Facioscapulohumeral muscular dystrophy (FSHD) is a prevalent myopathy, affecting 12/100,000 people (Deenen et al., 2014). Although clinical severity is highly variable, skeletal muscle weakness often appears first in the facial and shoulder girdle muscles, with muscles of the trunk and lower extremities becoming affected as the disease progresses. Distinctive features of FSHD include asymmetric skeletal muscle wasting and scapular winging (Tawil, 2008). Analysis of myoblasts from FSHD patients has revealed direct functional impairments, including increased susceptibility to oxidative stress (Winokur et al., 2003a, 2003b; Celegato et al., 2006; Macaione et al., 2007; Barro et al., 2010), up-regulation of apoptotic markers (Winokur et al., 2003a, 2003b; Sandri et al., 2001; Vanderplanck et al., 2011; Laoudj-Chenivesse et al., 2005) and repression of MYOD and MYOD-target genes (Winokur et al., 2003b; Celegato et al., 2006). Additionally, myogenic differentiation results in myotubes with either an atrophic, or hypertrophic and highly disorganised morphology (Barro et al., 2010; Vanderplanck et al., 2011; Tassin et al., 2012; Ansseau et al., 2009). Defects in myoblast function are likely to result in impaired muscle maintenance, directly contributing to clinical symptoms (Morgan and Zammit, 2010).

FSHD is divided into two clinically indistinguishable disorders. FSHD1 (1A) (OMIM #158900) is associated with the contraction of a macrosatellite repeat named D4Z4 in the subtelomeric region 4q35 (Tawil, 2008; van Deutekom et al., 1993). Embedded within each 3.3 kb D4Z4 repeat unit is an open reading frame (ORF) for Double homeodomain protein 4 (DUX4), an intron-less retrogene (Dixit et al., 2007; Gabriëls et al., 1999). Contraction of D4Z4 units to between 1–10 is associated with de-repression of chromatin (van Deutekom et al., 1993; Zeng et al., 2009; de Greef et al., 2009). If this occurs on a permissive chromosomal 4qA haplotype containing a polyadenylation site within the flanking pLAM region, DUX4 mRNA transcript from the distal D4Z4 repeat is stabilised and DUX4 protein translated (Dixit et al., 2007; Lemmers et al., 2010; Snider et al., 2010). In the rarer FSHD2 (1B) (OMIM #158901), de-repression of the D4Z4 macrosatellite occurs despite the presence of >11 D4Z4 repeats, but still requires the permissive 4qA haplotype containing the polyadenylation signal (de Greef et al., 2009, 2010). FSHD2 has recently been linked to mutations in the Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) gene, encoding a protein involved in DNA methylation (Lemmers et al., 2012). Recently, mutations in DNA methyltransferase 3B (DNMT3B) have also been implicated in perturbed epigenetic regulation in FSHD (van den Boogaard et al., 2016). Taken together, the consensus is that aberrant DUX4 expression underlies pathogenesis in both FSHD1 and FSHD2 (Tawil et al., 2014).

Two main DUX4 mRNA transcripts can be derived from the ORF in a D4Z4 repeat: DUX4-fl (full length) mainly expressed in germline cells, and the alternatively spliced DUX4-s (short) transcript expressed in some somatic cells. FSHD skeletal muscle expresses DUX4-fl whereas this transcript is not usually found in healthy control muscle (Snider et al., 2010), although very low levels have been reported in some studies (Jones et al., 2012; Tassin et al., 2013). Expression of DUX4 in myoblasts recapitulates the pathogenic phenotype of myoblasts from FSHD patients (Vanderplanck et al., 2011; Yao et al., 2014; Mitsuhashi et al., 2013; Bosnakovski et al., 2008a; Knopp, 2011; Geng et al., 2012; Bosnakovski et al., 2014; Kowaljow et al., 2007; Wallace et al., 2011; Geng et al., 2011; Banerji et al., 2015). Indeed, suppression of DUX4 expression using siRNA or anti-sense oligonucleotides rescues the atrophic phenotype of FSHD myotubes in vitro (Vanderplanck et al., 2011). DUX4 is a potent transcription factor and analysis of DUX4-expressing myoblasts has revealed that major transcriptional pathways such as cell cycle regulation, glutathione redox metabolism, myogenic differentiation and Wnt signalling are disrupted (Bosnakovski et al., 2008a; Geng et al., 2012; Banerji et al., 2015). In addition, many germline and neural genes are upregulated (Banerji et al., 2015; Dandapat et al., 2013). However, DUX4 is at very low levels in FSHD biopsies (Tassin et al., 2013). Despite this, we, and others, have shown that the transcriptional landscape of genes altered by DUX4 is much more prominent in FSHD patient-derived material (Yao et al., 2014; Banerji et al., 2015). Therefore, identification of DUX4-induced pathways that contribute to pathology in FSHD and are targetable with drugs provides a useful route for potential FSHD-specific therapies. We have focussed on the effects of DUX4 on myoblasts, to identify approaches to improve muscle repair and regeneration.

