Myoblast Fusion In Drosophila 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.

Drosophila melanogaster: A Model System to Study Distinct Genetic Programs in Myoblast Fusion.

Muscle fibers are multinucleated cells that arise during embryogenesis through the fusion of mononucleated myoblasts. Myoblast fusion is a lifelong process that is crucial for the growth and regeneration of muscles. Understanding the molecular mechanism of myoblast fusion may open the way for novel therapies in muscle wasting and weakness. Recent reports in Drosophila and mammals have provided new mechanistic insights into myoblast fusion. In Drosophila, muscle formation occurs twice: during embryogenesis and metamorphosis. A fundamental feature is the formation of a cell–cell communication structure that brings the apposing membranes into close proximity and recruits possible fusogenic proteins. However, genetic studies suggest that myoblast fusion in Drosophila is not a uniform process. The complexity of the players involved in myoblast fusion can be modulated depending on the type of muscle that is formed. In this review, we introduce the different types of multinucleated muscles that form during Drosophila development and provide an overview in advances that have been made to understand the mechanism of myoblast fusion. Finally, we will discuss conceptual frameworks in cell–cell fusion in Drosophila and mammals.

Ubiquitin-dependent remodeling of the actin cytoskeleton drives cell fusion.

Cell-cell fusion is a frequent and essential event during development, and its dysregulation causes diseases ranging from infertility to muscle weakness. Fusing cells need to repeatedly remodel their plasma membrane through orchestrated formation and disassembly of actin filaments, but how the dynamic reorganization of the cortical actin cytoskeleton is controlled is still poorly understood. Here, we identified a ubiquitin-dependent toggle switch that establishes reversible actin bundling during mammalian cell fusion. We found that EPS8-IRSp53 complexes stabilize cortical actin bundles at sites of cell contact to promote close membrane alignment. EPS8 monoubiquitylation by CUL3KCTD10 displaces EPS8-IRSp53 from membranes and counteracts actin bundling, a dual activity that restricts actin bundling to allow paired cells to progress with fusion. We conclude that cytoskeletal rearrangements during development are precisely controlled by ubiquitylation, raising the possibility of modulating the efficiency of cell-cell fusion for therapeutic benefit.

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.

DrosophilaMyoblast 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.

Group I Paks Promote Skeletal Myoblast Differentiation In Vivo and In Vitro.

Skeletal myogenesis is regulated by signal transduction, but the factors and mechanisms involved are not well understood. The group I Paks Pak1 and Pak2 are related protein kinases and direct effectors of Cdc42 and Rac1. Group I Paks are ubiquitously expressed and specifically required for myoblast fusion in Drosophila We report that both Pak1 and Pak2 are activated during mammalian myoblast differentiation. One pathway of activation is initiated by N-cadherin ligation and involves the cadherin coreceptor Cdo with its downstream effector, Cdc42. Individual genetic deletion of Pak1 and Pak2 in mice has no overt effect on skeletal muscle development or regeneration. However, combined muscle-specific deletion of Pak1 and Pak2 results in reduced muscle mass and a higher proportion of myofibers with a smaller cross-sectional area. This phenotype is exacerbated after repair to acute injury. Furthermore, primary myoblasts lacking Pak1 and Pak2 display delayed expression of myogenic differentiation markers and myotube formation. These results identify Pak1 and Pak2 as redundant regulators of myoblast differentiation in vitro and in vivo and as components of the promyogenic Ncad/Cdo/Cdc42 signaling pathway.

Myoblast Fusion: Playing Hard to Get

In Drosophila myoblast fusion, the fusing cell invades another by actin-enriched protrusion. In this issue of Developmental Cell, Kim etal. (2015) examine the myoblast fusion mechanism from the perspective of the "receiving" cell and report that fusion depends on the ability of this cell to stiffen its actomyosin cortex.

Live imaging provides new insights on dynamic F-actin filopodia and differential endocytosis during myoblast fusion in Drosophila.

The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 pathways. In the present study, we utilize the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. We also observed differential endocytosis of cell surface components at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the myotube.

