Deregulations of miR-1 and its target Multiplexin promote dilated cardiomyopathy associated with myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults. It is caused by the excessive expansion of noncoding CTG repeats, which when transcribed affects the functions of RNA-binding factors with adverse effects on alternative splicing, processing, and stability of a large set of muscular and cardiac transcripts. Among these effects, inefficient processing and down-regulation of muscle- and heart-specific miRNA, miR-1, have been reported in DM1 patients, but the impact of reduced miR-1 on DM1 pathogenesis has been unknown. Here, we use Drosophila DM1 models to explore the role of miR-1 in cardiac dysfunction in DM1. We show that miR-1 down-regulation in the heart leads to dilated cardiomyopathy (DCM), a DM1-associated phenotype. We combined in silico screening for miR-1 targets with transcriptional profiling of DM1 cardiac cells to identify miR-1 target genes with potential roles in DCM. We identify Multiplexin (Mp) as a new cardiac miR-1 target involved in DM1. Mp encodes a collagen protein involved in cardiac tube formation in Drosophila. Mp and its human ortholog Col15A1 are both highly enriched in cardiac cells of DCM-developing DM1 flies and in heart samples from DM1 patients with DCM, respectively. When overexpressed in the heart, Mp induces DCM, whereas its attenuation rescues the DCM phenotype of aged DM1 flies. Reduced levels of miR-1 and consecutive up-regulation of its target Mp/Col15A1 might be critical in DM1-associated DCM.

A polarized nucleus-cytoskeleton-ECM connection in migrating cardioblasts controls heart tube formation in Drosophila.

The formation of the cardiac tube is a remarkable example of complex morphogenetic processes conserved from invertebrates to humans. It involves coordinated collective migration of contralateral rows of cardiac cells. The molecular processes underlying the specification of cardioblasts (CBs) prior to migration are well established and significant advances have been made in understanding the process of lumen formation. However, the mechanisms of collective cardiac cells migration remain elusive. Here, we have identified CAP and MSP300 as novel actors involved during CB migration. They both exhibit highly similar temporal and spatial expression patterns in Drosophila migrating cardiac cells, and are necessary for the correct number and alignment of CBs, a prerequisite for the coordination of their collective migration. Our data suggest that CAP and MSP300 are part of a protein complex linking focal adhesion sites to nuclei via the actin cytoskeleton that maintains post-mitotic state and correct alignment of CBs.

Drosophila adult muscle precursor cells contribute to motor axon pathfinding and proper innervation of embryonic muscles.

Despites several decades of studies on the neuromuscular system, the relationship between muscle stem cells and motor neurons remains elusive. Using the Drosophila model, we provide evidence that adult muscle precursors (AMPs), the Drosophila muscle stem cells, interact with the motor axons during embryogenesis. AMPs not only hold the capacity to attract the navigating intersegmental (ISN) and segmental a (SNa) nerve branches, but are also mandatory to the innervation of muscles in the lateral field. This so-far-ignored AMP role involves their filopodia-based interactions with nerve growth cones. In parallel, we report the previously undetected expression of the guidance molecule-encoding genes sidestep and side IV in AMPs. Altogether, our data support the view that Drosophila muscle stem cells represent spatial landmarks for navigating motor neurons and reveal that their positioning is crucial for the muscles innervation in the lateral region. Furthermore, AMPs and motor axons are interdependent, as the genetic ablation of SNa leads to a specific loss of SNa-associated lateral AMPs.

Diversification of muscle types in Drosophila embryos.

Drosophila embryonic somatic muscles represent a simple and tractable model system to study the gene regulatory networks that control diversification of cell types. Somatic myogenesis in Drosophila is initiated by intrinsic action of the mesodermal master gene twist, which activates a cascade of transcriptional outputs including myogenic differentiation factor Mef2, which triggers all aspects of the myogenic differentiation program. In parallel, the expression of a combinatorial code of identity transcription factors (iTFs) defines discrete particular features of each muscle fiber, such as number of fusion events, and specific attachment to tendon cells or innervation, thus ensuring diversification of muscle types. Here, we take the example of a subset of lateral transverse (LT) muscles and discuss how the iTF code and downstream effector genes progressively define individual LT properties such as fusion program, attachment and innervation. We discuss new challenges in the field including the contribution of posttranscriptional and epitranscriptomic regulation of gene expression in the diversification of cell types.

The relationship between muscle stem cells and motor neurons.