At present, there is no single mammalian animal model that encompasses the genetic and pathophysiological spectrum of FSHD (Lek et al., 2015). To model FSHD, we have used retroviral-mediated expression of DUX4, in conjunction with constitutively active and dominant-negative versions, in primary murine satellite cells cultured ex vivo. Comparing gene expression data from DUX4-expressing murine satellite cell-derived myoblasts to a meta-analysis of published microarrays from FSHD patient biopsies and primary cultures (Banerji et al., 2015), we revealed a significant overlap between DUX4-expressing murine satellite cell-derived myoblasts and human FSHD muscle (Knopp et al., 2016).

We found that the receptor tyrosine kinase (RTK) Rearranged during transfection (RET) is up-regulated in DUX4-expressing myoblasts (Banerji et al., 2015; Dandapat et al., 2013). RET is a transmembrane RTK containing 4 cadherin-like repeats, a cysteine-rich area, a calcium-binding site on the extracellular region and several intracellular tyrosine kinase domains (Santoro et al., 2004; Airaksinen and Saarma, 2002). There are two main RET isoforms; RET9 and RET51, alternatively spliced in the 3’ region, resulting in an additional tyrosine at Y1096 on RET51 (Jain et al., 2006). These two main RET isoforms are differentially expressed, regulated and have diverse roles in development and tissue homeostasis (Yoong et al., 2005; Tsui-Pierchala et al., 2002; Richardson et al., 2012; Jain et al., 2010).

RET is activated by the secreted Glial cell-derived neurotrophic factor (GDNF) family ligands (GFL): GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN). GFLs bind with high affinity to one of four glycosylphosphatidylinositol (GPI)-anchored co-receptors of the GDNF Family Receptor (GFRα) family. GFL:GFRα complexes then recruit two RET molecules, triggering RET dimerisation and trans-phosphorylation of the intracellular region of both molecules. This leads to recruitment of adaptor proteins and activation of downstream signalling cascades such as MAPK, PI3/AKT, JAK/STAT, JNK, ERK5, SRC and PLCγ (Santoro et al., 2004; Airaksinen and Saarma, 2002; Runeberg-Roos and Saarma, 2007; Mulligan, 2014). RET9 and RET51 activate PI3K/AKT and ERK signalling with different dynamics and have distinct abilities to recruit Src or Frs2, suggesting differential activation of downstream targets by the two main RET isoforms (Tsui-Pierchala et al., 2002; Ishiguro et al., 1999; Besset et al., 2000).

Coordinated RET signalling is crucial for development of multiple tissues, including many neuronal cell types, kidneys and the thyroid gland (Runeberg-Roos and Saarma, 2007; Arighi et al., 2005). Indeed, RET knockout mice can be embryonic lethal with a wide range of organ defects (Jain et al., 2010; Schuchardt et al., 1994; de Graaff et al., 2001). RET signalling has also been shown to regulate spermatogonial and haematopoietic stem cell function (Naughton et al., 2006; Fonseca-Pereira et al., 2014). Dysregulated RET signalling is associated with disease: inactivating mutations of RET lead to a form of neurocristopathy called Hirschsprung’s disease (Amiel et al., 2008), whereas mutations of RET resulting in constitutively active isoforms are associated with several forms of inherited and somatic cancers (Arighi et al., 2005; Borrello et al., 2013). For example, mutations at codon 634 of RET are found in approximately 84% of multiple endocrine neoplasia (MEN2A) patients (Santoro et al., 2004; de Graaff et al., 2001; Mulligan et al., 1993).

