Antagonism Between DUX4 and DUX4c Highlights a Pathomechanism Operating Through ß-Catenin in Facioscapulohumeral Muscular Dystrophy.

Aberrant expression of the transcription factor DUX4 from D4Z4 macrosatellite repeats on chromosome 4q35, and its transcriptome, associate with pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). Forced DUX4 expression halts skeletal muscle cell proliferation and induces cell death. DUX4 binds DNA via two homeodomains that are identical in sequence to those of DUX4c (DUX4L9): a closely related transcriptional regulator encoded by a single, inverted, mutated D4Z4 unit located centromeric to the D4Z4 macrosatellite array on chromosome 4. However, the function and contribution of DUX4c to FSHD pathogenesis are unclear. To explore interplay between DUX4, DUX4c, and the DUX4-induced phenotype, we investigated whether DUX4c interferes with DUX4 function in human myogenesis. Constitutive expression of DUX4c rescued the DUX4-induced inhibition of proliferation and reduced cell death in human myoblasts. Functionally, DUX4 promotes nuclear translocation of ß-CATENIN and increases canonical WNT signalling. Concomitant constitutive expression of DUX4c prevents ß-CATENIN nuclear accumulation and the downstream transcriptional program. DUX4 reduces endogenous DUX4c levels, whereas constitutive expression of DUX4c robustly suppresses expression of DUX4 target genes, suggesting molecular antagonism. In line, DUX4 expression in FSHD myoblasts correlates with reduced DUX4c levels. Addressing the mechanism, we identified a subset of genes involved in the WNT/ß-CATENIN pathway that are differentially regulated between DUX4 and DUX4c, whose expression pattern can separate muscle biopsies from severely affected FSHD patients from healthy. Finally, blockade of WNT/ß-CATENIN signalling rescues viability of FSHD myoblasts. Together, our study highlights an antagonistic interplay whereby DUX4 alters cell viability via ß-CATENIN signalling and DUX4c counteracts aspects of DUX4-mediated toxicity in human muscle cells, potentially acting as a gene modifier for FSHD severity. Importantly, direct DUX4 regulation of the WNT/ß-CATENIN pathway informs future therapeutic interventions to ameliorate FSHD pathology.

DVL1 and DVL3 require nuclear localisation to regulate proliferation in human myoblasts.

WNT signalling is essential for regulating a diverse range of cellular processes. In skeletal muscle, the WNT pathway plays crucial roles in maintenance of the stem cell pool and myogenic differentiation. Focus is usually directed at examining the function of central components of the WNT pathway, including ß-CATENIN and the GSK3ß complex and TCF/LEF transcription factors, in tissue homeostasis and cancer. Other core components of the WNT pathway though, are three dishevelled (DVL) proteins: membrane associated proteins that propagate WNT signalling from membrane to nucleus. Here we examined DVL function in human myogenesis and the muscle-related cancer alveolar rhabdomyosarcoma. We demonstrate that DVL1 and DVL3 are necessary for efficient proliferation in human myoblasts and are important for timely myogenic differentiation. DVL1 and DVL3 also contribute to regulation of proliferation in rhabdomyosarcoma. DVL1 or DVL3 must be present in the nucleus to regulate proliferation, but they operate through different protein domains: DVL3 requires the DIX and PDZ domains, while DVL1 does not. Importantly, DVL1 and DVL3 activity is independent of markedly increased translocation of ß-CATENIN to the nucleus, normally a hallmark of active canonical WNT signalling.

DEPDC1B is a key regulator of myoblast proliferation in mouse and man.

