Skeletal muscle is the largest tissue in the body and loss of its function or its regenerative properties results in debilitating musculoskeletal disorders. Understanding the mechanisms that drive skeletal muscle formation will not only help to unravel the molecular basis of skeletal muscle diseases, but also provide a roadmap for recapitulating skeletal myogenesis in vitro from pluripotent stem cells (PSCs). PSCs have become an important tool for probing developmental questions, while differentiated cell types allow the development of novel therapeutic strategies. In this Review, we provide a comprehensive overview of skeletal myogenesis from the earliest premyogenic progenitor stage to terminally differentiated myofibers, and discuss how this knowledge has been applied to differentiate PSCs into muscle fibers and their progenitors in vitro.

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Making muscle: skeletal myogenesis in vivo and in vitro.

The fusion of myoblasts into multinucleate syncytia plays a fundamental role in muscle function, as it supports the formation of extended sarcomeric arrays, or myofibrils, within a large volume of cytoplasm. Principles learned from the study of myoblast fusion not only enhance our understanding of myogenesis, but also contribute to our perspectives on membrane fusion and cell-cell fusion in a wide array of model organisms and experimental systems. Recent studies have advanced our views of the cell biological processes and crucial proteins that drive myoblast fusion. Here, we provide an overview of myoblast fusion in three model systems that have contributed much to our understanding of these events: the Drosophila embryo; developing and regenerating mouse muscle; and cultured rodent muscle cells.

Myoblast fusion: lessons from flies and mice

Fusion of myoblasts is essential for the formation of multi-nucleated muscle fibres. However, the identity of muscle-specific proteins that directly govern this fusion process in mammals has remained elusive. Here we identify a muscle-specific membrane protein, named myomaker, that controls myoblast fusion. Myomaker is expressed on the cell surface of myoblasts during fusion and is downregulated thereafter. Overexpression of myomaker in myoblasts markedly enhances fusion, and genetic disruption of myomaker in mice causes perinatal death due to an absence of multi-nucleated muscle fibres. Remarkably, forced expression of myomaker in fibroblasts promotes fusion with myoblasts, demonstrating the direct participation of this protein in the fusion process. Pharmacological perturbation of the actin cytoskeleton abolishes the activity of myomaker, consistent with previous studies implicating actin dynamics in myoblast fusion. These findings reveal a long-sought myogenic fusion protein that controls mammalian myoblast fusion and provide new insights into the molecular underpinnings of muscle formation.

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Myomaker is a membrane activator of myoblast fusion and muscle formation

Netrins are secreted proteins that were first identified as guidance cues, directing cell and axon migration during neural development. Subsequent findings have demonstrated that netrins can influence the formation of multiple tissues, including the vasculature, lung, pancreas, muscle and mammary gland, by mediating cell migration, cell-cell interactions and cell-extracellular matrix adhesion. Recent evidence also implicates the ongoing expression of netrins and netrin receptors in the maintenance of cell-cell organisation in mature tissues. Here, we review the mechanisms involved in netrin signalling in vertebrate and invertebrate systems and discuss the functions of netrin signalling during the development of neural and non-neural tissues.

Netrins: versatile extracellular cues with diverse functions.

The cytoskeleton and its components — actin, microtubules and intermediate filaments — have been studied for decades, and multiple roles of the individual cytoskeletal substructures are now well established. However, in recent years it has become apparent that the three cytoskeletal elements also engage in extensive crosstalk that is important for core biological processes. Actin–microtubule crosstalk is particularly important for the regulation of cell shape and polarity during cell migration and division and the establishment of neuronal and epithelial cell shape and function. This crosstalk engages different cytoskeletal regulators and encompasses various physical interactions, such as crosslinking, anchoring and mechanical support. Thus, the cytoskeleton should be considered not as a collection of individual parts but rather as a unified system in which subcomponents co-regulate each other to exert their functions in a precise and highly adaptable manner. Actin and microtubules are essential for key cellular processes, including cell polarization, migration and division. Although their cellular roles are distinct, actin and microtubules also extensively influence each other’s dynamics and organization. This crosstalk encompasses multiple mechanisms and involves various crosslinkers, motors and regulatory proteins as well as mechanical cooperation.