The Rac-specific exchange factors Dock1 and Dock5 are dispensable for the establishment of the glomerular filtration barrier in vivo

Podocytes are specialized kidney cells that form the kidney filtration barrier through the connection of their foot processes. Nephrin and Neph family transmembrane molecules at the surface of podocytes interconnect to form a unique type of cell-cell junction, the slit diaphragm, which acts as a molecular sieve. The cytoplasmic tails of Nephrin and Neph mediate cytoskeletal rearrangement that contributes to the maintenance of the filtration barrier. Nephrin and Neph1 orthologs are essential to regulate cell-cell adhesion and Rac-dependent actin rearrangement during Drosophila myoblast fusion. We hypothesized here that molecules regulating myoblast fusion in Drosophila could contribute to signaling downstream of Nephrin and Neph1 in podocytes. We found that Nephrin engagement promoted recruitment of the Rac exchange factor Dock1 to the membrane. Furthermore, Nephrin overexpression led to lamellipodia formation that could be blocked by inhibiting Rac1 activity. We generated in vivo mouse models to investigate whether Dock1 and Dock5 contribute to the formation and maintenance of the kidney filtration barrier. Our results indicate that while Dock1 and Dock5 are expressed in podocytes, their functions are not essential for the development of the glomerular filtration barrier. Furthermore, mice lacking Dock1 were not protected from LPS-induced podocyte effacement. Our data suggest that Dock1 and Dock5 are not the important exchange factors regulating Rac activity during the establishment and maintenance of the glomerular barrier.

Actin-Propelled Invasive Membrane Protrusions Promote Fusogenic Protein Engagement During Cell-Cell Fusion

Cell-cell fusion is critical for the conception, development, and physiology of multicellular organisms. Although cellular fusogenic proteins and the actin cytoskeleton are implicated in cell-cell fusion, it remains unclear whether and how they coordinate to promote plasma membrane fusion. We reconstituted a high-efficiency, inducible cell fusion culture system in the normally nonfusing Drosophila S2R+ cells. Both fusogenic proteins and actin cytoskeletal rearrangements were necessary for cell fusion, and in combination they were sufficient to impart fusion competence. Localized actin polymerization triggered by specific cell-cell or cell-matrix adhesion molecules propelled invasive cell membrane protrusions, which in turn promoted fusogenic protein engagement and plasma membrane fusion. This de novo cell fusion culture system reveals a general role for actin-propelled invasive membrane protrusions in driving fusogenic protein engagement during cell-cell fusion.

Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles.

ADP-Ribosylation Factor 6 Regulates Mammalian Myoblast Fusion through Phospholipase D1 and Phosphatidylinositol 4,5-Bisphosphate Signaling Pathways

Myoblast fusion is an essential step during myoblast differentiation that remains poorly understood. M-cadherin-dependent pathways that signal through Rac1 GTPase activation via the Rho-guanine nucleotide exchange factor (GEF) Trio are important for myoblast fusion. The ADP-ribosylation factor (ARF)6 GTPase has been shown to bind to Trio and to regulate Rac1 activity. Moreover, Loner/GEP(100)/BRAG2, a GEF of ARF6, has been involved in mammalian and Drosophila myoblast fusion, but the specific role of ARF6 has been not fully analyzed. Here, we show that ARF6 activity is increased at the time of myoblast fusion and is required for its implementation in mouse C2C12 myoblasts. Specifically, at the onset of myoblast fusion, ARF6 is associated with the multiproteic complex that contains M-cadherin, Trio, and Rac1 and accumulates at sites of myoblast fusion. ARF6 silencing inhibits the association of Trio and Rac1 with M-cadherin. Moreover, we demonstrate that ARF6 regulates myoblast fusion through phospholipase D (PLD) activation and phosphatidylinositol 4,5-bis-phosphate production. Together, these data indicate that ARF6 is a critical regulator of C2C12 myoblast fusion and participates in the regulation of PLD activities that trigger both phospholipids production and actin cytoskeleton reorganization at fusion sites.

