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Cranial muscles have been the focus of many studies over the years because of their unique developmental programs and relative resistance to illnesses. In addition, head muscles possess clonal relationships with heart muscles and have been highly remodeled during vertebrate evolution. Here, we provide an overview of recent findings that have helped to redefine the boundaries and lineages of cranial mesoderm. These studies have important implications regarding the emergence of muscle connective tissues, which can share a common origin with skeletal muscle. We also highlight new regulatory networks of various muscle subgroups, particularly those derived from the most caudal arches, which remain poorly defined. Finally, we suggest future research avenues to characterize the nature of their intrinsic specificities and their emergence during evolution.

How distinct cell fates are manifested by direct lineage ancestry from bipotent progenitors, or by specification of individual cell types is a key question for understanding the emergence of tissues. The interplay between skeletal muscle progenitors and associated connective tissue cells provides a model for examining how muscle functional units are established. Most craniofacial structures originate from the vertebrate-specific neural crest cells except in the dorsal portion of the head, where they arise from cranial mesoderm. Here, using multiple lineage-tracing strategies combined with single cell RNAseq and in situ analyses, we identify bipotent progenitors expressing Myf5 (an upstream regulator of myogenic fate) that give rise to both muscle and juxtaposed connective tissue. Following this bifurcation, muscle and connective tissue cells retain complementary signalling features and maintain spatial proximity. Disrupting myogenic identity shifts muscle progenitors to a connective tissue fate. The emergence of Myf5-derived connective tissue is associated with the activity of several transcription factors, including Foxp2. Interestingly, this unexpected bifurcation in cell fate was not observed in craniofacial regions that are colonised by neural crest cells. Therefore, we propose that an ancestral bi-fated program gives rise to muscle and connective tissue cells in skeletal muscles that are deprived of neural crest cells.

Adult skeletal muscles are maintained during homeostasis and regenerated upon injury by muscle stem cells (MuSCs). A heterogeneity in self-renewal, differentiation and regeneration properties has been reported for MuSCs based on their anatomical location. Although MuSCs derived from extraocular muscles (EOM) have a higher regenerative capacity than those derived from limb muscles, the molecular determinants that govern these differences remain undefined. Here we show that EOM and limb MuSCs have distinct DNA methylation signatures associated with enhancers of location-specific genes, and that the EOM transcriptome is reprogrammed following transplantation into a limb muscle environment. Notably, EOM MuSCs expressed host-site specific positional Hox codes after engraftment and self-renewal within the host muscle. However, about 10% of EOM-specific genes showed engraftment-resistant expression, pointing to cell-intrinsic molecular determinants of the higher engraftment potential of EOM MuSCs. Our results underscore the molecular diversity of distinct MuSC populations and molecularly define their plasticity in response to microenvironmental cues. These findings provide insights into strategies designed to improve the functional capacity of MuSCs in the context of regenerative medicine., Author summary Adult skeletal muscles are regenerated upon injury by muscle stem cells (MuSCs). A heterogeneity in expression of key myogenic regulators and regeneration properties has been reported for MuSCs based on their anatomical location. Although MuSCs derived from extraocular muscles (EOM) have a higher regenerative capacity than those derived from limb muscles, the molecular determinants that govern these differences remain undefined. Here we show that EOM and limb MuSCs have distinct transcriptome and DNA methylation signatures, and that the EOM transcriptome is reprogrammed following transplantation into a limb muscle environment. Notably, EOM MuSCs adopted host-site specific positional Hox codes after engraftment within the host muscle. However, about 10% of EOM-specific genes were resistant to alterations following heterotopic engraftment, pointing to molecular determinants of the high engraftment potential of EOM MuSCs. Our results underscore the molecular diversity of distinct MuSC populations and molecularly define their plasticity in response to microenvironmental cues. These findings provide insights into strategies designed to improve the functional capacity of MuSCs in the context of regenerative medicine.

International audience; Pax7 expression marks stem cells in developing skeletal muscles and adult satellite cells during homeostasis and muscle regeneration. The genetic determinants that control the entrance into the myogenic program and the appearance of PAX7+ cells during embryogenesis are poorly understood. SIX homeoproteins are encoded by the Sine oculis homeobox related Six1-Six6 genes in vertebrates. Six1, Six2, Six4 and Six5 are expressed in the muscle lineage. Here we tested the hypothesis that Six1 and Six4 could participate in the genesis of myogenic stem cells. We show that fewer PAX7+ cells occupy a satellite cell position between the myofiber and its associated basal lamina in Six1 and Six4 (s1s4KO) at E18. However, PAX7+ cells are detected in remaining muscle masses present in the epaxial region of the double mutant embryos and are able to divide and contribute to muscle growth. To further characterize the properties of s1s4KO PAX7+ cells, we analyzed their transcriptome and tested their properties after transplantation in adult regenerating tibialis anterior (TA) muscle. Mutant stem cells form hypotrophic myofibers that are not innervated but retain the ability to self-renew.

