mechanisms of development 134 (2014) 1–15

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The RNA-binding protein Rbm24 is transiently expressed in myoblasts and is required for myogenic differentiation during vertebrate development Raphaëlle Grifone a,b, Xin Xie a,b, Adeline Bourgeois a,b, Audrey Saquet a,b, Delphine Duprez a,b, De-Li Shi a,b,* a b

Sorbonne Universités, UPMC Univ Paris 06, UMR 7622, Laboratory of Developmental Biology, Paris F-75005, France CNRS, UMR 7622, Laboratory of Developmental Biology, Paris F-75005, France

A R T I C L E

I N F O

A B S T R A C T

Article history:

RNA-binding proteins (RBP) contribute to gene regulation through post-transcriptional events.

Received 22 May 2014

Despite the important roles demonstrated for several RBP in regulating skeletal myogenesis

Received in revised form

in vitro, very few RBP coding genes have been characterized during skeletal myogenesis in

5 August 2014

vertebrate embryo. In the present study we report that Rbm24, which encodes the RNA-

Accepted 22 August 2014

binding motif protein 24, is required for skeletal muscle differentiation in vivo. We show

Available online 16 September 2014

that Rbm24 transcripts are expressed at all sites of skeletal muscle formation during embryogenesis of different vertebrates, including axial, limb and head muscles. Interestingly,

Keywords:

we find that Rbm24 protein starts to accumulate in MyoD-positive myoblasts and is tran-

Rbm24

siently expressed at the onset of muscle cell differentiation. It accumulates in myotomal

MyoD

and limb myogenic cells, but not in Pax3-positive progenitor cells. Rbm24 expression is under

Embryonic myogenesis

the direct regulation by MyoD, as demonstrated by in vivo chromatin immunoprecipita-

Myogenic differentiation

tion assay. Using morpholino knockdown approach, we further show that Rbm24 is required

Mouse

for somitic myogenic progenitor cells to differentiate into muscle cells during chick somitic

Chick

myogenesis. Altogether, these results highlight Rbm24 as a novel key regulator of the myogenic differentiation program during vertebrate development. © 2014 Elsevier Ireland Ltd. All rights reserved.

1.

Introduction

In the developing vertebrate embryo, skeletal muscles of the body are derived from transitory segmented structures called somites, budding off from unsegmented paraxial mesoderm alongside of the neural tube. Pax3-positive muscle progenitor cells are located in the dermomyotome, which constitutes the

dorsal epithelial layer of each somite. These cells fall down from the four edges of the dermomyotome to form the underneath myotome. This myotome, considered as the first skeletal muscle to form in the embryo, expands dorsally and ventrally to build all the musculature of the trunk. At the brachial and lumbar levels, Pax3-positive progenitor cells delaminate from the ventrolateral dermomyotome to migrate into the developing limb buds and differentiate into appendicular muscle

* Corresponding author. UPMC Univ Paris 06, UMR 7622, Laboratory of Developmental Biology, Sorbonne Universités, Paris F-75005, France. Tel.: +33 1 44272772; fax: +33 1 44273445. E-mail address: [email protected] (D-L. Shi). http://dx.doi.org/10.1016/j.mod.2014.08.003 0925-4773/© 2014 Elsevier Ireland Ltd. All rights reserved.

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mechanisms of development 134 (2014) 1–15

masses (Buckingham and Vincent, 2009). Somites are not the only site of myogenesis during vertebrate development; head muscles are also formed by cells originating from the prechordal and the pharyngeal mesoderm. Consistent with their different embryological origins, the genetic hierarchies operating upstream of myogenic specification are divergent in head and trunk mesoderm (Grifone and Kelly, 2007). However, at all sites of myogenesis, entry of specified progenitor cells into the myogenic program depends on the action of the myogenic regulatory factors (MRF) of the MyoD family of basic helix–loop– helix (bHLH) transcription factors: Myf5, MyoD and Mrf4 (Buckingham, 2006). Definitive muscle identity is acquired when threshold levels of these transcription factors are attained and downstream genes are activated (Weintraub, 1993). While upstream regulators that coordinate lineage specification through the activation of Myf5 and MyoD have been extensively characterized, the downstream mechanisms by which MyoD and Myf5 could specifically stabilize the muscle fate and promote muscle differentiation are not well understood. Myogenin, the fourth MRF member, whose expression is detected later during the myogenic program, is considered as a differentiation gene that promotes myoblasts to differentiate into functional mature myofibers by directly activating the expression of genes coding for contractile proteins (Davie et al., 2007). Myogenin-null mouse embryos indeed form Myf5- and MyoD-expressing myoblasts, but they are deficient in differentiated muscles (Venuti et al., 1995). Mrf4, which is expressed in myoblasts, also regulates their differentiation and subsequent myotube maturation (Braun and Arnold, 1995; Patapoutian et al., 1995). In addition, signals mediated by the evolutionarily conserved Notch pathway have been implicated downstream of MyoD in the regulation of myogenic differentiation in vertebrate embryos (Wittenberger et al., 1999). Delta1 is expressed in myoblasts as well as in differentiated myocytes and may provide signals that regulate and sustain skeletal muscle differentiation (Delfini et al., 2000; Schuster-Gossler et al., 2007; Vasyutina et al., 2007). As a downstream target of MyoD, Vgl2 has also been shown to be associated with skeletal muscle differentiation in chick myogenesis (Bonnet et al., 2010). Moreover, in addition to positively regulate Myf5 and MyoD expression in the myotome and the limbs, six homeoproteins have been shown to act downstream of MyoD to activate the expression of Myogenin and muscle specific genes during embryogenic myogenic differentiation (Grifone et al., 2005; Relaix et al., 2013; Richard et al., 2011). Global coordination of gene expression not only depends on transcriptional regulation, but also to a large extent on posttranscriptional events. RNA-binding proteins (RBP) are involved in modulating the metabolism of mRNAs at all stages of their lifetime thus appearing as key regulators of post-transcriptional gene expression. By controlling RNA metabolism, RBP have been implicated in different tissue-specific processes during embryonic development and the significance of post-transcriptional mechanisms for the regulation of cell differentiation becomes increasingly evident during embryogenesis (Graindorge et al., 2008; Huot et al., 2005; Spagnoli and Brivanlou, 2006). However, the possible contribution of post-transcriptional regulation by RBP to muscle development is only beginning to come to light. For example, HuR RNA-binding protein has been shown to enhance myogenic differentiation in vitro by binding to and