Neuromuscular system is constituted of multi-fibrillar muscles, tendons, motor neurons and associated muscle stem cells. Stereotyped pattern of muscle innervation and muscle-specific interactions with tendon cells suggest that neuromuscular system develops in a coordinated way. Remarkably, upon regeneration, coordinated assembly of all neuromuscular components is also critical to rebuild functional muscle. Thus, to ensure muscle function, the neuromuscular system components need to interact both during development and regeneration. Over the last decades, interactions between muscles and tendons, muscles and motor neurons and between muscles and muscle stem cells have been extensively analysed and documented. However, only recent evidence indicates that muscle stem cells interact with motor neurons and that these interactions contribute to building functional muscle both during development and regeneration. From this perspective, we discuss here the relationship between muscle stem cells and motor neurons during Drosophila neuromuscular system development and adverse impact of affected muscle stem cell-motor neuron interactions in regenerating vertebrate muscle.

Distinct subsets of Eve-positive pericardial cells stabilise cardiac outflow and contribute to Hox gene-triggered heart morphogenesis in Drosophila.

The Drosophila heart, composed of discrete subsets of cardioblasts and pericardial cells, undergoes Hox-triggered anterior-posterior morphogenesis, leading to a functional subdivision into heart proper and aorta, with its most anterior part forming a funnel-shaped cardiac outflow. Cardioblasts differentiate into Tin-positive 'working myocytes' and Svp-expressing ostial cells. However, developmental fates and functions of heart-associated pericardial cells remain elusive. Here, we show that the pericardial cells that express the transcription factor Even Skipped adopt distinct fates along the anterior-posterior axis. Among them, the most anterior Antp-Ubx-AbdA-negative cells form a novel cardiac outflow component we call the outflow hanging structure, whereas the Antp-expressing cells differentiate into wing heart precursors. Interestingly, Hox gene expression in the Even Skipped-positive cells not only underlies their antero-posterior diversification, but also influences heart morphogenesis in a non-cell-autonomous way. In brief, we identify a new cardiac outflow component derived from a subset of Even Skipped-expressing cells that stabilises the anterior heart tip, and demonstrate non-cell-autonomous effects of Hox gene expression in the Even Skipped-positive cells on heart morphogenesis.

Zebrafish as a Model for the Study of Lipid-Lowering Drug-Induced Myopathies.

Drug-induced myopathies are classified as acquired myopathies caused by exogenous factors. These pathological conditions develop in patients without muscle disease and are triggered by a variety of medicaments, including lipid-lowering drugs (LLDs) such as statins, fibrates, and ezetimibe. Here we summarise the current knowledge gained via studies conducted using various models, such as cell lines and mammalian models, and compare them with the results obtained in zebrafish (Danio rerio) studies. Zebrafish have proven to be an excellent research tool for studying dyslipidaemias as a model of these pathological conditions. This system enables in-vivo characterization of drug and gene candidates to further the understanding of disease aetiology and develop new therapeutic strategies. Our review also considers important environmental issues arising from the indiscriminate use of LLDs worldwide. The widespread use and importance of drugs such as statins and fibrates justify the need for the meticulous study of their mechanism of action and the side effects they cause.

Insulin-dependent Non-canonical Activation of Notch in Drosophila: A Story of Notch-Induced Muscle Stem Cell Proliferation.

Notch plays multiple roles both in development and in adult tissue homeostasis. Notch was first identified in Drosophila in which it has then been extensively studied. Among the flag-ship Notch functions we could mention its capacity to keep precursor and stem cells in a nondifferentiated state but also its ability to activate cell proliferation that in some contexts could led to cancer. In general, both these functions involve, canonical, ligand-dependent Notch activation. However, a ligand-independent Notch activation has also been described in a few cellular contexts. Here, we focus on one of such contexts, Drosophila muscle stem cells, called AMPs, and discuss how insulin-dependent noncanonical activation of Notch pushes quiescent AMPs to proliferation.

Beyond mice: Emerging and transdisciplinary models for the study of early-onset myopathies.

The use of the adapted models to decipher patho-physiological mechanisms of human diseases is always a great challenge. This is of particular importance for early-onset myopathies, in which pathological mutations often impact not only on muscle structure and function but also on developmental processes. Mice are currently the main animal model used to study neuromuscular disorders including the early-onset myopathies. However strategies based on simple animal models and on transdisciplinary approaches exploring mechanical muscle cell properties emerge as attractive, non-exclusive alternatives. These new ways provide valuable opportunities to improve our knowledge on how mechanical, biochemical, and genetic/epigenetic cues modulate the formation, organization and function of muscle tissues. Here we provide an overview of how single cell and micro-tissue engineering in parallel to non-mammalian, Drosophila and zebrafish models could contribute to filling gaps in our understanding of pathogenic mechanisms underlying early-onset myopathies. We also discuss their potential impact on designing new diagnostic and therapeutic strategies.