Summary MyoD and Myf5 are fundamental regulators of skeletal muscle lineage determination in the embryo, and their expression is induced in satellite cells following muscle injury. MyoD and Myf5 are also expressed by satellite cell precursors developmentally, although the relative contribution of historical and injury-induced expression to satellite cell function is unknown. We show that satellite cells lacking both MyoD and Myf5 (double knockout [dKO]) are maintained with aging in uninjured muscle. However, injured muscle fails to regenerate and dKO satellite cell progeny accumulate in damaged muscle but do not undergo muscle differentiation. dKO satellite cell progeny continue to express markers of myoblast identity, although their myogenic programming is labile, as demonstrated by dramatic morphological changes and increased propensity for non-myogenic differentiation. These data demonstrate an absolute requirement for either MyoD or Myf5 in muscle regeneration and indicate that their expression after injury stabilizes myogenic identity and confers the capacity for muscle differentiation., Highlights • MyoD or Myf5 expression in satellite cells is essential for muscle regeneration • Satellite cells lacking both regulatory genes exhibit labile myogenic programming • A single functional allele of either MyoD or Myf5 can support muscle regeneration • Satellite cells lacking both MyoD and Myf5 are maintained with aging, In this article, Goldhamer and colleagues show that loss of both MyoD and Myf5 in skeletal muscle satellite cells results in regenerative failure following injury. Satellite cell progeny accumulate in injured muscle and continue to express markers of myoblast identity, but do not undergo muscle differentiation, and exhibit a propensity for non-myogenic differentiation.

Summary Satellite cells are responsible for skeletal muscle regeneration. Upon activation, they proliferate as transient amplifying myoblasts, most of which fuse into regenerating myofibers. Despite their remarkable differentiation potential, these cells have limited migration capacity, which curtails clinical use for widespread forms of muscular dystrophy. Conversely, skeletal muscle perivascular cells have less myogenic potential but better migration capacity than satellite cells. Here we show that modulation of Notch and PDGF pathways, involved in developmental specification of pericytes, induces perivascular cell features in adult mouse and human satellite cell-derived myoblasts. DLL4 and PDGF-BB-treated cells express markers of perivascular cells and associate with endothelial networks while also upregulating markers of satellite cell self-renewal. Moreover, treated cells acquire trans-endothelial migration ability while remaining capable of engrafting skeletal muscle upon intramuscular transplantation. These results extend our understanding of muscle stem cell fate plasticity and provide a druggable pathway with clinical relevance for muscle cell therapy., Graphical Abstract, Highlights • DLL4 and PDGF-BB change satellite cell morphology, proliferation, and differentiation • DLL4 and PDGF-BB induce both perivascular and satellite cell gene expression • Treated satellite cells acquire perivascular cell properties and improved migration • Human satellite cell-derived myoblasts respond to DLL4 and PDGF-BB treatment, Gerli and Moyle and colleagues show that treatment with molecules involved in developmental specification of pericytes (DLL4 and PDGF-BB) alters satellite cell fate and provides them with features potentially relevant for novel cell therapy protocols.

In vertebrates, head and trunk muscles develop from different mesodermal populations and are regulated by distinct genetic networks. Neck muscles at the head-trunk interface remain poorly defined due to their complex morphogenesis and dual mesodermal origins. Here, we use genetically modified mice to establish a 3D model that integrates regulatory genes, cell populations and morphogenetic events that define this transition zone. We show that the evolutionary conserved cucullaris-derived muscles originate from posterior cardiopharyngeal mesoderm, not lateral plate mesoderm, and we define new boundaries for neural crest and mesodermal contributions to neck connective tissue. Furthermore, lineage studies and functional analysis ofTbx1-andPax3-null mice reveal a unique genetic program for somitic neck muscles that is distinct from that of somitic trunk muscles. Our findings unveil the embryological and developmental requirements underlying tetrapod neck myogenesis and provide a blueprint to investigate how muscle subsets are selectively affected in some human myopathies.

During growth, homeostasis and regeneration, stem cells are exposed to different energy demands. Here, we characterise the metabolic pathways that mediate the commitment and differentiation of mouse skeletal muscle stem cells, and how their modulation can influence the cell state. We show that quiescent satellite stem cells have low energetic demands and perturbed oxidative phosphorylation during ageing, which is also the case for cells from post-mortem tissues. We show also that myogenic fetal cells have distinct metabolic requirements compared to those proliferating during regeneration, with the former displaying a low respiration demand relying mostly on glycolysis. Furthermore, we show distinct requirements for peroxisomal and mitochondrial fatty acid oxidation (FAO) in myogenic cells. Compromising peroxisomal but not mitochondrial FAO promotes early differentiation of myogenic cells. Acute muscle injury and pharmacological block of peroxisomal and mitochondrial FAO expose differential requirements for these organelles during muscle regeneration. Taken together, these observations indicate that changes in myogenic cell state lead to significant alterations in metabolic requirements. In addition, perturbing specific metabolic pathways impacts on myogenic cell fates and the regeneration process., Summary: Distinct energy metabolism pathways act during mouse skeletal muscle stem cell commitment and differentiation in different physiological states.

The work described in this article is partially covered by patent application PCT/EP2012/066793 (publication number WO2013030243 A1). O.P., J.C. and M.K. are co-founders and shareholders of Anagenesis Biotechnologies, a start-up company specializing in the production of muscle cells in vitro for cell therapy and drug screenin; International audience; Body skeletal muscles derive from the paraxial mesoderm, which forms in the posterior region of the embryo. Using microarrays, we characterize novel mouse presomitic mesoderm (PSM) markers and show that, unlike the abrupt transcriptome reorganization of the PSM, neural tube differentiation is accompanied by progressive transcriptome changes. The early paraxial mesoderm differentiation stages can be efficiently recapitulated in vitro using mouse and human pluripotent stem cells. While Wnt activation alone can induce posterior PSM markers, acquisition of a committed PSM fate and efficient differentiation into anterior PSM Pax3+ identity further requires BMP inhibition to prevent progenitors from drifting to a lateral plate mesoderm fate. When transplanted into injured adult muscle, these precursors generated large numbers of immature muscle fibers. Furthermore, exposing these mouse PSM-like cells to a brief FGF inhibition step followed by culture in horse serum-containing medium allows efficient recapitulation of the myogenic program to generate myotubes and associated Pax7+ cells. This protocol results in improved in vitro differentiation and maturation of mouse muscle fibers over serum-free protocols and enables the study of myogenic cell fusion and satellite cell differentiation.

