Dual-Color Live Imaging of Adult Muscle Stem Cells in the Embryonic Tissues of Drosophila melanogaster.

Adult muscle stem cells (MuSCs) show remarkable capability in repairing injured tissues. Studying MuSCs in suitable model organisms, which show strong homology with vertebrate counterparts, helps in dissecting the mechanisms regulating their behavior. Additionally, ease of handling and access to technological tools make model organisms well suited for studying biological processes that are conserved across species. MuSCs quiescence, proliferation, and migration are regulated by various input of signals from the surrounding tissues that constitute the MuSCs niche. Observing MuSCs along with their niche in vivo through live imaging provides key information on how MuSCs behave in quiescent and activated states. Drosophila melanogaster is well known for its genetic tool arsenal and the similarity of its different biological processes with vertebrates. Hence, it is widely used to study different types of stem cells. Gained knowledge could then be extrapolated to the vertebrate/mammalian homologous systems to enhance our knowledge in stem cell fields. In this protocol, we discuss how to perform live cell imaging of Drosophila MuSCs, called adult muscle precursors (AMPs) at embryonic stages, using dual-color labelling to visualize both AMPs and the surrounding tissues. This dual-color fluorescent labelling enables the observation of in vivo behavior of two types of cells simultaneously and provides key information on their interactions. The originality of this protocol resides in its biological application to MuSCs and their niche.

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.

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.

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.

Straightjacket/α2δ3 deregulation is associated with cardiac conduction defects in myotonic dystrophy type 1.

Cardiac conduction defects decrease life expectancy in myotonic dystrophy type 1 (DM1), a CTG repeat disorder involving misbalance between two RNA binding factors, MBNL1 and CELF1. However, how DM1 condition translates into conduction disorders remains poorly understood. Here we simulated MBNL1 and CELF1 misbalance in the Drosophila heart and performed TU-tagging-based RNAseq of cardiac cells. We detected deregulations of several genes controlling cellular calcium levels, including increased expression of straightjacket/α2δ3, which encodes a regulatory subunit of a voltage-gated calcium channel. Straightjacket overexpression in the fly heart leads to asynchronous heartbeat, a hallmark of abnormal conduction, whereas cardiac straightjacket knockdown improves these symptoms in DM1 fly models. We also show that ventricular α2δ3 expression is low in healthy mice and humans, but significantly elevated in ventricular muscles from DM1 patients with conduction defects. These findings suggest that reducing ventricular straightjacket/α2δ3 levels could offer a strategy to prevent conduction defects in DM1.

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.

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.

Characterization of Drosophila Muscle Stem Cell-Like Adult Muscle Precursors.

Uncovering how muscle stem cells behave in quiescent and activated states is central to understand the basic rules governing normal muscle development and regeneration in pathological conditions. Specification of mesodermal lineages including muscle stemlike adult muscle precursors (AMPs) has been extensively studied in Drosophila providing an attractive framework for investigating muscle stem cell properties. Restricted number of AMP cells, relative ease in following their behavior, and large number of genetic tools available make fruit fly an attractive model system for studying muscle stem cells. In this chapter, we describe the recently developed tools to visualize and target the body wall and imaginal AMPs.

Muscle niche-driven Insulin-Notch-Myc cascade reactivates dormant Adult Muscle Precursors in Drosophila.

How stem cells specified during development keep their non-differentiated quiescent state, and how they are reactivated, remain poorly understood. Here, we applied a Drosophila model to follow in vivo behavior of adult muscle precursors (AMPs), the transient fruit fly muscle stem cells. We report that emerging AMPs send out thin filopodia that make contact with neighboring muscles. AMPs keep their filopodia-based association with muscles throughout their dormant state but also when they start to proliferate, suggesting that muscles could play a role in AMP reactivation. Indeed, our genetic analyses indicate that muscles send inductive dIlp6 signals that switch the Insulin pathway ON in closely associated AMPs. This leads to the activation of Notch, which regulates AMP proliferation via dMyc. Altogether, we report that Drosophila AMPs display homing behavior to muscle niche and that the niche-driven Insulin-Notch-dMyc cascade plays a key role in setting the activated state of AMPs.