Drosophila Hsp67Bc hot-spot variants alter muscle structure and function.

The Drosophila Hsp67Bc gene encodes a protein belonging to the small heat-shock protein (sHSP) family, identified as the nearest functional ortholog of human HSPB8. The most prominent activity of sHSPs is preventing the irreversible aggregation of various non-native polypeptides. Moreover, they are involved in processes such as development, aging, maintenance of the cytoskeletal architecture and autophagy. In larval muscles Hsp67Bc localizes to the Z- and A-bands, which suggests its role as part of the conserved chaperone complex required for Z-disk maintenance. In addition, Hsp67Bc is present at neuromuscular junctions (NMJs), which implies its involvement in the maintenance of NMJ structure. Here, we report the effects of muscle-target overexpression of Drosophila Hsp67Bc hot-spot variants Hsp67BcR126E and Hsp67BcR126N mimicking pathogenic variants of human HSPB8. Depending on the substitutions, we observed a different impact on muscle structure and performance. Expression of Hsp67BcR126E affects larval motility, which may be caused by impairment of mitochondrial respiratory function and/or by NMJ abnormalities manifested by a decrease in the number of synaptic boutons. In contrast, Hsp67BcR126N appears to be an aggregate-prone variant, as reflected in excessive accumulation of mutant proteins and the formation of large aggregates with a lesser impact on muscle structure and performance compared to the Hsp67BcR126E variant.

Drosophila small heat shock protein CryAB ensures structural integrity of developing muscles, and proper muscle and heart performance.

Molecular chaperones, such as the small heat shock proteins (sHsps), maintain normal cellular function by controlling protein homeostasis in stress conditions. However, sHsps are not only activated in response to environmental insults, but also exert developmental and tissue-specific functions that are much less known. Here, we show that during normal development the Drosophila sHsp CryAB [L(2)efl] is specifically expressed in larval body wall muscles and accumulates at the level of Z-bands and around myonuclei. CryAB features a conserved actin-binding domain and, when attenuated, leads to clustering of myonuclei and an altered pattern of sarcomeric actin and the Z-band-associated actin crosslinker Cheerio (filamin). Our data suggest that CryAB and Cheerio form a complex essential for muscle integrity: CryAB colocalizes with Cheerio and, as revealed by mass spectrometry and co-immunoprecipitation experiments, binds to Cheerio, and the muscle-specific attenuation of cheerio leads to CryAB-like sarcomeric phenotypes. Furthermore, muscle-targeted expression of CryAB(R120G), which carries a mutation associated with desmin-related myopathy (DRM), results in an altered sarcomeric actin pattern, in affected myofibrillar integrity and in Z-band breaks, leading to reduced muscle performance and to marked cardiac arrhythmia. Taken together, we demonstrate that CryAB ensures myofibrillar integrity in Drosophila muscles during development and propose that it does so by interacting with the actin crosslinker Cheerio. The evidence that a DRM-causing mutation affects CryAB muscle function and leads to DRM-like phenotypes in the fly reveals a conserved stress-independent role of CryAB in maintaining muscle cell cytoarchitecture.

Dissecting Pathogenetic Mechanisms and Therapeutic Strategies in Drosophila Models of Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1), the most common cause of adult-onset muscular dystrophy, is autosomal dominant, multisystemic disease with characteristic symptoms including myotonia, heart defects, cataracts and testicular atrophy. DM1 disease is being successfully modelled in Drosophila allowing to identify and validate new pathogenic mechanisms and potential therapeutic strategies. Here we provide an overview of insights gained from fruit fly DM1 models, either: (i) fundamental with particular focus on newly identified gene deregulations and their link with DM1 symptoms; or (ii) applied via genetic modifiers and drug screens to identify promising therapeutic targets.

Developmental Expression and Functions of the Small Heat Shock Proteins in Drosophila.

