Louise A. Moyle

Isolation, culture and immunostaining of skeletal muscle fibres to study myogenic progression in satellite cells.

Satellite cells are the resident stem cells of skeletal muscle, located on the surface of a myofibre, beneath the surrounding basal lamina. Satellite cells are responsible for the homeostasis, hypertrophy and repair of skeletal muscle fibres, being activated to enter proliferation and generate myoblasts that either fuse to existing myofibres, or fuse together for de novo myofibre formation. Isolating muscle fibres allows the associated satellite cells to be obtained while remaining in their anatomical niche beneath the basal lamina, free of interstitial and vascular tissue. Myofibres can then be immunostained to examine gene expression in quiescent satellite cells, or cultured to activate satellite cells before immunostaining to investigate gene expression dynamics during myogenic progression and self-renewal. Here, we describe methods for the isolation, culture and immunostaining of muscle fibres for examining satellite cell biology.

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.

DUX4 induces a transcriptome more characteristic of a less-differentiated cell state and inhibits myogenesis

ABSTRACT Skeletal muscle wasting in facioscapulohumeral muscular dystrophy (FSHD) results in substantial morbidity. On a disease-permissive chromosome 4qA haplotype, genomic and/or epigenetic changes at the D4Z4 macrosatellite repeat allows transcription of the DUX4 retrogene. Analysing transgenic mice carrying a human D4Z4 genomic locus from an FSHD-affected individual showed that DUX4 was transiently induced in myoblasts during skeletal muscle regeneration. Centromeric to the D4Z4 repeats is an inverted D4Z4 unit encoding DUX4c. Expression of DUX4, DUX4c and DUX4 constructs, including constitutively active, dominant-negative and truncated versions, revealed that DUX4 activates target genes to inhibit proliferation and differentiation of satellite cells, but that it also downregulates target genes to suppress myogenic differentiation. These transcriptional changes elicited by DUX4 in mouse have significant overlap with genes regulated by DUX4 in man. Comparison of DUX4 and DUX4c transcriptional perturbations revealed that DUX4 regulates genes involved in cell proliferation, whereas DUX4c regulates genes engaged in angiogenesis and muscle development, with both DUX4 and DUX4c modifing genes involved in urogenital development. Transcriptomic analysis showed that DUX4 operates through both target gene activation and repression to orchestrate a transcriptome characteristic of a less-differentiated cell state. Summary: DUX4 underlies pathogenesis in facioscapulohumeral muscular dystrophy. DUX4 acts mainly as a transcriptional activator that inhibits myogenesis by orchestrating a gene expression profile representative of a more stem-cell-like state.

β-Catenin is central to DUX4-driven network rewiring in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an incurable disease, characterized by skeletal muscle weakness and wasting. Genetically, FSHD is characterized by contraction or hypomethylation of repeat D4Z4 units on chromosome 4, which causes aberrant expression of the transcription factor DUX4 from the last repeat. Many genes have been implicated in FSHD pathophysiology, but an integrated molecular model is currently lacking. We developed a novel differential network methodology, Interactome Sparsification and Rewiring (InSpiRe), which detects network rewiring between phenotypes by integrating gene expression data with known protein interactions. Using InSpiRe, we performed a meta-analysis of multiple microarray datasets from FSHD muscle biopsies, then removed secondary rewiring using non-FSHD datasets, to construct a unified network of rewired interactions. Our analysis identified β-catenin as the main coordinator of FSHD-associated protein interaction signalling, with pathways including canonical Wnt, HIF1-α and TNF-α clearly perturbed. To detect transcriptional changes directly elicited by DUX4, gene expression profiling was performed using microarrays on murine myoblasts. This revealed that DUX4 significantly modified expression of the genes in our FSHD network. Furthermore, we experimentally confirmed that Wnt/β-catenin signalling is affected by DUX4 in murine myoblasts. Thus, we provide the first unified molecular map of FSHD signalling, capable of uncovering pathomechanisms and guiding therapeutic development.

Ret function in muscle stem cells points to tyrosine kinase inhibitor therapy for facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) involves sporadic expression of DUX4, which inhibits myogenesis and is pro-apoptotic. To identify target genes, we over-expressed DUX4 in myoblasts and found that the receptor tyrosine kinase Ret was significantly up-regulated, suggesting a role in FSHD. RET is dynamically expressed during myogenic progression in mouse and human myoblasts. Constitutive expression of either RET9 or RET51 increased myoblast proliferation, whereas siRNA-mediated knockdown of Ret induced myogenic differentiation. Suppressing RET activity using Sunitinib, a clinically-approved tyrosine kinase inhibitor, rescued differentiation in both DUX4-expressing murine myoblasts and in FSHD patient-derived myoblasts. Importantly, Sunitinib also increased engraftment and differentiation of FSHD myoblasts in regenerating mouse muscle. Thus, DUX4-mediated activation of Ret prevents myogenic differentiation and could contribute to FSHD pathology by preventing satellite cell-mediated repair. Rescue of DUX4-induced pathology by Sunitinib highlights the therapeutic potential of tyrosine kinase inhibitors for treatment of FSHD. DOI: http://dx.doi.org/10.7554/eLife.11405.001

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering

Summary Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development.