Summary Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types., Graphical Abstract, Highlights • MyoD and small molecules reprogram fibroblasts to myogenic progenitors termed iMPCs • iMPCs self-renew and express key satellite cell and myoblast markers • iMPC growth is driven by Pax7+ cells and requires Pax7 gene function • Transplanted iMPCs engraft and sustain muscle regeneration in vivo, In this article, Hochedlinger and colleagues reprogrammed mouse fibroblasts into induced myogenic progenitors (iMPCs) by transient expression of MyoD and treatment with small molecules. iMPCs can be extensively propagated in vitro and exhibit skeletal muscle stem/progenitor cell characteristics, including the requirement for Pax7 function as well as the ability to sustain muscle regeneration upon repeated injury.

Cell fate decisions occur through the action of multiple factors, including signalling molecules and transcription factors. Recently, the regulation of translation has emerged as an important step for modulating cellular function and fate, as exemplified by ribosomes that play distinct roles in regulating cell behaviour. Notchless (Nle) is a conserved nuclear protein that is involved in a crucial step in ribosome biogenesis, and is required for the maintenance of adult haematopoietic and intestinal stem/progenitor cells. Here, we show that activated skeletal muscle satellite cells in conditional Nle mutant mice are arrested in proliferation; however, deletion of Nle in myofibres does not impair myogenesis. Furthermore, conditional deletion of Nle in satellite cells during homeostasis did not impact on their fate for up to 3 months. In contrast, loss of Nle function in primary myogenic cells blocked proliferation because of major defects in ribosome formation. Taken together, we show that muscle stem cells undergo a stage-specific regulation of ribosome biogenesis, thereby underscoring the importance of differential modulation of mRNA translation for controlling cell fate decisions.

International audience; One of the most fascinating questions in regenerative biology is why some animals can regenerate injured structures while others cannot. Skeletal muscle has a remarkable capacity to regenerate even after repeated traumas, yet limited information is available on muscle repair mechanisms and how they have evolved. For decades, the main focus in the study of muscle regeneration was on muscle stem cells, however, their interaction with their progeny and stromal cells is only starting to emerge, and this is crucial for successful repair and re-establishment of homeostasis after injury. In addition, numerous murine injury models are used to investigate the regeneration process, and some can lead to discrepancies in observed phenotypes. This review addresses these issues and provides an overview of the some of the main regulatory cellular and molecular players involved in skeletal muscle repair.

Isolation of muscle stem cells from skeletal muscle is a critical step for the study of skeletal myogenesis and regeneration. Although stem cell isolation has been performed for decades, the emergence of flow cytometry with defined cell surface markers, or transgenic mouse models, has allowed the efficient isolation of highly enriched stem cell populations. Here, we describe the isolation of mouse muscle stem cells using two different combinations of enzyme treatments allowing the release of mononucleated muscle stem cells from their niche. Mouse muscle stem cells can be further isolated as a highly enriched population by flow cytometry using fluorescent reporters or cell surface markers. We will present advantages and drawbacks of these different approaches.

The cell microenvironment, which is critical for stem cell maintenance, contains both cellular and non-cellular components, including secreted growth factors and the extracellular matrix1–3. Although Notch and other signalling pathways have previously been reported to regulate quiescence of stem cells4–9, the composition and source of molecules that maintain the stem cell niche remain largely unknown. Here we show that adult muscle satellite (stem) cells in mice produce extracellular matrix collagens to maintain quiescence in a cell-autonomous manner. Using chromatin immunoprecipitation followed by sequencing, we identified NOTCH1/RBPJ-bound regulatory elements adjacent to specific collagen genes, the expression of which is deregulated in Notch-mutant mice. Moreover, we show that Collagen V (COLV) produced by satellite cells is a critical component of the quiescent niche, as depletion of COLV by conditional deletion of the Col5a1 gene leads to anomalous cell cycle entry and gradual diminution of the stem cell pool. Notably, the interaction of COLV with satellite cells is mediated by the Calcitonin receptor, for which COLV acts as a surrogate local ligand. Systemic administration of a calcitonin derivative is sufficient to rescue the quiescence and self-renewal defects found in COLV-null satellite cells. This study reveals a Notch–COLV–Calcitonin receptor signalling cascade that maintains satellite cells in a quiescent state in a cell-autonomous fashion, and raises the possibility that similar reciprocal mechanisms act in diverse stem cell populations. Muscle stem cell quiescence in mice is maintained by a Notch–Collagen V–CALCR signalling pathway that is activated and sustained in a cell-autonomous fashion.

In eukaryote cells, chromatin appears in several forms and is composed of genomic DNA, protein and RNA. The protein content of chromatin is composed primarily of core histones that are packaged into nucleosomes resulting in the condensation of the DNA. Several epigenetic mechanisms regulate the stability of the nucleosomes and the protein–protein interactions that modify the transcriptional activity of the DNA. Interestingly, epigenetic control of gene expression has recently emerged as a relevant mechanism involved in the regulation of many different biological processes including that of muscle development, muscle mass maintenance, function, and phenotype in health and disease. Recent investigations have shed light into the epigenetic control of biological mechanisms that are key regulators of embryonic muscle development and postnatal myogenesis. In the present review article, we provide a summary of the contents discussed in session 08, titled “Epigenetics of muscle regeneration”, during the course of the 45th European Muscle Conference, which was celebrated in Montpellier (France) in September 2016. The main theme of that session was to highlight the most recent progress on the role of epigenetics in the regulation of muscle development and regeneration. The current mini-review has been divided into two major sections. On the one hand, a brief introduction on the topic of myogenesis is offered for the non-specialized reader. On the other, a brief overview of the most relevant epigenetic players that have been shown to control muscle development and regeneration is given.