stabilizing MyoD and Myogenin mRNA (Figueroa et al., 2003). The Lin-28 RNA-binding protein has also been identified as an essential regulator of differentiation of cultured myoblasts, by increasing IGF2 mRNA translation efficiency (Polesskaya et al., 2007). Moreover, IMP2 RNA-binding protein influences C2C12 myoblasts motility by post-transcriptional regulation of cell adhesion proteins during myogenic differentiation (Boudoukha et al., 2010). More recently, RNA biogenesis defect caused by aberrant expression of RBP coding genes has emerged as a new pathogenic mechanism underlying a number of inherited diseases including myotonic dystrophies. Facioscapulohumeral muscular dystrophy (FSHD) has been linked to an altered expression of FXR1P, an RNA-binding protein highly expressed in skeletal muscle and whose inactivation in mice leads to a reduced musculature phenotype at birth (Mientjes et al., 2004). The FRG1 RNA-binding protein, whose upregulation has been demonstrated in FSHD muscle, is another candidate for this human muscle disease (Davidovic et al., 2008). Transgenic mice overexpressing FGR1 display a post-natal muscle growth defect and exhibit impaired muscle regeneration (Xynos et al., 2013). Whereas the involvement of RBP during myogenic differentiation has been mostly investigated in vitro, no expression and functional analyses have been carefully conducted in vivo during somitic myogenesis in vertebrates. Recently, the RNAbinding protein Hoi Polloi has been reported to play a role in the regulation of myotube elongation during Drosophila embryogenesis. This study highlights the essential role of posttranscriptional gene regulation during embryonic myogenesis (Johnson et al., 2013). Rbm24 gene encodes an RNA-binding protein harboring in its N-terminal part a single RNA-Recognition Motif (RRM) that is highly conserved in C. elegans, Xenopus, mice and human (Fetka et al., 2000). Seb4, the orthologous gene of Rbm24 in Xenopus, was shown to be expressed in the early gastrula, concomitantly with MyoD, in presomitic paraxial mesoderm and later developing somites (Fetka et al., 2000). We have reported that Seb4, as a target gene of MyoD, is required for Xenopus primary myogenesis, which takes place in presomitic mesoderm. This first wave of myogenesis is a Xenopus specificity that is not shared by avian and mammalian system (Li et al., 2010). Our results conferring a myogenic potential to Seb4 protein during early Xenopus development was supported by an in vitro study showing that mouse Rbm24 protein binds to the 3′-UTR region of Myogenin mRNA. This interaction stabilizes Myogenin mRNA and promotes myogenic differentiation in C2C12 mouse myoblast cell line (Jin et al., 2010). In the present study, we have carried out a detailed analysis of Rbm24 expression during development in representative animal models including Xenopus, chick and mice. By in situ hybridization we show that Rbm24 is expressed in all sites of skeletal muscle formation, including head, trunk and limbs. Using specific antibodies we have compared the pattern of Rbm24 protein accumulation with that of known muscle markers during mouse embryonic myogenesis and established that Rbm24 is transiently expressed in a narrow time window in differentiated myogenic cells, which have entered in the myogenic program as assayed by the expression of MyoD. By chromatin immunoprecipitation assays we have demonstrated that MyoD is recruited in vivo to the conserved Rbm24 5′-untranslated region in mouse embryo. Moreover, we show that Rbm24 ex-

mechanisms of development 134 (2014) 1–15

pression is partly affected in MyoD mutant embryos. Functional analysis using morpholino knockdown approach demonstrated that Rbm24 is required for somitic myogenic progenitor cells to differentiate in vivo during chick somitic embryogenesis. Taken together, our results establish that Rbm24, partially under the control of MyoD, is required for somitic myogenic differentiation during vertebrate embryogenesis.

2.

Results

2.1. Rbm24 is expressed at all sites of skeletal muscle formation during vertebrate development We have first performed whole-mount in situ hybridization to determine the expression pattern of Rbm24 mRNA during development of different vertebrate embryos. We found that Seb4, the orthologous gene of Rbm24 in Xenopus, is strongly expressed in all developing somites at larval stages 28 and 37 (Fig. 1A and B) and especially in the myotomal compartment of the somites that can be visualized on transverse vibratome sections (Fig. 1A′). Seb4 transcripts also strongly accumulate in hypaxial migrating cells (Fig. 1B) that delaminate from the ventrolateral edge of the dermomyotome to give rise to the most ventrolateral musculature (Martin and Harland, 2001). At the level of the cranial muscle anlagen, Seb4 gene is only expressed in a population of cells developing from the visceral arches namely branchiomeric muscles. Seb4 transcripts are indeed strongly detected in the levatores mandibulae muscle anlagen, which are partly mandibular arch-derived muscles, as well as in the quadrato-hyoangularis and orbitohyoidus muscle anlagen that are partly hyoid arch-derived muscles (Fig. 1B and C). Interestingly, most of these Seb4-positive muscles specifically express MyoD, but not Myf5 during Xenopus development (Fig. 1C–E). It is of note that Seb4 is not expressed in Myf5positive/MyoD-negative head muscles such as extraoccular muscles, intermandibularis muscle and branchial muscle anlagen (Fig. 1C–E). In addition to skeletal muscles, Seb4 expression is also detected in the lens, the otic vesicle, as well as in the developing heart (Fig. 1A–C). This analysis, which extends previous observation (Fetka et al., 2000), shows that Seb4 is highly expressed in developing muscles. In order to examine how Seb4 expression pattern depicted during Xenopus embryogenesis is conserved among vertebrates, Rbm24 expression was investigated during mouse and chick embryonic development. Whole-mount in situ hybridization carried out on mouse embryos at embryonic days (E) E10.5, E11.5 and E12.5 shows that Rbm24 is expressed throughout the dorsoventral extent of the myotome of all somites that will give rise to the overall truncal musculature (Fig. 1F, I and L). It is not detected in prospective forelimbs and hindlimbs at E10.5 (Fig. 1F–H), but is strongly expressed in developing forelimb and hindlimb skeletal muscle masses at E11.5 and E12.5 (Fig. 1I–N). Rbm24 is also expressed in a stream of cells that emerge from the somites at the anterior border of the forelimb and further move towards the septum transversum to form the diaphragm muscle at E12.5 (Fig. 1M). At E10.5, Rbm24 is weakly expressed in the mesodermal core of the first and second branchial arches (Fig. 1F). These mesodermal cells, originating from cranial paraxial and occipital anterior mesoderm,

