Myomaker mediates fusion of fast myocytes in zebrafish embryos

During skeletal muscle development, myoblasts fuse to form multinucleated myofibers. Myomaker [Transmembrane protein 8c (TMEM8c)] is a muscle-specific protein that is essential for myoblast fusion and sufficient to promote fusion of fibroblasts with muscle cells; however, the structure and biochemical properties of this membrane protein have not been explored. Here, we used CRISPR/Cas9 mutagenesis to disrupt myomaker expression in the C2C12 muscle cell line, which resulted in complete blockade to fusion. To define the functional domains of myomaker required to direct fusion, we established a heterologous cell-cell fusion system, in which fibroblasts expressing mutant versions of myomaker were mixed with WT myoblasts. Our data indicate that the majority of myomaker is embedded in the plasma membrane with seven membrane-spanning regions and a required intracellular C-terminal tail. We show that myomaker function is conserved in other mammalian orthologs; however, related family members (TMEM8a and TMEM8b) do not exhibit fusogenic activity. These findings represent an important step toward deciphering the cellular components and mechanisms that control myoblast fusion and muscle formation.P lasma membrane fusion is a fundamental cellular process required for the conception, development, and physiology of multicellular organisms. Membrane fusion occurs during viral infection of a host cell, between intracellular membranes, and between two plasma membranes to form syncytial tissues (1). Cell-cell fusion is critical for a wide array of cellular processes, including sperm-egg fertilization, macrophage function, bone and placental development, and skeletal muscle formation. This form of fusion must be precisely controlled to prevent inappropriate cellular mixing.The fusion of myoblasts requires cell recognition, migration, adhesion, signaling, and finally, membrane coalescence (2). Much of our knowledge about myoblast fusion has originated from studies performed in Drosophila. In this system, intracellular signaling results in cytoskeletal alterations and actin polymerization, which drive the formation of cellular projections that invade neighboring cells to cause fusion (3). Recent evidence also indicates a critical function for branched actin polymerization during Drosophila indirect flight muscle fusion (4). The essential role of the cytoskeleton in fusion is conserved in mammals, in which the actin regulators Rac1, cdc42, and N-WASp are required for muscle development (5-8). In addition to actin dynamics, numerous proteins that regulate diverse cellular processes have been associated with myoblast fusion (9-13).We recently discovered a muscle-specific membrane protein named myomaker [annotated as Transmembrane protein 8c (TMEM8c)] that is absolutely required for skeletal myocyte fusion in the mouse (14,15). To begin to decipher the mechanisms whereby myomaker controls cell-cell fusion, we designed a heterologous cell-cell fusion assay to monitor the ability of a series of myomaker mutants to direct the fusion of lab...

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“…2A). Morpholino knockdown of myomaker in zebrafish revealed that myomaker is essential for fast myocyte fusion (17).…”

Section: Resultsmentioning

confidence: 99%

Structure–function analysis of myomaker domains required for myoblast fusion

Millay

Gamage

Quinn

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…It is expressed exclusively in the skeletal muscle lineage and at the times during muscle development and regeneration when fusion is occurring. Significantly, ectopic expression of myomaker in fibroblasts allows them to fuse with myoblasts, an activity not seen previously with any other protein (Millay et al 2013(Millay et al , 2016Landemaine et al 2014). Whether myomaker is the long sought direct fusogen, or somehow regulates the process in another fundamental way, is not yet clear.…”

Section: Cell -Cell Contact and Skeletal Myogenesismentioning

confidence: 95%

“…One possibility here is the recently discovered myomaker, a cell-surface protein with seven membrane-spanning regions (Millay et al 2013(Millay et al , 2016. Myomaker (also called TMEM8c) is essential for myoblast fusion in both mice and zebrafish (Millay et al 2013(Millay et al , 2014Landemaine et al 2014). It is expressed exclusively in the skeletal muscle lineage and at the times during muscle development and regeneration when fusion is occurring.…”