International audience; Cells of several metazoan species have been shown to non-randomly segregate their DNA such that older template DNA strands segregate to one daughter cell. The mechanisms that regulate this asymmetry remain undefined. Determinants of cell fate are polarized during mitosis and partitioned asymmetrically as the spindle pole orients during cell division. Chromatids align along the pole axis; therefore, it is unclear whether extrinsic cues that determine spindle pole position also promote non-random DNA segregation. To mimic the asymmetric divisions seen in the mouse skeletal stem cell niche, we used micropatterns coated with extracellular matrix in asymmetric and symmetric motifs. We show that the frequency of non-random DNA segregation and transcription factor asymmetry correlates with the shape of the motif and that these events can be uncoupled. Furthermore, regulation of DNA segregation by cell adhesion occurs within a defined time interval. Thus, cell adhesion cues have a major impact on determining both DNA segregation patterns and cell fates.

International audience; In vivo reprogramming is a promising approach for tissue regeneration in response to injury. Several examples of in vivo reprogramming have been reported in a variety of lineages, but some including skeletal muscle have so far proven refractory. Here, we show that acute and chronic injury enables transcription-factor-mediated reprogramming in skeletal muscle. Lineage tracing indicates that this response frequently originates from Pax7+ muscle stem cells. Injury is associated with accumulation of senescent cells, and advanced aging or local irradiation further enhanced in vivo reprogramming, while selective elimination of senescent cells reduced reprogramming efficiency. The effect of senescence appears to be, at least in part, due to the release of interleukin 6 (IL-6), suggesting a potential link with the senescence-associated secretory phenotype. Collectively, our findings highlight a beneficial paracrine effect of injury-induced senescence on cellular plasticity, which will be important for devising strategies for reprogramming-based tissue repair.

International audience; A longstanding goal in regenerative medicine is to reconstitute functional tissues or organs after injury or disease. Attention has focused on the identification and relative contribution of tissue specific stem cells to the regeneration process. Relatively little is known about how the physiological process is regulated by other tissue constituents. Numerous injury models are used to investigate tissue regeneration, however, these models are often poorly understood. Specifically, for skeletal muscle regeneration several models are reported in the literature, yet the relative impact on muscle physiology and the distinct cells types have not been extensively characterised. We have used transgenic Tg:Pax7nGFP and Flk1GFP/+ mouse models to respectively count the number of muscle stem (satellite) cells (SC) and number/shape of vessels by confocal microscopy. We performed histological and immunostainings to assess the differences in the key regeneration steps. Infiltration of immune cells, chemokines and cytokines production was assessed in vivo by Luminex®.We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl2), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a "dead zone" devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl2 but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models.We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl2), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a "dead zone" devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl2 but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models.We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl2), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a "dead zone" devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl2 but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models.

How muscle diversity is generated in the vertebrate body is poorly understood. In the limb, dorsal and ventral muscle masses constitute the first myogenic diversification, as each gives rise to distinct muscles. Myogenesis initiates after muscle precursor cells (MPCs) have migrated from the somites to the limb bud and populated the prospective muscle masses. Here, we show that Sonic hedgehog (Shh) from the zone of polarizing activity (ZPA) drives myogenesis specifically within the ventral muscle mass. Shh directly induces ventral MPCs to initiate Myf5 transcription and myogenesis through essential Gli-binding sites located in the Myf5 limb enhancer. In the absence of Shh signaling, myogenesis is delayed, MPCs fail to migrate distally, and ventral paw muscles fail to form. Thus, Shh production in the limb ZPA is essential for the spatiotemporal control of myogenesis and coordinates muscle and skeletal development by acting directly to regulate the formation of specific ventral muscles.

Summary Satellite cells are adult skeletal muscle stem cells that are quiescent and constitute a poorly defined heterogeneous population. Using transgenic Tg:Pax7-nGFP mice, we show that Pax7-nGFP Hi cells are less primed for commitment and have a lower metabolic status and delayed first mitosis compared to Pax7-nGFP Lo cells. Pax7-nGFP Hi can give rise to Pax7-nGFP Lo cells after serial transplantations. Proliferating Pax7-nGFP Hi cells exhibit lower metabolic activity, and the majority performs asymmetric DNA segregation during cell division, wherein daughter cells retaining template DNA strands express stem cell markers. Using chromosome orientation-fluorescence in situ hybridization, we demonstrate that all chromatids segregate asymmetrically, whereas Pax7-nGFP Lo cells perform random DNA segregation. Therefore, quiescent Pax7-nGFP Hi cells represent a reversible dormant stem cell state, and during muscle regeneration, Pax7-nGFP Hi cells generate distinct daughter cell fates by asymmetrically segregating template DNA strands to the stem cell. These findings provide major insights into the biology of stem cells that segregate DNA asymmetrically.