Heat shock proteins (Hsps) form a large family of evolutionarily conserved molecular chaperones that help balance protein folding and protect cells from various stress conditions. However, there is growing evidence that Hsps may also play an active role in developmental processes. Here, we take the example of developmental expression and function of one class of Hsps characterized by low molecular weight, the small Hsps (sHsps). We discuss recent reports and genome-wide datasets that support vital sHsps functions in the developing nervous system, reproductive system, and muscles. This tissue- and time-specific sHsp expression is developmentally regulated, so that the enhancer sequence of an sHsp gene expressed in developing muscle, in addition to stress-inducible elements, also carries binding sites for myogenic regulatory factors. One possible reason for sHsp genes to switch on during development and in non-stress conditions is to protect vital developing organs from environmental insults.

Glycolysis supports embryonic muscle growth by promoting myoblast fusion.

Muscles ensure locomotion behavior of invertebrate and vertebrate organisms. They are highly specialized and form using conserved developmental programs. To identify new players in muscle development we screened Drosophila and zebrafish gene expression databases for orthologous genes expressed in embryonic muscles. We selected more than 100 candidates. Among them is the glycolysis gene Pglym78/pgam2, the attenuated expression of which results in the formation of thinner muscles in Drosophila embryos. This phenotype is also observed in fast muscle fibers of pgam2 zebrafish morphants, suggesting affected myoblast fusion. Indeed, a detailed analysis of developing muscles in Pglym78 RNAi embryos reveals loss of fusion-associated actin foci and an inefficient Notch decay in fusion competent myoblasts, both known to be required for fusion. In addition to Pglym78, our screen identifies six other genes involved in glycolysis or in pyruvate metabolism (Pfk, Tpi, Gapdh, Pgk, Pyk, and Impl3). They are synchronously activated in embryonic muscles and attenuation of their expression leads to similar muscle phenotypes, which are characterized by fibers with reduced size and the presence of unfused myoblasts. Our data also show that the cell size triggering insulin pathway positively regulates glycolysis in developing muscles and that blocking the insulin or target of rapamycin pathways phenocopies the loss of function phenotypes of glycolytic genes, leading to myoblast fusion arrest and reduced muscle size. Collectively, these data suggest that setting metabolism to glycolysis-stimulated biomass production is part of a core myogenic program that operates in both invertebrate and vertebrate embryos and promotes formation of syncytial muscles.

Zebrafish: A Model for the Study of Toxicants Affecting Muscle Development and Function.

The rapid progress in medicine, agriculture, and allied sciences has enabled the development of a large amount of potentially useful bioactive compounds, such as drugs and pesticides. However, there is another side of this phenomenon, which includes side effects and environmental pollution. To avoid or minimize the uncontrollable consequences of using the newly developed compounds, researchers seek a quick and effective means of their evaluation. In achieving this goal, the zebrafish (Danio rerio) has proven to be a highly useful tool, mostly because of its fast growth and development, as well as the ability to absorb the molecules diluted in water through its skin and gills. In this review, we focus on the reports concerning the application of zebrafish as a model for assessing the impact of toxicants on skeletal muscles, which share many structural and functional similarities among vertebrates, including zebrafish and humans.

Novel Drosophila model of myotonic dystrophy type 1: phenotypic characterization and genome-wide view of altered gene expression.

Myotonic dystrophy type 1 (DM1) is a multisystemic RNA-dominant disorder characterized by myotonia and muscle degeneration. In DM1 patients, the mutant DMPK transcripts containing expanded CUG repeats form nuclear foci and sequester the Muscleblind-like 1 splicing factor, resulting in mis-splicing of its targets. However, several pathological defects observed in DM1 and their link with disease progression remain poorly understood. In an attempt to fill this gap, we generated inducible transgenic Drosophila lines with increasing number of CTG repeats. Targeting the expression of these repeats to the larval muscles recapitulated in a repeat-size-dependent manner the major DM1 symptoms such as muscle hypercontraction, splitting of muscle fibers, reduced fiber size or myoblast fusion defects. Comparative transcriptional profiling performed on the generated DM1 lines and on the muscleblind (mbl)-RNAi line revealed that nuclear accumulation of toxic CUG repeats can affect gene expression independently of splicing or Mbl sequestration. Also, in mblRNAi contexts, the largest portion of deregulated genes corresponded to single-transcript genes, revealing an unexpected impact of the indirect influence of mbl on gene expression. Among the single-transcript Mbl targets is Muscle protein 20 involved in myoblast fusion and causing the reduced number of nuclei in muscles of mblRNAi larvae. Finally, by combining in silico prediction of Mbl targets with mblRNAi microarray data, we found the calcium pump dSERCA as a Mbl splice target and show that the membrane dSERCA isoform is sufficient to rescue a DM1-induced hypercontraction phenotype in a Drosophila model.