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.

The omega-3 fatty acid docosahexaenoic acid favorably modulates the inflammatory pathways and macrophage polarization within aorta of LDLR(-/-) mice.

The omega-3 fatty acid docosahexaenoic acid (DHA) has potent anti-atherogenic properties but its mechanisms of action at the vascular level remain poorly explored. Knowing the broad range of molecular targets of omega-3 fatty acids, microarray analysis was used to open-mindedly evaluate the effects of DHA on aorta gene expression in LDLR(-/-) mice and better understand its local anti-atherogenic action. Mice were fed an atherogenic diet and received daily oral gavages with oils rich in oleic acid or DHA. Bioinformatics analysis of microarray data first identified inflammation and innate immunity as processes the most affected by DHA supplementation within aorta. More precisely, several down-regulated genes were associated with the inflammatory functions of macrophages (e.g., CCL5 and CCR7), cell movement (e.g., ICAM-2, SELP, and PECAM-1), and the major histocompatibility complex (e.g., HLA-DQA1 and HLA-DRB1). Interestingly, several genes were identified as specific biomarkers of macrophage polarization, and their changes suggested a preferential orientation toward a M2 reparative phenotype. This observation was supported by the upstream regulator analysis highlighting the involvement of three main regulators of macrophage polarization, namely PPARγ (z-score = 2.367, p = 1.50 × 10(-13)), INFγ (z-score = -2.797, p = 2.81 × 10(-14)), and NFκB (z-score = 2.360, p = 6.32 × 10(-9)). Moreover, immunohistological analysis of aortic root revealed an increased abundance of Arg1 (+111 %, p = 0.01), a specific biomarker of M2 macrophage. The present study showed for the first time that DHA supplementation during atherogenesis is associated with protective modulation of inflammation and innate immunity pathways within aorta putatively through the orientation of plaque macrophages toward a M2 reparative phenotype.

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.

Neprilysin 4, a novel endopeptidase from Drosophila melanogaster, displays distinct substrate specificities and exceptional solubility states.

Proteins belonging to the family of neprilysins are typically membrane bound M13 endopeptidases responsible for the inactivation and/or activation of peptide signaling events on cell surfaces. Mammalian neprilysins are known to be involved in the metabolism of various regulatory peptides especially in the nervous, immune, cardiovascular and inflammatory systems. Although there is still much to learn about their participation in various diseases, they are potential therapeutic targets. Here we report on the identification and first characterization of neprilysin 4 (NEP4) from Drosophila melanogaster. Reporter lines as well as in situ hybridization combined with immunolocalization demonstrated NEP4 expression during embryogenesis in pericardial cells, muscle founder cells, glia cells and male gonads. Western blot analysis confirmed the prediction of one membrane bound and one soluble isoform, a finding quite unusual among neprilysins with presumably strong physiological relevance. At least one NEP4 isoform was found in every developmental stage indicating protein activities required throughout the whole life cycle of Drosophila. Heterologously expressed NEP4 exhibited substrate preferences comparable to human neprilysin 2 with distinct cleavage of substance P and angiotensin I.

Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation.

Tubulogenesis is an essential component of organ development, yet the underlying cellular mechanisms are poorly understood. We analyze here the formation of the Drosophila melanogaster cardiac lumen that arises from the migration and subsequent coalescence of bilateral rows of cardioblasts. Our study of cell behavior using three-dimensional and time-lapse imaging and the distribution of cell polarity markers reveals a new mechanism of tubulogenesis in which repulsion of prepatterned luminal domains with basal membrane properties and cell shape remodeling constitute the main driving forces. Furthermore, we identify a genetic pathway in which roundabout, slit, held out wings, and dystroglycan control cardiac lumen formation by establishing nonadherent luminal membranes and regulating cell shape changes. From these data we propose a model for D. melanogaster cardiac lumen formation, which differs, both at a cellular and molecular level, from current models of epithelial tubulogenesis. We suggest that this new example of tube formation may be helpful in studying vertebrate heart tube formation and primary vasculogenesis.

