasts into muscle fibres. This process is driven by the recurrent recruitment of proteins to the cell membrane to induce F-actin nucleation at the fusion site. Although several proteins involved in myoblast fusion have been identified, knowledge about their subcellular regulation is rather elusive. We identified the anaphase-promoting complex (APC/C) adaptor Fizzy related (Fzr) as an essential regulator of heart and muscle development. We show that APC/CFzr regulates the fusion of myoblasts as well as the mitotic exit of pericardial cells, cardioblasts and myoblasts. Surprisingly, overproliferation is not causative for the observed fusion defects. Instead, fzr mutants exhibit smaller F-actin foci at the fusion site and display reduced membrane breakdown between adjacent myoblasts. We show that lack of APC/CFzr causes accumulation and mislocalisation of Rols and Duf, two proteins involved in the fusion process. Duf seems to serve as direct substrate of the APC/CFzr and its destruction depends on the presence of distinct degron sequences. These novel findings indicate that protein destruction and turnover constitute major events during myoblast fusion.