Postsynaptic BMP signaling regulates myonuclear properties in Drosophila larval muscles.

The syncytial mammalian muscle fiber contains a heterogeneous population of (myo)nuclei. At the neuromuscular junction (NMJ), myonuclei have specialized positioning and gene expression. However, it remains unclear how myonuclei are recruited and what regulates myonuclear output at the NMJ. Here, we identify specific properties of myonuclei located near the Drosophila larval NMJ. These synaptic myonuclei have increased size in relation to their surrounding cytoplasmic domain (scaling), increased DNA content (ploidy), and increased levels of transcription factor pMad, a readout for BMP signaling activity. Our genetic manipulations show local BMP signaling affects muscle size, nuclear size, ploidy, and NMJ size and function. In support, RNA sequencing analysis reveals that pMad regulates genes involved in muscle growth, ploidy (i.e., E2f1), and neurotransmission. Our data suggest that muscle BMP signaling instructs synaptic myonuclear output that then positively shapes the NMJ synapse. This study deepens our understanding of how myonuclear heterogeneity supports local signaling demands to fine tune cellular function and NMJ activity.

Drosophila Tropomodulin is required for multiple actin-dependent processes within developing myofibers.

Proper muscle contraction requires the assembly and maintenance of sarcomeres and myofibrils. Although the protein components of myofibrils are generally known, less is known about the mechanisms by which they individually function and together synergize for myofibril assembly and maintenance. For example, it is unclear how the disruption of actin filament (F-actin) regulatory proteins leads to the muscle weakness observed in myopathies. Here, we show that knockdown of Drosophila Tropomodulin (Tmod), results in several myopathy-related phenotypes, including reduction of muscle cell (myofiber) size, increased sarcomere length, disorganization and misorientation of myofibrils, ectopic F-actin accumulation, loss of tension-mediating proteins at the myotendinous junction, and misshaped and internalized nuclei. Our findings support and extend the tension-driven self-organizing myofibrillogenesis model. We show that, like its mammalian counterpart, Drosophila Tmod caps F-actin pointed-ends, and we propose that this activity is crucial for cellular processes in different locations within the myofiber that directly and indirectly contribute to the maintenance of muscle function. Our findings provide significant insights to the role of Tmod in muscle development, maintenance and disease.

Cofilin Loss in Drosophila Muscles Contributes to Muscle Weakness through Defective Sarcomerogenesis during Muscle Growth.

Sarcomeres, the fundamental contractile units of muscles, are conserved structures composed of actin thin filaments and myosin thick filaments. How sarcomeres are formed and maintained is not well understood. Here, we show that knockdown of Drosophila cofilin (DmCFL), an actin depolymerizing factor, disrupts both sarcomere structure and muscle function. The loss of DmCFL also results in the formation of sarcomeric protein aggregates and impairs sarcomere addition during growth. The activation of the proteasome delays muscle deterioration in our model. Furthermore, we investigate how a point mutation in CFL2 that causes nemaline myopathy (NM) in humans affects CFL function and leads to the muscle phenotypes observed in vivo. Our data provide significant insights to the role of CFLs during sarcomere formation, as well as mechanistic implications for disease progression in NM patients.

Nuclear Scaling Is Coordinated among Individual Nuclei in Multinucleated Muscle Fibers.

Optimal cell performance depends on cell size and the appropriate relative size, i.e., scaling, of the nucleus. How nuclear scaling is regulated and contributes to cell function is poorly understood, especially in skeletal muscle fibers, which are among the largest cells, containing hundreds of nuclei. Here, we present a Drosophila in vivo system to analyze nuclear scaling in whole multinucleated muscle fibers, genetically manipulate individual components, and assess muscle function. Despite precise global coordination, we find that individual nuclei within a myofiber establish different local scaling relationships by adjusting their size and synthetic activity in correlation with positional or spatial cues. While myonuclei exhibit compensatory potential, even minor changes in global nuclear size scaling correlate with reduced muscle function. Our study provides the first comprehensive approach to unraveling the intrinsic regulation of size in multinucleated muscle fibers. These insights to muscle cell biology will accelerate the development of interventions for muscle diseases.

Tbx6, Mesp-b and Ripply1 regulate the onset of skeletal myogenesis in zebrafish.

During embryonic development, the paraxial mesoderm becomes segmented into somites, within which proliferative muscle progenitors and muscle fibers establish the skeletal musculature. Here, we demonstrate that a gene network previously implicated in somite boundary formation, involving the transcriptional regulators Tbx6, Mesp-b and Ripply1, also confers spatial and temporal regulation to skeletal myogenesis in zebrafish. We show that Tbx6 directly regulates mesp-b and ripply1 expression in vivo, and that the interactions within the regulatory network are largely conserved among vertebrates. Mesp-b is necessary and sufficient for the specification of a subpopulation of muscle progenitors, the central proportion of the Pax3(+)/Pax7(+) dermomyotome. Conditional ubiquitous expression indicates that Mesp-b acts by inhibiting myogenic differentiation and by inducing the dermomyotome marker meox1. By contrast, Ripply1 induces a negative-feedback loop by promoting Tbx6 protein degradation. Persistent Tbx6 expression in Ripply1 knockdown embryos correlates with a deficit in dermomyotome and myotome marker gene expression, suggesting that Ripply1 promotes myogenesis by terminating Tbx6-dependent inhibition of myogenic maturation. Together, our data suggest that Mesp-b is an intrinsic upstream regulator of skeletal muscle progenitors and that, in zebrafish, the genes regulating somite boundary formation also regulate the development of the dermomyotome in the anterior somite compartment.

Immunocytochemistry to study myogenesis in zebrafish.

During myogenesis, cells gradually transition from mesodermal precursors to myoblasts, myocytes, and then to muscle fibers. The molecular characterization of this process requires the ability to identify each of these cell types and the factors that regulate the transitions between them. The most versatile technique for assaying cell identities in situ is immunocytochemistry, because multiple independent molecular markers of differentiation can be assayed simultaneously. The zebrafish has developed into a popular model for the study of myogenesis, and immunocytochemical techniques have been critical. We have adapted existing protocols to optimize immunocytochemistry in zebrafish, and have tested many antibodies developed against mouse, chick, and frog muscle antigens for their cross-reactivity in zebrafish. Here, we present protocols for whole mount immunocytochemistry on both formaldehyde and Carnoy's fixed embryos as well as on sectioned zebrafish tissue. We include a table of antibodies useful for experiments on the molecular biology of myogenesis in zebrafish.

Distinct modes of vertebrate hypaxial muscle formation contribute to the teleost body wall musculature.

The formation of the body wall musculature in vertebrates is assumed to be initiated by direct ventral extension of the somites/myotomes. This contrasts to the formation of limb muscles and muscles involved in feeding or respiration/ventilation, which are founded by migratory muscle precursors (MMPs) distant to the somites. Here, we present evidence from morphology and expression of molecular markers proposing that the formation of the two muscle layers of the teleost body wall involves both of the above mechanisms: (1) MMPs from somites 5 and 6 found an independent muscle primordium-the so-called posterior hypaxial muscle (PHM)-which subsequently gives rise to the most anterior two segments of the medial obliquus inferioris (OI) muscle. (2) Direct epithelial extension of the hypaxial myotomes generates the OI segments from somite 7 caudalward and the entire lateral obliquus superioris (OS) muscle. The findings are discussed in relation to the evolution of hypaxial myogenic patterning including functional considerations. We hypothesise that the potential of the most anterior somites to generate migratory muscle precursors is a general vertebrate feature that has been differently utilised in the evolution in vertebrate groups.