Skeletal muscles exert diverse functions, enabling both crushing with great force and movement with exquisite precision. A remarkably distinct repertoire of genes and ontological features characterise this tissue, and recent evidence has shown that skeletal muscles of the head, the craniofacial muscles, are evolutionarily, morphologically and molecularly distinct from those of the trunk. Here, we review the molecular basis of craniofacial muscle development and discuss how this process is different to trunk and limb muscle development. Through evolutionary comparisons of primitive chordates (such as amphioxus) and jawless vertebrates (such as lampreys) with jawed vertebrates, we also provide some clues as to how this dichotomy arose.

In genetically modified mice with abnormal skeletal muscle development, bones and joints are differentially affected by the lack of skeletal muscle. We hypothesise that unequal levels of biophysical stimuli in the developing humerus and femur can explain the differential effects on these rudiments when muscle is absent. We find that the expression patterns of four mechanosensitive genes important for endochondral ossification are differentially affected in muscleless limb mutants, with more extreme changes in the expression in the humerus than in the femur. Using finite element analysis, we show that the biophysical stimuli induced by muscle forces are similar in the humerus and femur, implying that the removal of muscle contractile forces should, in theory, affect the rudiments equally. However, simulations in which a displacement was applied to the end of the limb, such as could be caused in muscleless mice by movements of the mother or normal littermates, predicted higher biophysical stimuli in the femur than in the humerus. Stimuli induced by limb movement were much higher than those induced by the direct application of muscle forces, and we propose that movements of limbs caused by muscle contractions, rather than the direct application of muscle forces, provide the main mechanical stimuli for normal skeletal development. In muscleless mice, passive movement induces unequal biophysical stimuli in the humerus and femur, providing an explanation for the differential effects seen in these mice. The significance of these results is that forces originating external to the embryo may contribute to the initiation and progression of skeletal development when muscle development is abnormal.

International audience; OBJECTIVE:Skeletal muscle may be the site of a variety of poorly understood immune reactions, particularly after myofiber injury, which is typically observed in inflammatory myopathies. This study was undertaken to explore both the cell dynamics and functions of resident macrophages and dendritic cells (DCs) in damaged muscle, using a mouse model of notexin-induced myoinjury to study innate immune cell reactions.METHODS:The myeloid cell reaction to notexin-induced myoinjury was analyzed by microscopy and flow cytometry. Bone marrow (BM) transplantation studies were used to discriminate resident from exudate monocyte/macrophages. Functional tests included cytokine screening and an alloantigenic mixed leukocyte reaction to assess the antigen-presenting cell (APC) function. Selective resident macrophage depletion was obtained by injection of diphtheria toxin (DT) into CD11b-DT receptor-transgenic mice transplanted with DT-insensitive BM.RESULTS:The connective tissue surrounding mouse muscle/fascicle tissue (the epimysium/perimysium) after deep muscle injury displayed a resident macrophage population of CD11b+F4/80+CD11c-Ly-6C-CX3CR1- cells, which concentrated first in the epimysium. These resident macrophages were being used by leukocytes as a centripetal migration pathway, and were found to selectively release 2 chemokines, cytokine-induced neutrophil chemoattractant and monocyte chemoattractant protein 1, and to crucially contribute to massive recruitment of neutrophils and monocytes from the blood. Early epimysial inflammation consisted of a predominance of Ly-6C(high)CX3CR1(low)CD11c- cells that were progressively substituted by Ly-6C(low)CX3CR1(high) cells displaying an intermediate, rather than high, level of CD11c expression. These CD11c(intermediate) cells were derived from circulating CCR2+ monocytes, functionally behaved as immature APCs in the absence of alloantigenic challenge, and migrated to draining lymph nodes while acquiring the phenotype of mature DCs (CD11c+Ia+CD80+ cells, corresponding to an inflammatory DC phenotype).CONCLUSION:The results in this mouse model show that resident macrophages in the muscle epimysium/perimysium orchestrate the innate immune response to myoinjury, which is linked to adaptive immunity through the formation of inflammatory DCs.

Genetically engineered mice (Myf5nLacZ/+, Myf5GFP-P/+) allowing direct muscle satellite cell (SC) visualization indicate that, in addition to being located beneath myofiber basal laminae, SCs are strikingly close to capillaries. After GFP+ bone marrow transplantation, blood-borne cells occupying SC niches previously depleted by irradiation were similarly detected near vessels, thereby corroborating the anatomical stability of juxtavascular SC niches. Bromodeoxyuridine pulse-chase experiments also localize quiescent and less quiescent SCs near vessels. SCs, and to a lesser extent myonuclei, were nonrandomly associated with capillaries in humans. Significantly, they were correlated with capillarization of myofibers, regardless to their type, in normal muscle. They also varied in paradigmatic physiological and pathological situations associated with variations of capillary density, including amyopathic dermatomyositis, a unique condition in which muscle capillary loss occurs without myofiber damage, and in athletes in whom capillaries increase in number. Endothelial cell (EC) cultures specifically enhanced SC growth, through IGF-1, HGF, bFGF, PDGF-BB, and VEGF, and, accordingly, cycling SCs remained mainly juxtavascular. Conversely, differentiating myogenic cells were both proangiogenic in vitro and spatiotemporally associated with neoangiogenesis in muscular dystrophy. Thus, SCs are largely juxtavascular and reciprocally interact with ECs during differentiation to support angio-myogenesis.