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will migrate laterally into the pharyngeal region to form branchiomeric skeletal muscles of the head at later stages (Trainor et al., 1994). These branchiomeric muscles also express Rbm24 at E11.5 and E12.5 (Fig. 1I and L). Like Xenopus Seb4, mouse Rbm24 is expressed in the otic vesicle at E10.5, the developing heart at E10.5 and E11.5 (Fig. 1F and I) and the lens from E10.5 to E12.5 (Fig. 1F, I, L and M). Whole-mount in situ hybridization performed on chick embryos at 3 days (HH20) and 3.5 days (HH23) of development reveals a similar expression pattern of Rbm24 to that depicted in Xenopus and mouse embryos. Rbm24 is detected in the entire myotome of all somites (Fig. 1O and R), in migrating premyogenic cells that have reached the forelimb and hindlimb at 3.5 days of development (Fig. 1R and S), in the mesodermal core of the first and second branchial arches (Fig. 1T), as well as in the lens, the otic vesicle and the heart (Fig. 1O and T). Altogether, these results demonstrate that the expression pattern of Rbm24 gene is similar in all examined vertebrate species. Considering its expression at all sites of skeletal muscle development in Xenopus, mouse and chick, Rbm24 thus may play a conserved role in the skeletal myogenic program.

2.2. Rbm24 protein does not accumulate in premyogenic progenitors but is present at the onset of their differentiation The expression pattern of Rbm24 gene suggests that it may function as a potential regulator of the muscle differentiation program. The expression of Rbm24 protein was thus further examined together with that of Pax3 and MyoD at the early stage E10.5 to see whether Rbm24 is expressed in premyogenic cells that have not entered and/or just entered the myogenic program. The transcription factor Pax3 is a key regulator of myogenesis and a marker of somitic muscle progenitor cells (Relaix et al., 2004). Thus, we first used the mouse Pax3 reporter line Pax3nLacZ that faithfully reproduces the expression of the endogenous gene (Relaix et al., 2003) to compare with Rbm24 expression by in situ hybridization at the same stage of development. Whereas Pax3 myogenic expression in E10.5 Pax3nlacZ heterozygous embryos, revealed by XGal staining for nLacZ, is found in the dermomyotome of the somites as well as in cells that move away from the dermomyotome to colonize the limbs, Rbm24 expression is restricted to the myotome, but is absolutely not detected in limb buds at this stage of development (Fig. 2A and C). At the prospective limb level, comparison of Rbm24 expression with that of MyoD at the same stage of development revealed that the cells that have just entered the myogenic program through the activation of MyoD expression have not activated Rbm24 gene expression (Fig. 2A and B). However, Rbm24 and MyoD transcripts are expressed concomitantly in the myotome that is formed by dermomyotomal Pax3-expressing cells (Fig. 2A and B). To distinguish the cell-type localization of Rbm24, MyoD and Pax3 proteins, we performed double immunofluorescence staining on transverse sections of E11.5 mouse embryos at different levels of the anteroposterior axis: at the level of the hindlimb (Fig. 2D, G, J and M), the interlimb (Fig. 2E, H, K and N) and the forelimb (Fig. 2F, I, L and O). At all three levels examined, Rbm24 protein does not accumulate in Pax3-positive cells of the dermomyotome (Fig. 2G–I). In addition, Rbm24 protein is also not detected in Pax3-positive undifferentiated progenitor cells

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mechanisms of development 134 (2014) 1–15

mechanisms of development 134 (2014) 1–15

that become interspersed within the differentiated myogenic cells of the myotome (arrow in Fig. 2G). These cells, which originate in the central portion of the dermomyotome, have been shown to retain a proliferating, undifferentiated progenitor state throughout late fetal development, and subsequently become integrated into developing myofibers (Gros et al., 2005; Relaix et al., 2005). Moreover, Rbm24 protein is not detected neither in Pax3-positive hypaxial cells that delaminate from the dermomyotome, migrate and span the hindlimb and forelimb buds to ultimately form the appendicular muscles (Fig. 2J and L), nor in Pax3-positive hypaxial cells of the dermomyotome that proliferate and expand ventrally to later form the body wall musculature (Fig. 2K). Double immunofluorescence staining using Rbm24 and MyoD antibodies further confirmed the above observation. Indeed, Rbm24 protein accumulation is restricted to MyoDpositive cells in the myotome (Fig. 2M and N), as well as in myogenic cells spanning the hindlimb and the forelimb (Fig. 2M, O and O′). Altogether, these results lead us to conclude that Rbm24 is not expressed in Pax3-positive myogenic stem cells but is activated in cells that have entered the myogenic program as assayed by the expression of MyoD.

2.3. Rbm24 protein transiently accumulates in differentiated myogenic cells We then examined the expression of Rbm24 protein during the time course of somitic and limb muscle differentiation both in mouse and chick embryo (Fig. 3A–D). Analysis by immunofluorescence on transverse sections of E11.5 mouse embryos at the forelimb level indicated that Rbm24 protein accumulates throughout the dorsoventral extent of the myotome, in cells that have delaminated from the ventrolateral edge of the somite to invade the limb buds, as well as in myotomal hypaxial cells extending ventrally to form the body wall

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musculature (Fig. 3E). Rbm24 protein is also strongly detected in the myocardium (Fig. 3E). Close examination of myotomal cells revealed that Rbm24 protein accumulates heterogeneously in the cytoplasm and is not detected in the nucleus (Fig. 3F and F′). In addition, Rbm24 protein seems to be spatially segregated in several foci within the cytoplasm, but the precise nature of these subcellular foci needs further characterization (Fig. 3F′). To monitor the kinetics of Rbm24 accumulation during myogenic differentiation in vivo, we carried out double immunostaining using Rbm24 antibody and MF20 antibody, which labels myosin heavy chains, at different stages of development. The results show that Rbm24 protein accumulates at the beginning of myogenic differentiation at E11.5 in myosin-positive differentiated cells of myotomes (Fig. 3G–I′). In E11.5 forelimb muscle masses, Rbm24 is expressed before myosin (Fig. 3G–I). Its expression is lost in somites and forelimbs one day later, at E12.5 in differentiated muscle anlagen that can be visualized by MF20 immunostaining (Fig. 3J). No Rbm24 expression is detected at E14.5 (Fig. 3K). The accumulation of Rbm24 protein in differentiated myotomal muscle progenitors was further confirmed in chick embryos. We found that Rbm24 protein significantly co-localizes with myosins in the myotome at 3.5 days of development (Fig. 3L–N′). Altogether, these results demonstrate that Rbm24 protein transiently accumulates in differentiated myogenic cells of myotomes and in developing limbs of vertebrates.