Cellular components and signals required for the cardiac outflow tract assembly in Drosophila.

Specification of cardiac primordia and formation of the Drosophila heart tube is highly reminiscent of the early steps of vertebrate heart development. We previously reported that the final morphogenesis of the Drosophila heart involves a group of nonmesodermal cells called heart-anchoring cells and a pair of derived from the pharyngeal mesoderm cardiac outflow muscles. Like the vertebrate cardiac neural crest cells, heart-anchoring cells migrate, interact with the tip of the heart, and participate in shaping the cardiac outflow tract. To better understand this process, we performed an in-depth analysis of how the Drosophila outflow tract is formed. We found that the most anterior cardioblasts that form a central outflow tract component, the funnel-shaped heart tip, do not originate from the cardiac primordium. They are initially associated with the pharyngeal cardiac outflow muscles and join the anterior aorta during outflow tract assembly. The particular morphology of the heart tip is disrupted in embryos in which heart-anchoring cells were ablated, revealing their critical role in outflow tract morphogenesis. We also demonstrate that Slit and Robo are required for directed movements of heart-anchoring cells toward the heart tip and that the cell-cell contact between the heart-anchoring cells and the ladybird-expressing cardioblasts is critically dependent on DE-cadherin Shotgun. Our observations suggest that the similarities between Drosophila and vertebrate cardiogenesis extend beyond the early developmental events.

Localization of chicken muscle FBPase in cardiomyocyte nuclei.

Fructose 1,6-bisphosphatase (FBPase; EC 3.1.3.11) localization in cardiomyocyte nuclei has recently been investigated in mammals [FEBS Lett. 539 (2003) 51]. In this study, nuclear localization of FBPase in the cardiac muscle of the chicken was studied by immunohistochemistry and other methods. A result of the electron microscopic investigation was confirmed by immunoblotting analysis. Using MALDI Q-TOF mass spectrometry and Mascot program, the nuclear FBPase was identified as muscle chicken FBPase. FBPase activity in isolated cardiomyocyte nuclei was 5.9 mU/g. Nuclear FBPase was strongly inhibited by allosteric inhibitor AMP. I(0.5) for AMP was 0.16 microM and was the same as for the purified chicken muscle enzyme.

The interaction of FBPase with aldolase: a kinetic and fluorescence investigation on chicken muscle enzymes.

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) is strongly inhibited by AMP in vitro and, therefore, at physiological concentrations of substrate and AMP, FBPase should be completely inhibited. Desensitization of rabbit muscle FBPase against AMP inhibition was previously observed in the presence of rabbit muscle aldolase. In this study, we analysed the kinetics of an FBPase catalyzed reaction and interaction between chicken muscle FBPase and chicken muscle aldolase. The initial rate of FBPase reaction vs. substrate concentration shows a maximum activity at a concentration of 20 microM Fru-1,6P2 and then decreases. Assuming rapid equilibrium kinetics, the enzyme-catalyzed reaction was described by the substrate inhibition model, with Ks approximately 5 microM and Ksi approximately 39 microM and factor beta approximately 0.2, describing change in the rate constant (k) of product formation from the ES and ESSi complexes. Based on ultracentrifugation studies, aldolase and FBPase form a hetero-complex with approximately 1:1 stoichiometry with a dissociation constant (Kd) of 3.8 microM. The FBPase-aldolase interaction was confirmed via fluorescence investigation. The aldolase-FBPase interaction results in aldolase fluorescence quenching and its maximum emission spectrum shifting from 344 to 356 nm. The Kd of the FBPase-aldolase complex, determined on the basis of fluorescence changes, is 0.4 microM at 25 degrees C with almost 1:1 stoichiometry. This interaction increases the I(0.5) for the AMP inhibition of FBPase threefold, and slightly affects FBPase affinity to magnesium ions, increasing the Ka and Hill coefficient (n). No effect of aldolase on the FBPase pH optimum was observed. Thus, the decrease in FBPase sensitivity to AMP inhibition enables FBPase to function in vivo thanks to aldolase.