Regeneration relies on coordinated action of multiple cell types to reconstitute the damaged tissue. Here we inactivate the endocytic adaptor protein Numb in skeletal muscle stem cells prior to chronic or severe muscle injury in mice. We observe two types of senescence in regenerating muscle; a transient senescence in non-myogenic cells of control and Numb mutant mice that partly depends on INK4a/ARF activity, and a persistent senescence in myogenic cells lacking Numb. The senescence levels of Numb-deficient muscle is reduced to wild type levels by an anti-oxidant treatment or p53 ablation, resulting in functional rescue of the regenerative potential in Numb mutants. Ex vivo experiments suggest that Numb-deficient senescent cells recruit macrophages to sustain inflammation and drive fibrosis, two hallmarks of the impaired muscle regeneration in Numb mutants. These findings provide insights into previously reported developmental and oncogenic senescence that are also differentially regulated by p53., Regeneration of skeletal muscle relies on the function of muscle satellite cells. Here, Le Roux et al. show that the endocytic adaptor protein Numb promotes skeletal muscle regeneration after injury by preventing a p53-dependent senescence of satellite cells and consequent inflammation and fibrosis.

Skeletal muscle serves as a paradigm for the acquisition of cell fate, yet the relationship between primitive cell populations and emerging myoblasts has remained elusive. We identify a novel population of resident Pax3+/Pax7+, muscle marker-negative cells throughout development. Using mouse mutants that uncouple myogenic progression, we show that these Pax+ cells give rise to muscle progenitors. In the absence of skeletal muscle, they apoptose after down-regulation of Pax7. Furthermore, they mark the emergence of satellite cells during fetal development, and do not require Pax3 function. These findings identify critical cell populations during lineage restriction, and provide a framework for defining myogenic cell states for therapeutic studies.

In vertebrates, skeletal muscle is a model for the acquisition of cell fate from stem cells. Two determination factors of the basic helix-loop-helix myogenic regulatory factor (MRF) family, Myf5 and Myod, are thought to direct this transition because double-mutant mice totally lack skeletal muscle fibres and myoblasts. In the absence of these factors, progenitor cells remain multipotent and can change their fate. Gene targeting studies have revealed hierarchical relationships between these and the other MRF genes, Mrf4 and myogenin, where the latter are regarded as differentiation genes. Here we show, using an allelic series of three Myf5 mutants that differentially affect the expression of the genetically linked Mrf4 gene, that skeletal muscle is present in the new Myf5:Myod double-null mice only when Mrf4 expression is not compromised. This finding contradicts the widely held view that myogenic identity is conferred solely by Myf5 and Myod, and identifies Mrf4 as a determination gene. We revise the epistatic relationship of the MRFs, in which both Myf5 and Mrf4 act upstream of Myod to direct embryonic multipotent cells into the myogenic lineage.

Multiple waves of muscle precursors are released from skeletal muscle progenitor cells throughout developmental life, and this process is initiated in precise locations in the embryo. Skeletal muscle diversifies not only after the acquisition of muscle identity, but curiously heterogeneity is observed even in the stem cell population. Recent studies on cell lineage, cell fusion and the nature of post-natal satellite cells have expanded on our fundamental knowledge of the formation of this tissue, and how this tissue is replenished by resident and circulating regenerative stem cells during adult life.

Comment in : Response: Contributions of the Myf5-independent lineage to myogenesis. [Dev Cell. 2014]; International audience; The myogenic regulatory genes Myf5, Mrf4, Myod, and Myogenin likely arose by gene duplications during evolution, presumably to address the more demanding requirements of the vertebrate body plan. Two cell lineages were proposed to be regulated independently by Myf5 and Myod to safeguard against tissue failure. Here we report severe muscle loss following ablation of Myf5-expressing cells. Using both lineage-specific and ubiquitous reporter alleles, we show that the remaining muscles in Myf5Cre-DTA embryos arise mainly from Myf5+ escaper cells. Elimination of Myf5Cre-DTA cells on a Myod null background did not result in the total absence of skeletal muscles, as would be expected if a Myod+/Myf5-independent cell population played a major role in this scenario. Therefore, these observations are incompatible with a previously proposed functional two-lineage model. These findings will have an impact on the interpretation of phenotypes obtained using similar strategies in other tissues.

Skeletal muscles in vertebrates have a phenomenal regenerative capacity. A muscle that has been crushed can regenerate fully both structurally and functionally within a month. Remarkably, efficient regeneration continues to occur following repeated injuries. Thousands of muscle precursor cells are needed to accomplish regeneration following acute injury. The differentiated muscle cells, the multinucleated contractile myofibers, are terminally withdrawn from mitosis. The source of the regenerative precursors is the skeletal muscle stem cells-the mononucleated cells closely associated with myofibers, which are known as satellite cells. Satellite cells are mitotically quiescent or slow-cycling, committed to myogenesis, but undifferentiated. Disruption of the niche after muscle damage results in their exit from quiescence and progression towards commitment. They eventually arrest proliferation, differentiate, and fuse to damaged myofibers or make de novo myofibers. Satellite cells are one of the well-studied adult tissue-specific stem cells and have served as an excellent model for investigating adult stem cells. They have also emerged as an important standard in the field of ageing and stem cells. Several recent reviews have highlighted the importance of these cells as a model to understand stem cell biology. This chapter begins with the discovery of satellite cells as skeletal muscle stem cells and their developmental origin. We discuss transcription factors and signalling cues governing stem cell function of satellite cells and heterogeneity in the satellite cell pool. Apart from satellite cells, a number of other stem cells have been shown to make muscle and are being considered as candidate stem cells for amelioration of muscle degenerative diseases. We discuss these "offbeat" muscle stem cells and their status as adult skeletal muscle stem cells vis-a-vis satellite cells. The ageing context is highlighted in the concluding section.