2.4. Rbm24 is a direct downstream gene of MyoD in mouse embryo Since the expression of Rbm24 is restricted to MyoDpositive cells during somitic and limb myogenesis in mice, we wondered whether Rbm24 could be a direct target gene of MyoD in mouse myoblasts. We previously demonstrated that Seb4 could be a direct target of MyoD during Xenopus primary

Fig. 1 – Rbm24 is expressed at all sites of skeletal muscle formation during vertebrate development. (A–E) Detection of Seb4 (A–C), XMyoD (D) and XMyf5 (E) expression by whole-mount in situ hybridization on stage 28 (A) and stage 37 (B–E) Xenopus embryos. (A,A′) Seb4 is expressed in all somites (so) and specifically in the myotome (my) of the somites as shown on transverse vibratome section (A′) of the embryo shown in A at the level of the somite. It is also expressed in non-muscle territories including lens (le) and otic vesicle (ov), and is transiently expressed in developing heart at the stage 28 (he). (B,B′,C) Seb4 expression in hypaxial migrating cells (hyp) (B) as well as in the following branchiomeric cranial muscles: levatores mandibulae muscles anlagen (lev), quadrato-hyoangularis and orbitohyoideus muscles anlagen (qho), and interhyoideus muscle analgen (int.hy). B′ is a transverse vibratome section through the embryo in B, at the level of the head. (D,E) Cranial muscles also express XMyoD (D) but not XMyf5 except for the interhyoideus muscle analgen (E). (F–N) Detection of mouse Rbm24 expression by whole-mount in situ hybridization on E10.5 (F–H), E11.5 (I–K) and E12.5 (L–N) mouse embryos. Rbm24 is expressed in the entire myotomal compartment of all developing somites (so) at all stages. (F–H) At E10.5, it is also expressed in the lens (le), otic vesicle (ov), heart (he) and weakly in the mesodermal core of the first and second branchial arches (arrows in F). It is not detected in developing forelimbs (asterisks in F and G) and hindlimbs (asterisks in H). (I–K) At E11.5, Rbm24 is detected in all appendicular muscle masses at the forelimb (fl) and hindlimb (hl) levels (arrowheads in I) and in further developing head muscles (hm). (L–N) At E12.5, the expression of Rbm24 persists in appendicular muscle masses and developing head muscles. Strong expression can be observed in the limb (arrowheads in L). (M) Diaphragm muscle (dia) also expresses mRbm24 at this stage. (O–T) Analysis of chick Rbm24 expression by wholemount in situ hybridization on 3 days (stage HH20) (O–Q) and 3.5 days (stage HH23) (R–T) chick embryos. As in mouse embryos, chick Rbm24 is expressed in the entire myotomal compartment of all developing somites (so) at all stages. It is not detected in limb buds at 3 days of development (asterisks in O–Q) but is observed in developing forelimbs (fl) and hindlimbs (hl) at 3.5 days of development (arrowheads in R and S). Chick Rbm24 is expressed in the mesodermal core of the first and second branchial arches at 3 days of development (arrows in T). Other expression sites include otic vesicle (ov), lens (le) and heart (he).

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mechanisms of development 134 (2014) 1–15

mechanisms of development 134 (2014) 1–15

myogenesis in presomitic mesoderm (Li et al., 2010). However, unlike avian and mammalian embryo, Xenopus myogenesis is characterized by a wave of primary myogenesis taking place under the control of Myf5 and MyoD within the presomitic mesoderm of early gastrulae. The so formed mononucleated primary myotomal myofibers completely disappear during metamorphosis and are progressively replaced by secondary adult multinucleated myofibers (Chanoine and Hardy, 2003; Della Gaspera et al., 2012). By luciferase reporter gene assays in Xenopus ectodermal explants, we have demonstrated that MyoD ectopic expression could activate a 0.65 kb Rbm24 promoter sequence harboring six consensus (CANNTG) E-boxes, which are potential binding sites for MyoD and other bHLH protein (Li et al., 2010). To confirm that MyoD is a direct regulator of Rbm24 gene expression during somitic myogenic differentiation in vivo, we analyzed the recruitment of MyoD to the mouse Rbm24 gene in somites and limbs. Within the mouse Rbm24 gene, more than thirty E-boxes are present in the 5 kb regulatory region upstream of the transcription start site and 6 E-boxes are present in the 5′-untranslated region (UTR). However, multiple sequence analyses using the genome alignment portal of ECR browser revealed that only four E-boxes, namely E1 to E4, located in the 5′-UTR were perfectly conserved among different mammalian species (Fig. 4A and B). We thus decided to analyze whether this region could bind MyoD protein in vivo. Chromatin immunoprecipitation (ChIP) assays on E11.5 mouse embryos was performed using MyoD-specific antibody and PCR primers encompassing the four conserved E-boxes (Fig. 4A and B). The result indicates that endogenous MyoD binds to the 0.4 kb Rbm24 5′-UTR region containing the four E-boxes, while an anti-GFP antibody does not immunoprecipitate this sequence (Fig. 4C). The binding of MyoD to the Rbm24 5′-UTR correlates with an increased acetylated histone 4 mark, reflecting an opened state of chromatin in this region (Shogren-Knaak et al., 2006). Thus, this observation suggests that MyoD may directly activate Rbm24 gene expression during myogenesis in mouse. To further assess whether MyoD participates in the activation of Rbm24 transcription in vivo, we analyzed the expression of Rbm24 in MyoD–/– mutant mice during embryonic myogenesis. While homozygote MyoD–/– and heterozygote MyoD+/– mutant E11.5 embryos display a similar level of Rbm24 expression at the level of the myotomes, expression of Rbm24 in both forelimbs and hindlimbs appears reduced in the