Studies detailing expression of Ret, the GFL’s and GFRα’s in developing or adult rat, mouse and human skeletal muscle report expression of at least some RET signalling components, predominantly during development e.g. (Worby et al., 1998; Naveilhan et al., 1998; Russell et al., 2000; Lindahl et al., 2001; Yang et al., 2006; Mikaels et al., 2000; Golden et al., 1999; Yang and Nelson, 2004). Interestingly, Ret and Gdnf mRNA transcripts have been shown in mesenchymal cells of the second branchial arch in E9-9.5 mouse embryos (Golden et al., 1999; Natarajan et al., 2002), from which some facial muscles develop. We recently reported that ret is important for skeletal muscle development in zebrafish, where ret-gfrα3-artemin signalling is required to regulate myogenic differentiation in a subset of muscle precursors deriving from the first and second brachial arches (Knight et al., 2011).

Here we show that Ret is a novel mediator of satellite cell function, contributing to the regulation of proliferation and initiation of myogenic differentiation. Critically, DUX4-mediated Ret signalling is pathogenic to satellite cells, contributing to impaired differentiation. However, blocking DUX4-mediated Ret signalling using siRNA or the RTK inhibitor Sunitinib, leads to an increase in the myogenic capacity of DUX4-expressing murine satellite cells. Importantly, Sunitinib also improves proliferation and differentiation in FSHD patient-derived myoblasts both in vitro, and in vivo after grafting into regenerating muscle in mice. These observations highlight using RTK inhibitors as a potential therapeutic strategy for FSHD.

We report that Ret is a novel mediator of muscle stem cell function, is regulated by DUX4 and contributes to the pathogenic phenotype of FSHD. We focused on Ret signalling as a potential mediator of DUX4-induced muscle pathology for several reasons. FSHD is characterised by the often asymmetric wasting of facial, shoulder and upper arm muscles (Tawil, 2008), and can be associated with several non-muscle tissue pathologies including sensorineural hearing loss and retinal vasculopathies (Verhagen et al., 1995). Development of the auditory system, retinal patterning and facial muscles have been shown to depend on Ret signalling in a number of species. Ret is expressed in the second pharyngeal arch, from which some facial muscles arise, both in mouse and zebrafish (Knight et al., 2011; Pachnis et al., 1993). Additionally, Ret, GFRα1 and GFRα2 are expressed in the retina and the sensory epithelium of the cochlea, both of which can be affected in FSHD (Golden et al., 1999; Zordan et al., 2006; Yu et al., 1998). Phenotypic analysis of Ret, GFRa (Enomoto et al., 1998; Tomac et al., 2000; Rossi et al., 1999; Honma et al., 2002; Lindahl et al., 2000) and GFL (Honma et al., 2002; Moore et al., 1996; Heuckeroth et al., 1999; Tomac et al., 2002)-mutant mice have not reported a skeletal muscle phenotype, although this may have been overlooked due to the severe phenotype and perinatal lethality of some mutant lines. However, Ret is expressed in the indirect flight muscles of Drosophila, and overexpression of constitutively active Ret (RET^MEN2B) in muscle via a MEF2-GAL4 driver causes increased muscle fibre size, irregular myofibrils and abnormal actin deposits (Klein et al., 2014). Interestingly, irregular myotube morphology is often reported in FSHD patient-derived cultures (Barro et al., 2010; Tassin et al., 2012). As we found that Ret was activated by DUX4 in murine satellite cells, it becomes a potential candidate for contributing to DUX4-induced myopathy.

There has been no description of Ret function during myogenesis in mammals, despite the descriptions of Ret pathway genes expressed in specific muscle cell populations, including facial muscle precursors (Golden et al., 1999; Natarajan et al., 2002; Knight et al., 2011). We found that Ret and three of its co-receptors: Gfrα1, Gfrα2 and Gfrα4 are dynamically expressed in mammalian limb satellite cells. Intriguingly, Ret expression was higher during proliferation, whereas Gfrα1, Gfrα2 and Gfrα4 expression increased upon differentiation. This reciprocal expression pattern between Ret and its co-receptors also occurs in other cell types (Li et al., 2009) and so may regulate Ret signalling by feedback loops. Additionally, the ligands GDNF and PSPN, required to activate Gfrα1 and Gfrα4 (and to a lesser extent Gfrα2), are expressed by muscle fibres (Golden et al., 1999; Yang and Nelson, 2004; Lindahl et al., 2000), suggesting that myofibres may signal to satellite cells via Ret signalling. We also found that RET is expressed at low levels in primary human myoblasts, revealing a conserved expression in vertebrate muscle. Constitutive-expression and knockdown studies revealed that Ret signalling is required to maintain a proliferating myoblast population and prevent premature cell cycle exit and myogenic differentiation. Specifically, constitutively active RET increased satellite cell proliferation but did not affect terminal differentiation. Consistent with this, siRNA-mediated knockdown of Ret reduced Pax7 expression, concomitant with reduced satellite cell proliferation, whilst increasing differentiation. Knockdown of Gfrα1, Gfrα2 or Gfrα4 causes similar, if less severe, defects suggesting that Ret is interacting with its Gfrα co-receptors to control the switch from proliferation to differentiation in myoblasts.