The Complex Spatio-Temporal Regulation of the Drosophila Myoblast Attractant Gene duf/kirre

A key early player in the regulation of myoblast fusion is the gene dumbfounded (duf, also known as kirre). Duf must be expressed, and function, in founder cells (FCs). A fixed number of FCs are chosen from a pool of equivalent myoblasts and serve to attract fusion-competent myoblasts (FCMs) to fuse with them to form a multinucleate muscle-fibre. The spatial and temporal regulation of duf expression and function are important and play a deciding role in choice of fibre number, location and perhaps size. We have used a combination of bioinformatics and functional enhancer deletion approaches to understand the regulation of duf. By transgenic enhancer-reporter deletion analysis of the duf regulatory region, we found that several distinct enhancer modules regulate duf expression in specific muscle founders of the embryo and the adult. In addition to existing bioinformatics tools, we used a new program for analysis of regulatory sequence, PhyloGibbs-MP, whose development was largely motivated by the requirements of this work. The results complement our deletion analysis by identifying transcription factors whose predicted binding regions match with our deletion constructs. Experimental evidence for the relevance of some of these TF binding sites comes from available ChIP-on-chip from the literature, and from our analysis of localization of myogenic transcription factors with duf enhancer reporter gene expression. Our results demonstrate the complex regulation in each founder cell of a gene that is expressed in all founder cells. They provide evidence for transcriptional control—both activation and repression—as an important player in the regulation of myoblast fusion. The set of enhancer constructs generated will be valuable in identifying novel trans-acting factor-binding sites and chromatin regulation during myoblast fusion in Drosophila. Our results and the bioinformatics tools developed provide a basis for the study of the transcriptional regulation of other complex genes.

Rab11 is required for myoblast fusion in Drosophila

Rab11, an evolutionarily conserved, ubiquitously expressed subfamily of small monomeric Rab GTPases, has been implicated in regulating vesicular trafficking through the recycling of endosomal compartment. In order to gain an insight into the role of this gene in myogenesis during embryonic development, we have studied the expression pattern of Rab11 in mesoderm during muscle differentiation in Drosophila embryo. When dominant-negative or constitutively active Drosophila Rab11 proteins are expressed or Rab11 is reduced via double-stranded RNA in muscle precursors, they cause partial failure of myoblast fusion and show anomalies in the shape of the muscle fibres. Our results suggest that Rab11 plays no role in cell fate specification in muscle precursors but is required late in the process of myoblast fusion.

Actin regulators take the reins in Drosophila myoblast fusion

AbstractSkeletal muscle formation, growth and repair depend on myoblast fusion events. Therefore, in-depth understanding of the underlying molecular mechanisms controlling these events that ultimately lead to skeletal muscle formation may be fundamental for developing new therapies for tissue repair. To this end, the greatest advances in furthering understanding myoblast fusion has been made in Drosophila. Recent studies have shown that transient F-actin structures, so-called actin plugs or foci, are known to form at the site of contacting myoblasts. Indeed, actin regulators of the WASP family that control the activation of the Arp2/3 complex and thereby branched F-actin formation have been demonstrated to be crucial for myoblast fusion. Myoblast-specific cell adhesion molecules seem to be involved in the recruitment of WASP family members to the site of myoblast fusion and form a Fusion-Restricted Myogenic-Adhesive Structure (FuRMAS). Currently, the exact role of the FuRMAS is not completely understood. However, recent studies indicate that WASP-dependent F-actin regulation is required for fusion pore formation as well as for the correct integration of fusing myoblasts into the growing muscle. In this review, I discuss latest cellular studies, and recent genetic and biochemical analyses on actin regulation during myoblast fusion.

Wiskott-Aldrich Syndrome Protein Is a Key Regulator of the Phagocytic Cup Formation in Macrophages