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homozygote MyoD–/– mutants compared to the heterozygote MyoD+/– embryos (Fig. 4D). Moreover, Rbm24 expression in the head muscle anlagen is dramatically reduced in the homozygote mutants compared to the heterozygote embryos (Fig. 4D). We conclude that MyoD regulates Rbm24 expression, fully in head muscles, partially in limb muscles and not in myotomes. Thus, MyoD may not be the only upstream regulator of Rbm24 gene in myogenic cells of the myotome. It may participate with yet unknown transcription factors in the activation of the Rbm24 gene in the early differentiated myotomal cells. Concerning the genetic link between MyoD and Rbm24 during limb myogenesis, since MyoD–/– embryos exhibit a delayed differentiation of hypaxial musculature, resulting in the reduction of differentiation marker expression (Havis et al., 2012; Kablar et al., 1997), it remains unclear whether MyoD directly regulates Rbm24 expression in limb myogenic cells. However, the expression of Rbm24 in the head muscle anlagen of E11.5 MyoD–/– embyros is more dramatically reduced than the delay of differentiation reported at this developmental stage (Kablar et al., 1997). This observation suggests that Rbm24 is a direct downstream gene of MyoD in vivo.

2.5. Rbm24 is required for myogenic differentiation in vivo The Rbm24 expression pattern in MyoD-positive cells and the in vivo recruitment of MyoD to the Rbm24 5′-UTR region imply that Rbm24 plays an important role in the control of the myogenic differentiation program. We therefore directly tested whether Rbm24 is required for somitic myogenic differentiation in vivo using morpholino knockdown approach during chick somitic myogenesis. We used somite electroporation in chick embryos, which allows the targeting of dermomyotomal myogenic progenitor cells (Scaal et al., 2004). Right side interlimb somites of HH15 (2.5 days) chick embryos were electroporated with a fluorescein-labeled antisense morpholino oligonucleotide inhibiting the translation of chick Rbm24 mRNA (Fig. 5A–C), or with a control antisense morpholino oligonucleotide containing six mismatches, which was not able to bind to Rbm24 mRNA (Fig. 5D–F). Left side somites were not electroporated and served as an internal control. Twenty-four hours after electroporation, green fluorescence could be observed in most of the myotomal compartment of electroporated somites as

Fig. 2 – Rbm24 is not expressed in Pax3-positive premyogenic cells but starts to be expressed in MyoD-positive myoblasts. (A,B) Detection of mouse Rbm24 (A) and MyoD (B) expression by whole-mount in situ hybridization on E10.5 mouse embryos. Rbm24 is detected in the myotome (my) similarly to MyoD, but it is not detected in the hypaxial progenitors invading the forelimb (fl) bud. (C) Detection of LacZ expression by whole-mount X-Gal staining on an E10.5 Pax3nLacZ/+ transgenic mouse embryo. (D–F) The levels of the sections are indicated on the embryo by a red line. The areas focused in G–O are boxed on the transverse sections. (G–L) Double immunofluorescence staining of Rbm24 and Pax3 proteins on transverse sections of E11.5 mouse embryo at the level of the hindlimb (G,J), interlimb (H,K) and forelimb (I,L). At all levels Rbm24 protein accumulates in the myotome (my) but it is excluded from Pax3-expressing dermomyotomal cells (dm) and from Pax3-positive cells that are interspersed within the myotome (arrow in G). At the hindlimb (G), interlimb (H) and forelimb (I) levels, Rbm24 does not colocalize with Pax3 in migrating cells. (M–O) Double immunofluorescence staining of Rbm24 and MyoD proteins on transverse sections of E11.5 mouse embryos at the level of the hindlimb (M), interlimb (N) and forelimb (O). Rbm24 colocalizes with MyoD in the myotome (arrow in N) and in myogenic cells that have migrated into the limbs. (O′) Higher magnification of doubly labeled cells of the forelimb. nt, neural tube; fl, forelimb; hl, hindlimb; he, heart.

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mechanisms of development 134 (2014) 1–15

mechanisms of development 134 (2014) 1–15

well as in hypaxial and epaxial dermomyotomal lips (Fig. 5A, A′, D and D′). Differentiation of myotomal myogenic cells was examined 24 hours after electroporation by immunofluorescence on serial transverse sections at the level of electroporated somites. Embryos electroporated with a chicken Rbm24 antisense morpholino displayed a marked reduction of Rbm24 protein level in electroporated myotomal cells compared to the control sides, thus demonstrating the ability of the morpholino to inhibit mRNA translation (Fig. 5B). Myotomal cells prevented from translating Rbm24 mRNA exhibited a failure to differentiate as revealed by a strong decrease of MF20 immunostaining of the myosin heavy chain as compared to control sides (Fig. 5C). By contrast, myotomal cells targeted by the control morpholino and visualized by the green fluorescence (Fig. 5D and D′) did not show detectable decrease of Rbm24 protein (Fig. 5E) or myosin proteins (Fig. 5F) as compared to the non-electroporated contralateral somites. To overcome a bias in the plane of transverse sections, wholemount immunostaining on chick embryos that have been electroporated either with the Rbm24 morpholino (Fig. 5G, G′ and G″) or with the Rbm24 control morpholino (Fig. 5H, H′ and H″) has been performed. Detection of fluorescein on the whole embryo shows the accumulation of the morpholino in the interlimb somites that have been electroporated, with an obvious accumulation in the hypaxial lip (hyp) and in the myotome (my) (Fig. 5G and H). Immunodetection of myosin protein revealed that the myotome of the somites targeted by the Rbm24 morpholino are severely reduced with fewer and less elongated myofibers as compared to the non-electroporated somites at the same side (Fig. 5G′). Moreover, interlimb somites of the non-electroporated contralateral side of the embryo display normally differentiated myotomes (Fig. 5G″). The control experiment using the Rbm24 control morpholino revealed that the myotomes of interlimb somites differentiate similarly as those of the non-electroporated somites of the same side (Fig. 5H′) and those of the contralateral side of the embryo (Fig. 5H″). Altogether, these results demonstrate the specificity of the Rbm24 morpholino and indicate that Rbm24 is required for somitic myogenic differentiation in vivo.

3.