Having shown that Ret is involved in the control of myogenesis, we aimed to understand its role in mediating DUX4 pathology. DUX4 is pathogenic in both myoblasts and differentiated muscle (Vanderplanck et al., 2011; Bosnakovski et al., 2008a, 2014; Kowaljow et al., 2007; Wallace et al., 2011; Dandapat et al., 2014). Recent microarray and ChIP-seq datasets reveal that DUX4 causes wide-ranging transcriptional disruption in myogenic cells (Geng et al., 2012; Banerji et al., 2015; Rahimov et al., 2012; Knopp et al., 2016) , with its potent transcriptional activation through its C-terminal domain (Geng et al., 2012; Clapp et al., 2007; Kawamura-Saito et al., 2006). However, identifying which disrupted pathways directly contribute to DUX4-specific pathology is crucial when considering future drug design to treat FSHD.

Ret is genetically downstream of DUX4 in myoblasts, but does not appear to be a direct transcriptional target. Transgenic analysis has shown that in mouse, 1.9 kb 5’ of the Transcriptional Start Site (TSS) of Ret is sufficient to drive tissue specific expression (Zordan et al., 2006). However, analysing two published ChIP-Seq datasets of DUX4 overexpression in human myoblasts (Geng et al., 2012; Choi et al., 2016), we failed to identify significant peaks within 15 kb 5’ of the Ret TSS. In addition, sequence analysis also failed to find a DUX4 consensus binding site (TAATCTAATCA – [Zhang et al., 2016]) within this same 15 kb region. It is of note however, that the C-terminus of DUX4 can recruit acetyltransferases (p300/CBP) to histones to induce acetylation, hence promoting transcription at sites distant to DUX4 DNA binding (Choi et al., 2016), so DUX4 could still directly controlling Ret in an epigenetic manner. However, also arguing against Ret being a direct transcriptional target of DUX4 is our observation that Ret expression is not only enhanced by DUX4 and the constitutively active tMALDUX4-VP16, but also by our DUX4 dominant-negative tMALDUX4-ERD construct in the microarray analysis. The tMALDUX4-ERD construct contains the two DNA binding homeodomains of DUX4, so should select the same cohort of transcriptional targets as DUX4. However, if DUX4 binds directly to the Ret promoter, tMALDUX4-ERD should suppress transcription and so reduce Ret expression, but this is the opposite to what we observe. Together, these observations indicate that DUX4 affects Ret expression indirectly, via binding to genes that, in turn, control Ret expression.

Blocking Ret expression or signalling increases the probability of DUX4-expressing myoblasts to differentiate into multinucleate myotubes. Despite this, some aspects of DUX4-mediated pathologies were not rescued by Ret inhibition. For example, inhibition of Ret using Sunitinib did not alter DUX4-induced apoptosis. DUX4 transcriptionally dysregulates a large number of pathways, so it is unlikely that one pathway only is responsible for all pathological characteristics (Banerji et al., 2015). For example, we have recently shown that β-catenin signalling is important for DUX4-mediated transcriptional dysregulation in skeletal muscle (Banerji et al., 2015).

To investigate the relative importance of Ret in mediating DUX4 action, we adopted a pharmacological approach that allowed us to modify Ret signalling in a dose-dependent manner. As the readout, we used the suppression of differentiation caused by constitutive CA RET51 (RET51-MENA) expression in murine C2C12 myoblasts and asked at which doses we could inhibit Ret to rescue differentiation, without causing deleterious side-effects. Comparison of Ret inhibitors Zactima (Vandetanib/ZD6474) (Carlomagno et al., 2002), TG101209 (Pardanani et al., 2007) and Sunitinib (Sutent, SU11248) (Kim et al., 2006) revealed that Suntinib showed the most robust rescue of CA RET51-mediated inhibition of myogenesis. We further tested the ability of Sunitinib to rescue DUX4-mediated phenotypes in murine satellite cells and showed that we could again rescue differentiation, but not proliferation: consistent with the effects of siRNA-mediated knockdown of Ret. Zactima, TG101209 and Sunitinib also inhibit other receptor tyrosine kinases, including VEGFR, EGF and MET (Plaza-Menacho et al., 2014). Of note, Vegfr2 is also a DUX4 target identified in our microarray screen (Banerji et al., 2015), highlighting another potential mediator of DUX4 pathology in myoblasts. Since many of these receptors activate the same intracellular pathways as Ret, it will be important to understand their relative roles and redundancies, when activated in the context of FSHD.