OBJECTIVES: DISHEVELLED, EGL-10, PLECKSTRIN (DEP) domain-containing 1B (DEPDC1B) promotes dismantling of focal adhesions and coordinates detachment events during cell cycle progression. DEPDC1B is overexpressed in several cancers with expression inversely correlated with patient survival. Here, we analysed the role of DEPDC1B in the regulation of murine and human skeletal myogenesis. MATERIALS AND METHODS: Expression dynamics of DEPDC1B were examined in murine and human myoblasts and rhabdomyosarcoma cells in vitro by RT-qPCR and/or immunolabelling. DEPDC1B function was mainly tested via siRNA-mediated gene knockdown. RESULTS: DEPDC1B was expressed in proliferating murine and human myoblasts, with expression then decreasing markedly during myogenic differentiation. SiRNA-mediated knockdown of DEPDC1B reduced myoblast proliferation and induced entry into myogenic differentiation, with deregulation of key cell cycle regulators (cyclins, CDK, CDKi). DEPDC1B and ß-catenin co-knockdown was unable to rescue proliferation in myoblasts, suggesting that DEPDC1B functions independently of canonical WNT signalling during myogenesis. DEPDC1B can also suppress RHOA activity in some cell types, but DEPDC1B and RHOA co-knockdown actually had an additive effect by both further reducing proliferation and enhancing myogenic differentiation. DEPDC1B was expressed in human Rh30 rhabdomyosarcoma cells, where DEPDC1B or RHOA knockdown promoted myogenic differentiation, but without influencing proliferation. CONCLUSION: DEPDC1B plays a central role in myoblasts by driving proliferation and preventing precocious myogenic differentiation during skeletal myogenesis in both mouse and human.

Impairments in contractility and cytoskeletal organisation cause nuclear defects in nemaline myopathy.

Nemaline myopathy (NM) is a skeletal muscle disorder caused by mutations in genes that are generally involved in muscle contraction, in particular those related to the structure and/or regulation of the thin filament. Many pathogenic aspects of this disease remain largely unclear. Here, we report novel pathological defects in skeletal muscle fibres of mouse models and patients with NM: irregular spacing and morphology of nuclei; disrupted nuclear envelope; altered chromatin arrangement; and disorganisation of the cortical cytoskeleton. Impairments in contractility are the primary cause of these nuclear defects. We also establish the role of microtubule organisation in determining nuclear morphology, a phenomenon which is likely to contribute to nuclear alterations in this disease. Our results overlap with findings in diseases caused directly by mutations in nuclear envelope or cytoskeletal proteins. Given the important role of nuclear shape and envelope in regulating gene expression, and the cytoskeleton in maintaining muscle fibre integrity, our findings are likely to explain some of the hallmarks of NM, including contractile filament disarray, altered mechanical properties and broad transcriptional alterations.

VGLL3 operates via TEAD1, TEAD3 and TEAD4 to influence myogenesis in skeletal muscle.

VGLL proteins are transcriptional co-factors that bind TEAD family transcription factors to regulate events ranging from wing development in fly, to muscle fibre composition and immune function in mice. Here, we characterise Vgll3 in skeletal muscle. We found that mouse Vgll3 was expressed at low levels in healthy muscle but that its levels increased during hypertrophy or regeneration; in humans, VGLL3 was highly expressed in tissues from patients with various muscle diseases, such as in dystrophic muscle and alveolar rhabdomyosarcoma. Interaction proteomics revealed that VGLL3 bound TEAD1, TEAD3 and TEAD4 in myoblasts and/or myotubes. However, there was no interaction with proteins from major regulatory systems such as the Hippo kinase cascade, unlike what is found for the TEAD co-factors YAP (encoded by YAP1) and TAZ (encoded by WWTR1). Vgll3 overexpression reduced the activity of the Hippo negative-feedback loop, affecting expression of muscle-regulating genes including Myf5, Pitx2 and Pitx3, and genes encoding certain Wnts and IGFBPs. VGLL3 mainly repressed gene expression, regulating similar genes to those regulated by YAP and TAZ. siRNA-mediated Vgll3 knockdown suppressed myoblast proliferation, whereas Vgll3 overexpression strongly promoted myogenic differentiation. However, skeletal muscle was overtly normal in Vgll3-null mice, presumably due to feedback signalling and/or redundancy. This work identifies VGLL3 as a transcriptional co-factor operating with the Hippo signal transduction network to control myogenesis.