9

Discussion

3.1. Rbm24 expression is associated with all sites of muscle differentiation in vertebrate embryos This study established that the Rbm24 gene is expressed at all sites of myogenesis including head, trunk and limb in Xenopus, mouse and chick embryos. Furthermore, by analyzing the precise distribution of the protein during myogenesis in mice embryos using specific antibodies, we have demonstrated that Rbm24 protein does not accumulate in dermomyotomal and migrating Pax3-positive premyogenic progenitors, but starts to accumulate in MyoD-expressing myoblasts of myotomes and limbs at E11.5 stage of development. Thus, it appears that the expression of Rbm24 protein is associated with a differentiated state of myogenic cells in a narrow time window since we could not detect the protein one day later, at E12.5 and even later at E14.5 in developing truncal and appendicular musculature. This short period of accumulation of Rbm24 protein around the time of MyoD expression, to potentially control the entry into differentiation of the myoblasts, is not transcriptionally regulated since we showed that Rbm24 transcripts are present until E14.5 in all developing muscle masses. Whether the protein level decreases after E11.5 to become undetectable by classical immunofluorescence analysis needs further investigation. Moreover, since Rbm24 transcripts and protein strongly accumulate in heart and skeletal muscles in adult, it is necessary to draw a detailed kinetics of its expression during late embryogenesis and after birth. The results of this study represent the first report of a detailed expression of an RNA-binding protein during vertebrate embryonic myogenesis. Our expression analysis suggests that Rbm24 could function in the differentiation step of skeletal muscle progenitors. This is in agreement with previous observations demonstrating a role for Rbm24 in promoting myogenic differentiation through post-transcriptional regulation of Myogenin stability in the C2C12 mouse myoblast cell line (Jin et al., 2010; Miyamoto et al., 2009). In addition, we have extended previous observation by showing that the expression

Fig. 3 – Rbm24 protein transiently accumulates in myotomal and limb myogenic cells. (A–D) Mouse and chick embryos showing the levels of sections. (E-F′) Detection of mouse Rbm24 protein by immunofluorescence analysis on transverse sections of E11.5 mouse embryos at the forelimb level as indicated in the embryo in A by a red line. (E) Rbm24 protein accumulates in myotomes (my) as well as in forelimb (fl) myogenic cells. It is also strongly expressed in the heart (he). (F) Higher magnification of myotomal cells shows the cytoplasmic accumulation of Rbm24 protein. Nuclei were labeled by DAPI staining. (F′) Localization of Rbm24 protein in the cytoplasm of a myotomal cell as heterogenous and punctuated foci. (G–I′) Double immunofluoresecence staining of Rbm24 and myosin proteins on transverse sections of mouse embryos at the forelimb level as indicated in A. At E11.5, Rbm24 protein accumulates in myosin-positive differentiated muscle cells within the myotome. Rbm24 protein accumulates in myogenic cells that have reached the limb but have not initiated their differentiation as assessed by the lack of myosin expression. (I′) Higher magnification of doubly labeled myotomal cells. (J,K) Double immunofluorescence staining of Rbm24 and myosin proteins on transverse sections of E12.5 and E14.5 mouse embryos at the forelimb level as indicated in B and C, respectively. At E12.5 and E14.5, Rbm24 protein is absent in differentiating skeletal muscles and in myocardial cells. (L–N′) Double immunofluorescence staining of chick Rbm24 and myosin proteins on transverse sections of 3 days (HH20) chick embryo at the interlimb level as indicated in D. Rbm24 colocalizes with myosin proteins in differentiated muscle cells in the myotome. (N′) Higher maginification of doubly labeled myotomal cells. nt, neural tube; ve, vertebrae; hu, humerus; hl, hindlimb; he, heart.

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Fig. 4 – The 5′-UTR of the mouse Rbm24 gene contains conserved E-boxes that are directly bound and potentially activated by MyoD in vivo. (A) Schematic representation of the 0.75 kb 5′-UTR and the 5 kb regulatory sequences upstream the transcription start site of the mouse Rbm24 gene. Red boxes represent the conserved E-boxes while grey boxes identify other non-conserved E-boxes. Arrows annotated F and R indicate the location of primers used for PCR in the ChIP experiment. (B) Nucleotide sequence alignments of cow, chimpanzee and human E-boxes (namely E1 to E4) located in the 0.75 kb 5′-UTR region. (C) ChIP assays were performed from E11.5 mice embryos with antibodies against MyoD, acetylhistone H4 (AcH4) or GFP as a negative control. ChIP products and input chromatin as a positive control were amplified by PCR using primers encompassing the four conserved E-boxes (E1 to E4) located in the 5′-UTR region. The binding of MyoD to the mouse Rbm24 5′-UTR is detected. (D) Analysis of Rbm24 expression by whole-mount in situ hybridization on E11.5 MyoD+/– and MyoD–/– embryos. Although Rbm24 is similarly expressed in the myotome of the homozygote and the heterozygote mutant embryos, its expression is dramatically decreased in the forelimb, the hindlimb and the head muscle anlagen (arrows) of homozygote MyoD–/– embryos.

mechanisms of development 134 (2014) 1–15

of Seb4, the orthologous gene of Rbm24 in Xenopus, is restricted to MyoD-positive branchiomeric cranial muscles, which are known to be the first muscles to differentiate in the head (Della Gaspera et al., 2012), this also reinforces the role for Rbm24 during muscle differentiation.