Phagocytosis is a vital first-line host defense mechanism against infection involving the ingestion and digestion of foreign materials such as bacteria by specialized cells, phagocytes. For phagocytes to ingest the foreign materials, they form an actin-based membrane structure called phagocytic cup at the plasma membranes. Formation of the phagocytic cup is impaired in phagocytes from patients with a genetic immunodeficiency disorder, Wiskott-Aldrich syndrome (WAS). The gene defective in WAS encodes Wiskott-Aldrich syndrome protein (WASP). Mutation or deletion of WASP causes impaired formation of the phagocytic cup, suggesting that WASP plays an important role in the phagocytic cup formation. However, the molecular details of its formation remain unknown. We have shown that the WASP C-terminal activity is critical for the phagocytic cup formation in macrophages. We demonstrated that WASP is phosphorylated on tyrosine 291 in macrophages, and the WASP phosphorylation is important for the phagocytic cup formation. In addition, we showed that WASP and WASP-interacting protein (WIP) form a complex at the phagocytic cup and that the WASP.WIP complex plays a critical role in the phagocytic cup formation. Our results indicate that the phosphorylation of WASP and the complex formation of WASP with WIP are the essential molecular steps for the efficient formation of the phagocytic cup in macrophages, suggesting a possible disease mechanism underlying phagocytic defects and recurrent infections in WAS patients.

Expression and functional analysis of a novel Fusion Competent Myoblast specific GAL4 driver

In the Drosophila embryo, body wall muscles are formed by the fusion of two cell types, Founder Cells (FCs) and Fusion Competent Myoblasts (FCMs). Using an enhancer derived from the Dmef2 gene ([C/D] ∗), we report the first GAL4 driver specifically expressed in FCMs. We have determined that this GAL4 driver causes expression in a subset of FCMs and, upon fusion, in developing myotubes from stage 14 onwards. In addition, we have shown that using this Dmef2-5x[C/D] ∗-GAL4 driver to express dominant negative Rac in only FCMs causes a partial fusion block. This novel GAL4 driver will provide a useful reagent to study Drosophila myoblast fusion and muscle differentiation.

Actin on Multiple Fronts to Generate a Muscle Fiber

How the actin cytoskeleton is harnessed to fulfill its diverse cellular functions is a recurrent and intriguing question. In this issue of Developmental Cell, Kim et al. and Massarwa et al. describe a new role for F-actin, and more specifically the actin regulator WASp, in myoblast fusion in Drosophila.

The Wiskott–Aldrich syndrome protein (WASP) is essential for myoblast fusion in Drosophila

In higher organisms, mononucleated myoblasts fuse to form multinucleated myotubes. During this process, myoblasts undergo specific changes in cell morphology and cytoarchitecture. Previously, we have shown that the actin regulator Kette (Hem-2/Nap-1) is essential for myoblast fusion. In this study, we describe the role of the evolutionary conserved Wiskott–Aldrich syndrome protein that serves as a regulator for the Arp2/3 complex for myoblast fusion. By screening an EMS mutagenesis collection, we discovered a new wasp allele that does not complete fusion during myogenesis. Interestingly, this new wasp 3D3-035 allele is characterized by a disruption of fusion after precursor formation. The molecular lesion in this wasp allele leads to a stop codon preventing translation of the CA domain. Usually, the WASP protein exerts its function through the Arp2/3-interacting CA domain. Accordingly, a waspΔCA that is expressed in a wild-type background acts as dominant-negative during the fusion process. Furthermore, we show that the myoblast fusion phenotype of kette mutant embryos can be suppressed by reducing the gene dose of wasp 3D3-035 . Thus, Kette antagonizes WASP function during myoblast fusion.

Myoblasts incompetent encodes a zinc finger transcription factor required to specify fusion-competent myoblasts in Drosophila.

We report a new gene, myoblasts incompetent, essential for normal myogenesis and myoblast fusion in Drosophila. myoblasts incompetent encodes a putative zinc finger transcription factor related to vertebrate Gli proteins and to Drosophila Cubitus interruptus. myoblasts incompetent is expressed in immature somatic and visceral myoblasts. Expression is predominantly in fusion-competent myoblasts and a loss-of-function mutation in myoblasts incompetent leads to a failure in the normal differentiation of these cells and a complete lack of myoblast fusion. In the mutant embryos, founder myoblasts differentiate normally and form mononucleate muscles, but genes that are specifically expressed in fusion-competent cells are not activated and the normal downregulation of twist expression in these cells fails to occur. In addition, fusion-competent myoblasts fail to express proteins characteristic of the general pathway of myogenesis such as myosin and Dmef2. Thus myoblasts incompetent appears to function specifically in the general pathway of myogenesis to control the differentiation of fusion-competent myoblasts.