Actin–microtubule crosstalk in cell biology

Dock1 (also known as Dock180) is a prototypical member of a new family of atypical Rho GTPase activators. Genetic studies in Drosophila and Caenorhabditis elegans have demonstrated that Dock1 orthologues in these organisms have a crucial role in activating Rac GTPase signaling. We generated mutant alleles of the closely related Dock1 and Dock5 genes to study their function in mammals. We report that while Dock5 is dispensable for normal mouse embryogenesis, Dock1 has an essential role in embryonic development. A dramatic reduction of all skeletal muscle tissues is observed in Dock1-null embryos. Mechanistically, this embryonic defect is attributed to a strong deficiency in myoblast fusion, which is detectable both in vitro and in vivo. Furthermore, we have uncovered a contribution of Dock5 toward myofiber development. These studies identify Dock1 and Dock5 as critical regulators of the fusion step during primary myogenesis in mammals and demonstrate that a specific component of the myoblast fusion machinery identified in Drosophila plays an evolutionarily conserved role in higher vertebrates.

https://www.pnas.org/content/pnas/105/40/15446.full.pdf

The atypical Rac activator Dock180 (Dock1) regulates myoblast fusion in vivo

The mammalian DOCK180 protein belongs to an evolutionarily conserved protein family, which together with ELMO proteins, is essential for activation of Rac GTPase-dependent biological processes. Here, we have analyzed the DOCK180-ELMO1 interaction, and map direct interaction interfaces to the N-terminal 200 amino acids of DOCK180, and to the C-terminal 200 amino acids of ELMO1, comprising the ELMO1 PH domain. Structural and biochemical analysis of this PH domain reveals that it is incapable of phospholipid binding, but instead structurally resembles FERM domains. Moreover, the structure revealed an N-terminal amphiphatic alpha-helix, and point mutants of invariant hydrophobic residues in this helix disrupt ELMO1-DOCK180 complex formation. A secondary interaction between ELMO1 and DOCK180 is conferred by the DOCK180 SH3 domain and proline-rich motifs at the ELMO1 C-terminus. Mutation of both DOCK180-interaction sites on ELMO1 is required to disrupt the DOCK180-ELMO1 complex. Significantly, although this does not affect DOCK180 GEF activity toward Rac in vivo, Rac signaling is impaired, implying additional roles for ELMO in mediating intracellular Rac signaling.

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An alpha-helical extension of the ELMO1 pleckstrin homology domain mediates direct interaction to DOCK180 and is critical in Rac signaling.

An Evolutionarily Conserved Autoinhibitory Molecular Switch in ELMO Proteins Regulates Rac Signaling

ELMO Recruits Actin Cross-linking Family 7 (ACF7) at the Cell Membrane for Microtubule Capture and Stabilization of Cellular Protrusions

*lnstitut de Recherches Cliniques de Montreal, Montreal, QC, Canada H2W 1R7; +Departement de Medecine, Universite de Montreal, Montreal, QC, Canada H3C 3J7; *Centre de Recherche en Biochimie Macromoleculaire, Centre National de la Recherche Scientifique, 34293 Montpellier, France; ?Memorial Sloan Kettering Cancer Center, New York, NY 10065; 'Burnham Institute for Medical Research, La Jolla, CA 92037; and **Division of Experimental Medicine, McGill University, Montreal, QC, Canada H3A 1A3

Nadine Fradet

Papers

The atypical Rac activator Dock180 (Dock1) regulates myoblast fusion in vivo

An alpha-helical extension of the ELMO1 pleckstrin homology domain mediates direct interaction to DOCK180 and is critical in Rac signaling.

An Evolutionarily Conserved Autoinhibitory Molecular Switch in ELMO Proteins Regulates Rac Signaling

ELMO Recruits Actin Cross-linking Family 7 (ACF7) at the Cell Membrane for Microtubule Capture and Stabilization of Cellular Protrusions

The atypical Rac activator Dock 180 (Dockl) regulates myoblast fusion in vivo