Dynamic transcriptomic analysis reveals suppression of PGC1α/ERRα drives perturbed myogenesis in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent, incurable myopathy, linked to epigenetic derepression of D4Z4 repeats on chromosome 4q, leading to ectopic DUX4 expression. FSHD patient myoblasts have defective myogenic differentiation, forming smaller myotubes with reduced myosin content. However, molecular mechanisms driving such disrupted myogenesis in FSHD are poorly understood. We performed high-throughput morphological analysis describing FSHD and control myogenesis, revealing altered myogenic differentiation results in hypotrophic myotubes. Employing polynomial models and an empirical Bayes approach, we established eight critical time points during which human healthy and FSHD myogenesis differ. RNA-sequencing at these eight nodal time points in triplicate, provided temporal depth for a multivariate regression analysis, allowing assessment of interaction between progression of differentiation and FSHD disease status. Importantly, the unique size and structure of our data permitted identification of many novel FSHD pathomechanisms undetectable by previous approaches. For further analysis here, we selected pathways that control mitochondria: of interest considering known alterations in mitochondrial structure and function in FSHD muscle, and sensitivity of FSHD cells to oxidative stress. Notably, we identified suppression of mitochondrial biogenesis, in particular via peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), the cofactor and activator of oestrogen-related receptor α (ERRα). PGC1α knock-down caused hypotrophic myotubes to form from control myoblasts. Known ERRα agonists and safe food supplements biochanin A, daidzein or genistein, each rescued the hypotrophic FSHD myotube phenotype. Together our work describes transcriptomic changes in high resolution that occur during myogenesis in FSHD ex vivo, identifying suppression of the PGC1α-ERRα axis leading to perturbed myogenic differentiation, which can effectively be rescued by readily available food supplements.

Satellite cells delivered in their niche efficiently generate functional myotubes in three-dimensional cell culture.

Biophysical/biochemical cues from the environment contribute to regulation of the regenerative capacity of resident skeletal muscle stem cells called satellites cells. This can be observed in vitro, where muscle cell behaviour is influenced by the particular culture substrates and whether culture is performed in a 2D or 3D environment, with changes including morphology, nuclear shape and cytoskeletal organization. To create a 3D skeletal muscle model we compared collagen I, Fibrin or PEG-Fibrinogen with different sources of murine and human myogenic cells. To generate tension in the 3D scaffold, biomaterials were polymerised between two flexible silicone posts to mimic tendons. This 3D culture system has multiple advantages including being simple, fast to set up and inexpensive, so providing an accessible tool to investigate myogenesis in a 3D environment. Immortalised human and murine myoblast lines, and primary murine satellite cells showed varying degrees of myogenic differentiation when cultured in these biomaterials, with C2 myoblasts in particular forming large multinucleated myotubes in collagen I or Fibrin. However, murine satellite cells retained in their niche on a muscle fibre and embedded in 3D collagen I or Fibrin gels generated aligned, multinucleated and contractile myotubes.

Coordinated action of Axin1 and Axin2 suppresses ß-catenin to regulate muscle stem cell function.

The resident stem cells of skeletal muscle are satellite cells, which are regulated by both canonical and non-canonical Wnt pathways. Canonical Wnt signalling promotes differentiation, and is controlled at many levels, including via Axin1 and Axin2-mediated ß-catenin degradation. Axin1 and Axin2 are thought equivalent suppressors of canonical Wnt signalling, although Axin2 is also a Wnt target gene. We show that Axin1 expression was higher in proliferating satellite cells, while Axin2 was up-regulated during differentiation. siRNA-mediated Axin1 knockdown changed cell morphology, suppressed proliferation and promoted myogenic differentiation. Simultaneous knockdown of both Axin1 and ß-catenin rescued proliferation and partially, premature differentiation. Surprisingly, retroviral-mediated overexpression of Axin2 was unable to compensate for knockdown of Axin1 in satellite cells, indicating that Axin1 and Axin2 are not fully redundant. Isolated satellite cells from Axin2-null mice also had no major phenotype. However, siRNA-mediated knockdown of Axin1 in Axin2-null cells strongly inhibited proliferation, while inducing differentiation, clear nuclear localisation of ß-catenin, up-regulation of canonical Wnt target genes (Axin2, Lef1, Tcf4, Pitx2c and Lgr5) and activation of a TCF reporter construct. Again, concomitant knockdown of Axin1 and ß-catenin in Axin2-null satellite cells rescued morphology and proliferation, but only partially prevented precocious differentiation. Thus, Axin1 and Axin2 do not have equivalent functions in satellite cells, but are both involved in repression of Wnt/ß-catenin signalling to maintain proliferation and contribute to controlling timely myogenic differentiation.