Reversible immortalisation enables genetic correction of human muscle progenitors and engineering of next-generation human artificial chromosomes for Duchenne muscular dystrophy.

Transferring large or multiple genes into primary human stem/progenitor cells is challenged by restrictions in vector capacity, and this hurdle limits the success of gene therapy. A paradigm is Duchenne muscular dystrophy (DMD), an incurable disorder caused by mutations in the largest human gene: dystrophin. The combination of large‐capacity vectors, such as human artificial chromosomes (HACs), with stem/progenitor cells may overcome this limitation. We previously reported amelioration of the dystrophic phenotype in mice transplanted with murine muscle progenitors containing a HAC with the entire dystrophin locus (DYS‐HAC). However, translation of this strategy to human muscle progenitors requires extension of their proliferative potential to withstand clonal cell expansion after HAC transfer. Here, we show that reversible cell immortalisation mediated by lentivirally delivered excisable hTERT and Bmi1 transgenes extended cell proliferation, enabling transfer of a novel DYS‐HAC into DMD satellite cell‐derived myoblasts and perivascular cell‐derived mesoangioblasts. Genetically corrected cells maintained a stable karyotype, did not undergo tumorigenic transformation and retained their migration ability. Cells remained myogenic in vitro (spontaneously or upon MyoD induction) and engrafted murine skeletal muscle upon transplantation. Finally, we combined the aforementioned functions into a next‐generation HAC capable of delivering reversible immortalisation, complete genetic correction, additional dystrophin expression, inducible differentiation and controllable cell death. This work establishes a novel platform for complex gene transfer into clinically relevant human muscle progenitors for DMD gene therapy.

Generation of a mouse model of FSHD to reveal the DUX4 expression profile and dynamics

Methods: A subset of 20 adults (10 males, 10 females) from PHENODM1 have been scanned using 3.0T MRI scanner and bilateral lower limb axial T1weighted and T2-weighted STIR images have been analysed. All pelvic girdle, thigh and lower leg muscles were scored bilaterally according to the Mercuri scale on axial T1-weighted sequences. In addition to the OMMYD-2 measures described we compared our fi ndings to quantitative strength assessments in the ankle dorsifl exors and plantifl exors, knee extensors and hip fl exors. The average of the quadriceps and ankle dorsifl exors Mercuri scores were considered for the non-parametric correlations against muscle strength. Kruskal-Wallis Test was utilised to compare the diff erent mean values between the stratifi ed sample. Results: The mean age at MRI was 41.2±9.9 years with a mean disease duration of 22.2±10.5 years. On T1-weighted images the most severely aff ected muscles were: gastrocnemius medialis (Mercuri median score: 3), soleus (2b), peroneus longus (1), tibialis anterior (1), fl exor digitorum (1), vastus (1), sartorius (1) and biceps femoris (1). STIR abnormalities were detected in 9 patients, mainly in gastrocnemius medialis and soleus. Signifi cant correlation values were identifi ed between sartorius (MRI score) and hip fl exors strength and between ankle dorsifl exors strength and MRI score for dorsifl exors and plantifl exors. Tibialis anterioris’ Mercuri score gave indications of discrimination between patient performance on functional outcomes. Conclusions: This study provides the foundation for MRI assessments as noninvasive biomarker for DM1. Future work on larger cohorts and longitudinal assessments will be required for validation.

P31 Role of Ret in satellite cell myogenesis and facioscapulohumeral muscular dystrophy

disease and Walker-Warburg syndrome. Here we investigate the structural changes in muscle, eye and notochord of 3dpf zebrafish larvae, with particular emphasis on the basement membrane. Antisense oligonucleotide morpholinos were used to knock down FKRP and fukutin in wild type zebrafish. The morphants had abnormal muscle fibres, disrupted vertical myosepta and sarcolemma. The area of the notochord was observed to be smaller in all morphants when compared to controls. Electron microscopy revealed disturbances in all three layers that form the peri-notochord sheath including the basement membrane. Toluidine blue staining showed disorganised retinal layering in both morphants. Dysplasia of the lens was observed in most fukutin morphants and two FKRP morphants with a severe phenotype. Transmission electron microscopy studies revealed a homogenous perturbation across the inner limiting membranes of both morphants which may account for the lens dysplasia. The rod and cones in the photoreceptor cell layer were found in lower density in both morphants with the least density in fukutin knock-downs which may be as a result of a disrupted external limiting membrane. We therefore conclude that FKRP and fukutin are essential for the integrity of membranes in the eye, muscle and notochord of developing zebrafish larvae.