Since the seminal discovery of the cell-fate regulator Myod, studies in skeletal myogenesis have inspired the search for cell-fate regulators of similar potential in other tissues and organs. It was perplexing that a similar transcription factor for other tissues was not found; however, it was later discovered that combinations of molecular regulators can divert somatic cell fates to other cell types. With the new era of reprogramming to induce pluripotent cells, the myogenesis paradigm can now be viewed under a different light. Here, we provide a short historical perspective and focus on how the regulation of skeletal myogenesis occurs distinctly in different scenarios and anatomical locations. In addition, some interesting features of this tissue underscore the importance of reconsidering the simple-minded view that a single stem cell population emerges after gastrulation to assure tissuegenesis. Notably, a self-renewing long-term Pax7+ myogenic stem cell population emerges during development only after a first wave of terminal differentiation occurs to establish a tissue anlagen in the mouse. How the future stem cell population is selected in this unusual scenario will be discussed. Recently, a wealth of information has emerged from epigenetic and genome-wide studies in myogenic cells. Although key transcription factors such as Pax3, Pax7, and Myod regulate only a small subset of genes, in some cases their genomic distribution and binding are considerably more promiscuous. This apparent nonspecificity can be reconciled in part by the permissivity of the cell for myogenic commitment, and also by new roles for some of these regulators as pioneer transcription factors acting on chromatin state.

Summary Precise control of the thyroid hormone (T3)-dependent transcriptional program is required by multiple cell systems, including muscle stem cells. Deciphering how this is achieved and how the T3 signal is controlled in stem cell niches is essentially unknown. We report that in response to proliferative stimuli such as acute skeletal muscle injury, type 3 deiodinase (D3), the thyroid hormone-inactivating enzyme, is induced in satellite cells where it reduces intracellular thyroid signaling. Satellite cell-specific genetic ablation of dio3 severely impairs skeletal muscle regeneration. This impairment is due to massive satellite cell apoptosis caused by exposure of activated satellite cells to the circulating TH. The execution of this proapoptotic program requires an intact FoxO3/MyoD axis, both genes positively regulated by intracellular TH. Thus, D3 is dynamically exploited in vivo to chronically attenuate TH signaling under basal conditions while also being available to acutely increase gene programs required for satellite cell lineage progression., Graphical Abstract, Highlights • D3 is induced in proliferating satellite cells thereby reducing thyroid signaling • D3 depletion causes massive cell apoptosis in vitro and in vivo • Apoptosis requires FoxO3, a TH target gene • Satellite cells customize TH signature and adapt it to their functional needs, Deiodinases inactivate thyroid hormone (TH) signaling, allowing a precise control of TH action at the cellular level. Dentice et al. find that type 3 deiodinase acts as a survival factor during skeletal muscle repair, and attenuation of TH signaling is required to prevent muscle stem cell apoptosis and promote lineage progression in vivo.

The myogenic factor Myf5 plays a key role in muscle cell determination, in response to signalling cascades that lead to the specification of muscle progenitor cells. We have adopted a YAC transgenic approach to identify regulatory sequences that direct the complex spatiotemporal expression of this gene during myogenesis in the mouse embryo. Important regulatory regions with distinct properties are distributed over 96 kb upstream of the Myf5 gene. The proximal 23 kb region directs early expression in the branchial arches, epaxial dermomyotome and in a central part of the myotome, the epaxial intercalated domain. Robust expression at most sites in the embryo where skeletal muscle forms depends on an enhancer-like sequence located between −58 and −48 kb from the Myf5 gene. This element is active in the epaxial and hypaxial myotome, in limb muscles, in the hypoglossal chord and also at the sites of Myf5 transcription in prosomeres p1 and p4 of the brain. However later expression of Myf5 depends on a more distal region between −96 and −63 kb, which does not behave as an enhancer. This element is necessary for expression in head muscles but strikingly only plays a role in a subset of trunk muscles, notably the hypaxially derived ventral body muscles and also those of the diaphragm and tongue. Transgene expression in limb muscle masses is not affected by removal of the −96/−63 region. Epaxially derived muscles and some hypaxial muscles, such as the intercostals and those of the limb girdles, are also unaffected. This region therefore reveals unexpected heterogeneity between muscle masses, which may be related to different facets of myogenesis at these sites. Such regulatory heterogeneity may underlie the observed restriction of myopathies to particular muscle subgroups.

Sonic hedgehog (Shh), produced by the notochord and floor plate, is proposed to function as an inductive and trophic signal that controls somite and neural tube patterning and differentiation. To investigate Shh functions during somite myogenesis in the mouse embryo, we have analyzed the expression of the myogenic determination genes, Myf5 and MyoD, and other regulatory genes in somites of Shh null embryos and in explants of presomitic mesoderm from wild-type and Myf5 null embryos. Our findings establish that Shh has an essential inductive function in the early activation of the myogenic determination genes, Myf5 and MyoD, in the epaxial somite cells that give rise to the progenitors of the deep back muscles. Shh is not required for the activation of Myf5 and MyoD at any of the other sites of myogenesis in the mouse embryo, including the hypaxial dermomyotomal cells that give rise to the abdominal and body wall muscles, or the myogenic progenitor cells that form the limb and head muscles. Shh also functions in somites to establish and maintain the medio-lateral boundaries of epaxial and hypaxial gene expression. Myf5, and not MyoD, is the target of Shh signaling in the epaxial dermomyotome, as MyoD activation by recombinant Shh protein in presomitic mesoderm explants is defective in Myf5 null embryos. In further support of the inductive function of Shh in epaxial myogenesis, we show that Shh is not essential for the survival or the proliferation of epaxial myogenic progenitors. However, Shh is required specifically for the survival of sclerotomal cells in the ventral somite as well as for the survival of ventral and dorsal neural tube cells. We conclude, therefore, that Shh has multiple functions in the somite, including inductive functions in the activation of Myf5, leading to the determination of epaxial dermomyotomal cells to myogenesis, as well as trophic functions in the maintenance of cell survival in the sclerotome and adjacent neural tube.