ErbB3 binding protein-1 (Ebp1) controls proliferation and myogenic differentiation of muscle stem cells.

Satellite cells are resident stem cells of skeletal muscle, supplying myoblasts for post-natal muscle growth, hypertrophy and repair. Many regulatory networks control satellite cell function, which includes EGF signalling via the ErbB family of receptors. Here we investigated the role of ErbB3 binding protein-1 (Ebp1) in regulation of myogenic stem cell proliferation and differentiation. Ebp1 is a well-conserved DNA/RNA binding protein that is implicated in cell growth, apoptosis and differentiation in many cell types. Of the two main Ebp1 isoforms, only p48 was expressed in satellite cells and C2C12 myoblasts. Although not present in quiescent satellite cells, p48 was strongly induced during activation, remaining at high levels during proliferation and differentiation. While retroviral-mediated over-expression of Ebp1 had only minor effects, siRNA-mediated Ebp1 knockdown inhibited both proliferation and differentiation of satellite cells and C2C12 myoblasts, with a clear failure of myotube formation. Ebp1-knockdown significantly reduced ErbB3 receptor levels, yet over-expression of ErbB3 in Ebp1 knockdown cells did not rescue differentiation. Ebp1 was also expressed by muscle cells during developmental myogenesis in mouse. Since Ebp1 is well-conserved between mouse and chick, we switched to chick to examine its role in muscle formation. In chick embryo, Ebp1 was expressed in the dermomyotome, and myogenic differentiation of muscle progenitors was inhibited by specific Ebp1 down-regulation using shRNA electroporation. These observations demonstrate a conserved function of Ebp1 in the regulation of embryonic muscle progenitors and adult muscle stem cells, which likely operates independently of ErbB3 signaling.

Sphingosine-1-phosphate receptor 3 influences cell cycle progression in muscle satellite cells.

Skeletal muscle retains a resident stem cell population called satellite cells, which are mitotically quiescent in mature muscle, but can be activated to produce myoblast progeny for muscle homeostasis, hypertrophy and repair. We have previously shown that satellite cell activation is partially controlled by the bioactive phospholipid, sphingosine-1-phosphate, and that S1P biosynthesis is required for muscle regeneration. Here we investigate the role of sphingosine-1-phosphate receptor 3 (S1PR3) in regulating murine satellite cell function. S1PR3 levels were high in quiescent myogenic cells before falling during entry into cell cycle. Retrovirally-mediated constitutive expression of S1PR3 led to suppressed cell cycle progression in satellite cells, but did not overtly affect the myogenic program. Conversely, satellite cells isolated from S1PR3-null mice exhibited enhanced proliferation ex-vivo. In vivo, acute cardiotoxin-induced muscle regeneration was enhanced in S1PR3-null mice, with bigger muscle fibres compared to control mice. Importantly, genetically deleting S1PR3 in the mdx mouse model of Duchenne muscular dystrophy produced a less severe muscle dystrophic phenotype, than when signalling though S1PR3 was operational. In conclusion, signalling though S1PR3 suppresses cell cycle progression to regulate function in muscle satellite cells.

Pitx genes are redeployed in adult myogenesis where they can act to promote myogenic differentiation in muscle satellite cells.

Skeletal muscle retains a resident stem cell population called satellite cells. Although mitotically quiescent in mature muscle, satellite cells can be activated to produce myoblast progeny to generate myonuclei for skeletal muscle homoeostasis, hypertrophy and repair. Regulation of satellite cell function in adult requires redeployment of many of the regulatory networks fundamental to developmental myogenesis. Involved in such control of muscle stem cell fate in embryos are members of the Pitx gene family of bicoid-class homeodomain proteins. Here, we investigated the expression and function of all three Pitx genes in muscle satellite cells of adult mice. Endogenous Pitx1 was undetectable, whilst Pitx2a, Pitx2b and Pitx2c were at low levels in proliferating satellite cells, but increased during the early stages of myogenic differentiation. By contrast, proliferating satellite cells expressed robust amounts of Pitx3, with levels then decreasing as cells differentiated, although Pitx3 remained expressed in unfused myoblasts. To examine the role of Pitx genes in satellite cell function, retroviral-mediated expression of Pitx1, all Pitx2 isoforms or Pitx3, was used. Constitutive expression of any Pitx isoform suppressed satellite cell proliferation, with the cells undergoing enhanced myogenic differentiation. Conversely, myogenic differentiation into multinucleated myotubes was decreased when Pitx2 or Pitx3 levels were reduced using siRNA. Together, our results show that Pitx genes play a role in regulating satellite cell function during myogenesis in adult.