3.2. Rbm24 is required for myogenic differentiation in vivo downstream of MyoD We have demonstrated by ChIP that MyoD is recruited to the Rbm24 5′-UTR of the Rbm24 gene in E11.5 mouse embryos, suggesting that MyoD may be directly involved in regulating Rbm24 expression in myogenic cells during mouse embryogenesis. Moreover, examination of Rbm24 expression in MyoD mutant embryo indicates that MyoD participates with other unknown transcription factors in the activation of Rbm24 expression. These results are consistent with our previous study in which we have demonstrated that MyoD could activate a 0.65 kb Xenopus Seb4 regulatory region harboring six core consensus (CANNTG) E-boxes (Li et al., 2010). They are also in agreement with a recent observation showing that MyoD binds to genomic sequence of Xenopus Seb4 (Maguire et al., 2012). Thus, our finding suggests that the regulation of Rbm24 by MyoD during muscle differentiation may be conserved among vertebrate species. Mouse mutant analyses indicate that MyoD function is associated with the muscle differentiation step (Hasty et al., 1993; Nabeshima et al., 1993; Rawls et al., 1998), while Myf5 is involved in muscle specification (Tajbakhsh et al., 1997). Moreover, Myf5 does not support muscle differentiation in the absence of the other three MRFs (Valdez et al., 2000). Therefore, the fact that Rbm24 is under the control of MyoD reinforces our hypothesis that Rbm24 is involved in the differentiation step of myogenesis. Seb4, the Xenopus ortholog of Rbm24, plays a role during myogenesis activation at gastrula stages (Li et al., 2010). This first wave of myogenesis, which takes place in the presomitic mesoderm, is a Xenopus specificity that is not shared by amniotes and mammals. As a result, early downregulation of Seb4 in Xenopus embryo impairs early mesoderm formation, thus precluding further investigation of Seb4 during somitic myogenic differentiation. We have therefore examined, in the present study, the functional role of Rbm24 in myogenic differentiation by using a morpholino knockdown approach during chick somitic myogenesis. We have demonstrated that Rbm24 loss of function impairs the differentiation of somitic myogenic progenitor cells since myotomal cells, prevented from translating Rbm24 mRNA, fail to express myosin protein. In zebrafish embryo, Rbm24 gene exists as two variants, Rbm24a and Rbm24b. A previous study indicates that knockdown of Rbm24a variant leads to impaired contractility of the heart due to a decrease in the expression of several components of the sarcomere. These results reveal an important role played by this RNA-binding protein in cardiogenic differentiation process (Poon et al., 2012). However, somitic myogenic differentiation, which could not be determined by looking overall somite morphology, has not been examined in this study. Thus, our functional analysis combined with expression data provides a direct evidence of the requirement of Rbm4 in the differentiation step of myogenesis.

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3.3. Possible mechanisms of Rbm24 function during myogenic differentiation in vivo The underlying mechanism by which Rbm24 functions during myogenic differentiation in vivo has to be investigated. Using a RIP-Chip assay from Flag-tagged Rbm24overexpressing C2C12 myogenic cells, it was found that Rbm24 protein interacts with the 3′-UTR of Myogenin mRNA and regulates its stability (Jin et al., 2010). Similar analysis needs to be carried out in vivo to determine the RNA targets of endogenous Rbm24 protein. In addition, close examination of Rbm24 protein in myotomal cells revealed that it accumulates heterogenously in the cytoplasm and is not detected in the nucleus. Indeed, Rbm24 protein seems to segregate in several foci within the cytoplasm. In vitro studies of the co-localization of Rbm24 and Ge-1 indicated that the heterogenous cytoplasmic accumulation of Rbm24 is not associated to processingbodies (P-bodies), which are dynamic and reversible nonmembrane-bound cytoplasmic structures that accumulate a fraction of translationally silent mRNAs (data not shown). P-bodies could be defined as the sites of mRNA sequestration, reversible mRNA repression and mRNA decay (Olszewska et al., 2012). The exclusion of Rbm24 protein from P-bodies is consistent with the fact that Rbm24 has never been associated to translation repression. In C. elegans, the Rbm24related protein SUP-12 was shown to regulate the musclespecific splicing of unc-60 pre-mRNA, which gives rise to unc60A and unc-60B encoding non-muscle and muscle isoforms of actin depolymerizing factor, respectively (Anyanful et al., 2004). However, in vertebrates, these two proteins are encoded by separate genes and exhibit distinct tissue distribution; thus, whether vertebrate Rbm24 plays a role in mRNA splicing during myogenic differentiation remains to be determined. Although muscle lineage specification has been studied for decades, the mechanism that regulates overall muscle differentiation program is still an area of intense study to further understand muscle developmental processes. Moreover, compared to other fields, research on RNA-binding proteins in skeletal muscle biology is still in its infancy. In particular, the role of RNA-binding proteins in controlling muscle development during embryogenesis remains largely unknown. Our results demonstrating that Rbm24 regulates somitic myogenic differentiation and stabilize muscle phenotype in vertebrate embryos should help to further understand the molecular pathway underlying muscle cell specification and differentiation.

4.

Materials and methods

4.1.

Xenopus, chick and mouse embryos

Xenopus embryos were obtained from females injected with 500 UI of human chorionic gonadotropin (Sigma) and artificially fertilized. Fertilized chick eggs from commercial sources, JA 57 strain (Morizeau, Dangers) or White Leghorn (HAAS, Kaltenhouse), were incubated at 37 °C. Embryos were staged according to Hamburger and Hamilton (HH) stages (Hamburger, 1992). MyoD–/– (Kablar et al., 1997), Pax3IRESnLacZ (Relaix et al., 2004)

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or C57bl6 wild-type mouse embryos were collected after natural overnight mating.

4.2. Whole-mount in situ hybridization and X-Gal staining Xenopus whole-mount in situ hybridization was performed using standard protocol (Harland, 1991). Probes including Seb4,

XMyoD and XMyf5 were previously described (Li et al., 2010) and were labeled using digoxygenin-11-UTP and appropriate RNA polymerase (Roche). Mouse and chick embryos were fixed in 4% paraformaldehyde and processed for whole-mount in situ hybridization as previously described (Grifone et al., 2004). The mouse and chicken Rbm24 probes were synthetized using T7 RNA polymerase after linearization of pGEM-T-mRbm24 and pGEM-T-cRbm24 by Sac I. Vibratome sections (100 µm) of wholemount stained embryos were made after inclusions of the

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embryos in 4% agarose. Sections were then mounted using Kaiser’s glycerol gelatin solution (Merck). Pax3nLacZ mouse embryos were fixed in 4% paraformaldehyde for 1 hour, washed in PBS and stained in X-Gal-staining solution (1 mg/ml X-Gal, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6 and 2 mM MgCl2 in PBS) at 37 °C for 2 hours. Images of wholemount stained embryos were taken using a stereomicroscope (S8 APO, Leica).