Model organisms in the fight against muscular dystrophy: lessons from drosophila and Zebrafish.

Muscular dystrophies (MD) are a heterogeneous group of genetic disorders that cause muscle weakness, abnormal contractions and muscle wasting, often leading to premature death. More than 30 types of MD have been described so far; those most thoroughly studied are Duchenne muscular dystrophy (DMD), myotonic dystrophy type 1 (DM1) and congenital MDs. Structurally, physiologically and biochemically, MDs affect different types of muscles and cause individual symptoms such that genetic and molecular pathways underlying their pathogenesis thus remain poorly understood. To improve our knowledge of how MD-caused muscle defects arise and to find efficacious therapeutic treatments, different animal models have been generated and applied. Among these, simple non-mammalian Drosophila and zebrafish models have proved most useful. This review discusses how zebrafish and Drosophila MD have helped to identify genetic determinants of MDs and design innovative therapeutic strategies with a special focus on DMD, DM1 and congenital MDs.

Drosophila adult muscle precursors form a network of interconnected cells and are specified by the rhomboid-triggered EGF pathway.

In Drosophila, a population of muscle-committed stem-like cells called adult muscle precursors (AMPs) keeps an undifferentiated and quiescent state during embryonic life. The embryonic AMPs are at the origin of all adult fly muscles and, as we demonstrate here, they express repressors of myogenic differentiation and targets of the Notch pathway known to be involved in muscle cell stemness. By targeting GFP to the AMP cell membranes, we show that AMPs are tightly associated with the peripheral nervous system and with a subset of differentiated muscles. They send long cellular processes running along the peripheral nerves and, by the end of embryogenesis, form a network of interconnected cells. Based on evidence from laser ablation experiments, the main role of these cellular extensions is to maintain correct spatial positioning of AMPs. To gain insights into mechanisms that lead to AMP cell specification, we performed a gain-of-function screen with a special focus on lateral AMPs expressing the homeobox gene ladybird. Our data show that the rhomboid-triggered EGF signalling pathway controls both the specification and the subsequent maintenance of AMP cells. This finding is supported by the identification of EGF-secreting cells in the lateral domain and the EGF-dependent regulatory modules that drive expression of the ladybird gene in lateral AMPs. Taken together, our results reveal an unsuspected capacity of embryonic AMPs to form a cell network, and shed light on the mechanisms governing their specification and maintenance.

Tailup plays multiple roles during cardiac outflow assembly in Drosophila.

The Drosophila LIM-homeodomain transcription factor Tailup and its vertebrate counterpart Islet1 are expressed in cardiac progenitor cells where they play a specification role. Loss of function of Islet1 leads to a complete absence of the right ventricle and affects the development of the cardiac outflow tract in mouse embryos. Similarly, tailup mutant embryos display a reduced number of cardiac cells but the role of tailup in cardiac outflow formation in Drosophila remains unknown. Here, we show that tailup is expressed in the main Drosophila cardiac outflow components, i.e., heart anchoring cells (HANC) and cardiac outflow muscles (COM) and that loss of its function and/or tissue-specific knockdowns dramatically affect cardiac outflow morphogenesis. Our data demonstrate that tailup plays many roles and is required for the acquisition of HANC and COM properties. We also show that tailup regulates HANC motility, COM shapes and their attachment to the heart tip and genetically interacts with ladybird, shotgun and slit, which are known to be involved in cardiac outflow assembly. Furthemore, using tissue-specific overexpression of dominant negative tailup constructs lacking sequences encoding either the homeodomain or the LIM domain, we demonstrate that tailup can exert its function not only in transcription factor mode but also via its protein-protein interaction domain. We identify Tailup as an evolutionarily-conserved regulator of cardiac outflow formation and provide further evidence for its conserved role in heart development.

Developmental origin of tendon diversity in Drosophila melanogaster.

Myogenesis is a developmental process that is largely conserved in both Drosophila and higher organisms. Consequently, the fruit fly is an excellent in vivo model for identifying the genes and mechanisms involved in muscle development. Moreover, there is growing evidence indicating that specific conserved genes and signaling pathways govern the formation of tissues that connect the muscles to the skeleton. In this review, we present an overview of the different stages of tendon development, from the specification of tendon progenitors to the assembly of a stable myotendinous junction across three different myogenic contexts in Drosophila: larval, flight and leg muscle development. We underline the different aspects of tendon cell specification and differentiation in embryo and during metamorphosis that result into tendon morphological and functional diversity.