Since DUX4 up-regulates Ret, and Sunitinib can clearly rescue the differentiation block caused by CA-RET51 in C2C12 myoblasts, we investigated intracellular signalling events. ERK and AKT are two key intracellular mediators of Ret signalling e.g. (Mograbi et al., 2001). ERK1/2 phosphorylation was inhibited by Sunitinib in DUX4-expressing iC2C12-DUX4 myoblasts, but not in control myoblasts not expressing DUX4, suggesting a potential mechanism by which DUX4-mediated Ret signalling could inhibit differentiation. In contrast, AKT phosphorylation was unaltered by inducing DUX4. Although Ret can operate through ERK1/2 signalling, it is possible that Sunitinib could also be acting on ERK1/2 phosphorylation activated by other pathways perturbed by, or in response to, DUX4.

To assess whether Sunitinib could be used to ameliorate FSHD pathologies in man, we used human myoblast lines isolated from a mosaic FSHD1 patient (Krom et al., 2012). We assessed whether Sunitinib could rescue cell morphology, proliferation and the differentiation capacity of human myoblasts displaying FSHD pathologies. Using statistical models we showed that Sunitinib could induce the FSHD myoblasts to adopt the phenotype of isogenic myoblasts lacking the D4Z4 contraction. Transplantation assays of FSHD myoblasts into cryodamaged immunodeficient mice showed increased engraftment of FSHD myoblasts occurred in response to daily systemic Sunitinib treatment. Therefore, this pre-clinical model for evaluating modifiers of FSHD pathology identifies Sunitinib as a potential therapeutic agent for FSHD.

In conclusion, we have shown that Ret is a novel regulator of satellite cell function in mammalian muscle, and contributes to the FSHD phenotype. Specifically, blocking DUX4-mediated Ret signalling increases myogenic differentiation, giving further insight into the molecular pathology of DUX4 and highlighting Ret signalling as potential drug target. Crucially, the clinically-approved RET inhibitor Sunitinib (Widmer et al., 2014) rescues cell morphology, proliferation and myogenic differentiation in human FSHD myoblasts both in vitro and in vivo. Given that FSHD is currently incurable, our findings highlight RTK inhibitors as a potential novel therapeutic strategy to treat muscle wasting in FSHD.

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Author details

1. Louise A Moyle

   1. Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom
   2. Department of Cell and Developmental Biology, University College London, London, United Kingdom

   Contribution

   LAM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Eric Blanc

   1. Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom
   2. Core Unit Bioinformatics, Berlin Institute of Health, Berlin, Germany
   3. Institute of Pathology, Charite Universitatsmedizin Berlin, Berlin, Germany

   Contribution

   EB, Conception and design, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Oihane Jaka

   Centre of Human and Aerospace Physiological Sciences, King's College London, London, United Kingdom

   Contribution

   OJ, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Johanna Prueller

   Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom

   Contribution

   JP, Helped design, perform and analyse the Western Blots to examine the effects of Sunitinib on ERK and AKT mediated signalling in response to DUX4, and presented as Figures 7H-J in the revised manuscript

   Competing interests

   The authors declare that no competing interests exist.

5. Christopher RS Banerji

   Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom

   Contribution

   CRSB, Wrote software to analyse cell eccentricity, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

6. Francesco Saverio Tedesco

   Department of Cell and Developmental Biology, University College London, London, United Kingdom

   Contribution

   FST, Helped design, perform and analyse the grafting experiments to test the effects of Sunitnib in vivo and presented as Figure 12 in the revised manuscript

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-5321-7682

7. Stephen DR Harridge

   Centre of Human and Aerospace Physiological Sciences, King's College London, London, United Kingdom

   Contribution

   SDRH, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

8. Robert D Knight

   Craniofacial Development and Stem Cell Biology, King's College London, London, United Kingdom

   Contribution

   RDK, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   robert.knight@kcl.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-9920-836X

9. Peter S Zammit

   Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom

   Contribution

   PSZ, Conception and design, Analysis and interpretation of data, Drafting and revising the article

   For correspondence

   peter.zammit@kcl.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-9562-3072

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