Specification and behavior of AMPs, muscle-committed transient Drosophila stem cells.

During development, transient stem cells play critical roles in the formation of specific tissues. Adult Muscle Precursors (AMPs) are at the origin of all adult Drosophila muscles and as we report here represent a novel population of muscle-committed transient stem cells. Similar to vertebrate muscle stem cells, AMPs keep Notch signaling active and express Enhancer of split m6 (E(spl)m6) gene, a read-out of Notch pathway. To get insights into AMP cell specification we performed a gain-of-function screen and found that the rhomboid-triggered Epidermal Growth Factor (EGF) signaling pathway controls both the specification and the subsequent maintenance of AMPs. Our findings are 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. Interestingly, by targeting GFP to the AMP cell membranes we also demonstrated that AMPs send long cellular processes and form a network of interconnected cells. As revealed by laser ablation experiments, the main role of AMP cell connections is to maintain their correct spatial positioning.

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.

Muscle development and regeneration in normal and pathological conditions: learning from Drosophila.

The recent demonstration that, throughout evolution, many molecular mechanisms have been highly conserved is fundamental to the advancement of our knowledge on muscle development and regeneration. Research has provided new insights into genetic cascades governing early steps of embryonic myogenesis and the regeneration of adult muscle in normal and pathological conditions, thus revealing significant similarity of both processes. Here we provide a current view on genetic mechanisms underlying muscle regeneration with a special focus on regeneration processes that take place in diseased and aging human muscle. Through examples of Drosophila models of human muscular diseases, we discuss potential impact they might have on uncovering molecular bases and identifying new treatments of muscle disorders. Taking advantage of evolutionarily conserved aspects of muscle development and the relative ease by which molecular pathways can be uncovered and dissected in a simple animal model, the fruit fly, we provide a comprehensive analysis of muscle development in Drosophila. Importantly, identification of muscle stem cell like adult muscle precursors in Drosophila makes fruit fly an attractive model system for studying muscle stem cell biology and muscle regeneration. In support of this assumption, recent studies in our laboratory provide arguments that important insights into the biology of vertebrate muscle stem cells can be gained from genetic analysis in Drosophila.

Muscle stem cells and model systems for their investigation.

Stem cells are characterized by their clonal ability both to generate differentiated progeny and to undergo self-renewal. Studies of adult mammalian organs have revealed stem cells in practically every tissue. In the adult skeletal muscle, satellite cells are the primary muscle stem cells, responsible for postnatal muscle growth, hypertrophy, and regeneration. In the past decade, several molecular markers have been found that identify satellite cells in quiescent and activated states. However, despite their prime importance, surprisingly little is known about the biology of satellite cells, as their analysis was for a long time hampered by a lack of genetically amenable experimental models where their properties can be dissected. Here, we review how the embryonic origin of satellite cells was discovered using chick and mouse model systems and discuss how cells from other sources can contribute to muscle regeneration. We present evidence for evolutionarily conserved properties of muscle stem cells and their identification in lower vertebrates and in the fruit fly. In Drosophila, muscle stem cells called adult muscle precursors (AMP) can be identified in embryos and in larvae by persistent expression of a myogenic basic helix-loop-helix factor Twist. AMP cells play a crucial role in the Drosophila life cycle, allowing de novo formation and regeneration of adult musculature during metamorphosis. Based on the premise that AMPs represent satellite-like cells of the fruit fly, important insight into the biology of vertebrate muscle stem cells can be gained from genetic analysis in Drosophila.