4.3. Immunohistochemistry on paraffin-embedded embryo sections Mouse and chicken embryos were fixed in 4% paraformaldehyde (PFA) for 2 hours, washed in PBS, dehydrated in ethanol, cleared in xylene and immersed in warm liquid paraffin before being embedded in a paraffin block. Sections of 10 µm thickness were made on a microtome (Leica). For immunohistochemistry, sections were deparaffinized in xylene, rehydrated in ethanol and rinsed in PBS. Antigen retrieval was performed to unmask the antigen epitope in 10 mM citrate buffer (pH 6.0) at 95–100 °C for 15 minutes. Sections are rinsed in PBS, blocked for 1 hour in saturation solution (3% BSA, 0.1% Triton in PBS) and incubated overnight at 4 °C with primary antibodies (Rbm24, ProteinTech, 1/1000; Pax3, DSHB, 1/250; MyoD, Dako, 1/100; MF20, DSHB, 1/500). After three washes in PBT (0.1% Tween 20 in PBS), sections were incubated for 1 hour in alexa-488 conjugated anti-rabbit (1/400) or alexa-596 conjugated anti-mouse (1/400) secondary antibodies (Interchim), washed in PBT prior to mounting in Dako Fluorescent Mounting medium. Confocal images were taken using a confocal microscope (TCS SP5 II, Leica).

4.4.

Whole-mount immunostaining of chick embryos

Chicken embryos were fixed in 4% paraformaldehyde (PFA) overnight, washed in PBS and permeabilized in a PBS-0.1% Triton X-100 solution for 1 hour. Embryos are then blocked in a 20% fetal bovine serum solution for 2 hours before an overnight incubation at 4 °C with the MF20 antibody (1/500) diluted in the same blocking solution. Embryos are rinsed in PBS and incu-

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bated for 2 hours with horseradish peroxidase (HRP)-conjugated antibody, rinsed and processed to HRP detection using 3,3′diaminobenzidine (DAB) tablets (Sigma).

4.5.

Chromatin immunoprecipitation (ChIP)

ChIP assays were performed as previously described (Havis et al., 2006; Li et al., 2013). Twelve E11.5 mouse embryos were homogenized using a mechanical disruption device (Lysing Matrix A, Fast Prep MP1) for 40 seconds. Ten micrograms of the rabbit polyclonal MyoD antibody (Santa Cruz, sc-304x) or 8 µg of the acetylated histone H4 (AcH4) antibody (Upstate Biotechnology) as a positive control were used to immunoprecipitate 30 µg of sonicated chromatin. ChIP products were analyzed by PCR to amplify a 395 bp Rbm24 5′-UTR containing 5 (CANNTG) E-boxes using the forward primer (5′-AGAGCCCCAGCCA GGCGACC-3′) and reverse primer (5′-CAGAGAGGGTG GTGGTCTCC-3′). The ChIP experiments were performed three times.

4.6. Morpholino antisense oligonucleotides in ovo somite electroporation Fluorescein-labeled morpholino oligonucleotide against chicken Rbm24 (5′-TGGTGTCCTTCTGCGTCGTGTGCAT-3′) and the corresponding control morpholino containing six mismatches, indicated by lower cases (5′-TGcTGTgCTTgTG CcTCGTcTGgAT-3′) were from Gene Tools. They were suspended in PBS and used at a concentration of 500 µM. At 2.5 days of development (HH15 stage), which corresponds to 25-somites chick embryo, interlimb somites were made visible by removing vitelline membrane overlying the embryo. A glass capillary was carefully inserted into the somitocoele of five interlimb somites. Fluorescein-labeled morpholino solution was smoothly pushed by injector pressure into the somitocoele of the somites. After injection, dermomyotomes were quickly electroporated with hand-made electrodes at the level of the injected interlimb somites. The electric pulses generated by the electric current will cause a breakdown of somitic cell plasma membrane and drive the negatively charged fluorescein-

Fig. 5 – Rbm24 is required for somitic myogenic differentiation in vivo. Analysis of chick Rbm24 and myosin expression in chick embryos electroporated with fluorescein-labeled Rbm24 morpholino or Rbm24 control morpholino. (A–C) Representative images from serial sections of chick embryos electroporated with Rbm24 morpholino. (A) Localization of Rbm24 morpholino in almost all the myotome (my) as well as in epaxial (ep) and hypaxial (hyp) lips of the dermomyotome as revealed by the presence of fluorescein. (A′) Higher magnification image corresponding to A. (B,C) Rbm24 morpholino dramatically decreases the level of Rbm24 (B) and myosin (C) proteins in electroporated myotome compared to the non-electroporated contralateral one. (D–F) Representative images from serial sections of chick embryos electroporated with a control Rbm24 morpholino containing six mismatches. (D) Distribution of the control morpholino. (D′) Higher magnification image corresponding to D. (E,F) The protein level of Rbm24 (E) and myosin (F) is similar in the targeted and non-targeted myotomes of the somites. nt, neural tube. (G-H″) Chick embryos electroporated with Rbm24 morpholino (G-G″) or Rbm24 control morpholino (H-H″). (G,H) Fluorescein detection shows the electroporated somites at the right side of the embryo, indicated by brackets. The left side is not electroporated. Fluorescein is detected in the hypaxial lip (hyp) as well as in the myotome (my). (G′,H′) Myosin immunodetection on the right side of the embryo shown in G and H. Brackets indicate the electroporated somites. The somites targeted by the Rbm24 morpholino obviously display a failure of differentiation of the myotome compared to the non-electroporated somites at the same side of the embryo. The somites targeted by the Rbm24 control morpholino differentiate similarly as the non-electroporated somites at the same side of the embryo. (G″,H″) Myosin immunodetection on the left side of the same embryos as shown in G and H. Non-electroporated contralateral somites differentiate normally as assessed by the immunodetection of myosin protein.

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labeled morpholino oligonucleotide into the dermomyotomal cells that lie adjacent to the positive electrode and will contribute to the underlying myotome. The immunodetection of Rbm24 and myosin proteins were analyzed on 6 different electroporated embryos for each condition.

Author contributions R.G, D.D. and D.L.S designed the study. R.G, X.X and A.S carried out the in situ hybridization and immunohistochemistry experiments. R.G. and X.X performed the ChIP experiments. A.B. performed the chick somite electroporations. R.G and D.L.S contributed to the data analysis and interpretation and wrote the manuscript.

Acknowledgements We are grateful to Frédéric Relaix for providing Pax3nLacZ/+ mice. We thank Pascal Maire and Clémence Carron for discussion and critical reading of the manuscript. This work was supported by grants from Association Française contre les Myopathies (AFM) [N°13737] and Agence Nationale de la Recherche (ANR) [ANR-09-BLAN-0262-03] to D.L. Shi.

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