Section: Cell -Cell Contact and Skeletal Myogenesismentioning

confidence: 99%

Keep Your Friends Close: Cell–Cell Contact and Skeletal Myogenesis

Krauss

Joseph

Goel

2017

Cold Spring Harb Perspect Biol

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“…Over the course of 4 days culturing in the low serum condition (DM; days 7 to 10), the mRNA levels of several analyzed genes increased (MRF4, MYOG, MEF2C, DES and MYH1), starting on day 7 (P < 0.05), and these levels remained constant until the end of the experimental model (Figure 3). The novel muscle-specific gene TMEM8C, also named myomaker, was recently described in mouse and zebrafish models and demonstrated to promote myoblast fusion (Millay et al, 2013;Landemaine et al, 2014). In BoSCs, TMEM8C mRNA expression was significantly up-regulated (P < 0.05), starting on the 1st day of cultivation under differentiating conditions (day 7) and until day 10, by which time the myoblasts had fused (Figure 3).…”

Section: Resultsmentioning

confidence: 97%

“…Bars = 250 μm. Landemaine et al, 2014;Luo et al, 2015). However, its expression pattern and roles in bovine species have not been investigated.…”

Section: Discussionmentioning

confidence: 99%

Temporal correlation between differentiation factor expression and microRNAs in Holstein bovine skeletal muscle

Miretti

Volpe

Martignani

et al. 2017

Animal

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Satellite cells are adult stem cells located between the basal lamina and sarcolemma of muscle fibers. Under physiological conditions, satellite cells are quiescent, but they maintain a strong proliferative potential and propensity to differentiate, which underlies their critical role in muscle preservation and growth. MicroRNAs (miRNAs) play essential roles during animal development as well as in stem cell self-renewal and differentiation regulation. MiRNA-1, miRNA-133a and miRNA-206 are closely related muscle-specific miRNAs, and are thus defined myomiRNAs. MyomiRNAs are integrated into myogenic regulatory networks. Their expression is under the transcriptional and post-transcriptional control of myogenic factors and, in turn, they exhibit widespread control of muscle gene expression. Very little information is available about the regulation and behavior of satellite cells in large farm animals, in particular during satellite cell differentiation. Here, we study bovine satellite cells (BoSCs) undergoing a differentiation process and report the expression pattern of selected genes and miRNAs involved. Muscle samples of longissimus thoracis from Holstein adult male animals were selected for the collection of satellite cells. All satellite cell preparations demonstrated myotube differentiation. To characterize the dynamics of several transcription factors expressed in BoSCs, we performed real-time PCR on complementary DNA generated from the total RNA extracted from BoSCs cultivated in growth medium (GM) or in differentiation medium (DM) for 4 days. In the GM condition, BoSCs expressed the satellite cell lineage markers as well as transcripts for the myogenic regulatory factors. At the time of isolation from muscle, PAX7 was expressed in nearly 100% of BoSCs; however, its messenger RNA (mRNA) levels dramatically decreased between 3 and 6 days post isolation ( P < 0.01). MyoD mRNA levels increased during the 1st day of cultivation in DM (day 7; P < 0.02), showing a gradual activation of the myogenic gene program. During the subsequent 4 days of culture in DM, several tested genes, including MRF4, MYOG, MEF2C, TMEM8C, DES and MYH1, showed increased expression ( P < 0.05), and these levels remained high throughout the culture period investigated. Meanwhile, the expression of genes involved in the differentiation process also miRNA-1, miRNA-133a and miRNA-206 were strongly up-regulated on the 1st day in DM (day 7; P < 0.05). Analysis revealed highly significant correlations between myomiRNAs expression and MEF2C, MRF4, TMEM8C, DES and MYH1 gene expression ( P < 0.001). Knowledge about the transcriptional changes correlating with the growth and differentiation of skeletal muscle fibers could be helpful for developing strategies to improve production performance in livestock.

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Myomaker mediates fusion of fast myocytes in zebrafish embryos

Cited by 61 publications

References 15 publications

Structure–function analysis of myomaker domains required for myoblast fusion

Structure–function analysis of myomaker domains required for myoblast fusion

Keep Your Friends Close: Cell–Cell Contact and Skeletal Myogenesis

Temporal correlation between differentiation factor expression and microRNAs in Holstein bovine skeletal muscle

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