Over the past year, interest has focused on identifying signalling molecules--including Wnts, Sonic hedgehog, BMP-4, and noggin--that divert somitic mesodermal cells into the muscle lineage, either by induction or derepression. New mouse mutants have also provided insights into somite formation and differentiation, as well as pointing to novel differences between head, trunk, and limb myogenic programmes. In addition, recent genetic, embryological, and molecular studies have shed new light on somite formation and the establishment of muscle progenitor cells.

International audience; The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs.

Vertebrate muscles are defined and patterned at the stage of primary myotube formation, but there is no clear description of how these cells form in vivo. Of particular interest is whether primary myotubes are "seeded" by a unique myoblast population that differentiates as mononucleated myocytes, similar to the founder myoblasts of insects.We analyzed the cell populations and processes leading to initiation of primary myogenesis in limb buds of rats and mice. Pax3(+ve) myogenic precursors migrate into the limb bud and initially consolidate into dorsal and ventral muscle masses in the absence of Pax7 expression. Approximately a day later, Pax7(+ve) cells appear in the central aspect of the limb base and subsequently throughout the limb muscle masses. Primary myogenesis is initiated within each muscle mass at a time when only Pax3, and not Pax7, protein can be detected. Primary myotubes form initially as elongate mononucleated myocytes, well before cleavage of the muscle masses has occurred. Multinucleate myotubes appear approximately a day later. A similar process is seen during initiation of chick limb primary myogenesis.Primary myotubes of vertebrate limb muscles are initiated by mononucleated myocytes, that appear structurally analogous to the founder myoblasts of insects.

International audience; In recent years, our views on how DNA and genes are organised and regulated have evolved significantly. One example is provided by reports that single DNA strands in the double helix could carry distinct forms of information. That chromatids carrying old and nascently replicated DNA strands are recognised by the mitotic machinery, then segregated in a concerted way to distinct daughter cells after cell division is remarkable. Notably, this phenomenon in several cases has been associated with the cell fate choice of resulting daughter cells. Here, we review the evidence for asymmetric or template DNA strand segregation in mammals with a focus on skeletal muscle.

The myosin light chain IF/3F locus contains two independent promoters, MLC1F and MLC3F, which are differentially activated during skeletal muscle development. Transcription at this locus is regulated by a 3' skeletal muscle enhancer element, which directs correct temporal and tissue-specific expression from the MLC1F promoter in transgenic mice. To investigate the role of this enhancer in regulation of the MLC3F promoter in vivo, we have analyzed reporter gene expression in transgenic mice containing lacZ under transcriptional control of the mouse MLC3F promoter and 3' enhancer element. Our results show that these regulatory elements direct strong expression of lacZ in skeletal muscle; the transgene, however, is activated 4-5 d before the endogenous MLC3F promoter, at the time of initiation of MLC1F transcription. In adult mice, transgene activity is downregulated in muscles that have reduced contributions of type IIB fibers (soleus and diaphragm). The rostrocaudal positional gradient of transgene expression documented for MLC1F transgenic mice (Donoghue, M., J. P. Merlie, N. Rosenthal, and J. R. Sanes. 1991. Proc. Natl. Acad. Sci. USA. 88:5847-5851) is not seen in MLC3F transgenic mice. Although MLC3F was previously thought to be restricted to skeletal striated muscle, the MLC3F-lacZ transgene is expressed in cardiac muscle from 7.5 d of development in a spatially restricted manner in the atria and left ventricular compartments, suggesting that transcriptional differences exist between cardiomyocytes in left and right compartments of the heart. We show here that transgene-directed expression of the MLC3F promoter reflects low level expression of endogenous MLC3F transcripts in the mouse heart.

Skeletal muscle regeneration mainly depends on satellite cells, a population of resident muscle stem cells. However, our understanding of the molecular mechanisms underlying satellite cell activation is still largely undefined. Here, we show that Cripto, a regulator of early embryogenesis, is a novel regulator of muscle regeneration and satellite cell progression toward the myogenic lineage. Conditional inactivation of cripto in adult satellite cells compromises skeletal muscle regeneration, whereas gain of function of Cripto accelerates regeneration, leading to muscle hypertrophy. Moreover, we provide evidence that Cripto modulates myogenic cell determination and promotes proliferation by antagonizing the TGF-β ligand myostatin. Our data provide unique insights into the molecular and cellular basis of Cripto activity in skeletal muscle regeneration and raise previously undescribed implications for stem cell biology and regenerative medicine.

Six1 in satellite cells is important for muscle regeneration and homeostasis of the stem cell niche by regulating MyoD, Myogenin, and Dusp6-ERK signaling., Satellite cells (SCs) are stem cells that mediate skeletal muscle growth and regeneration. Here, we observe that adult quiescent SCs and their activated descendants expressed the homeodomain transcription factor Six1. Genetic disruption of Six1 specifically in adult SCs impaired myogenic cell differentiation, impaired myofiber repair during regeneration, and perturbed homeostasis of the stem cell niche, as indicated by an increase in SC self-renewal. Six1 regulated the expression of the myogenic regulatory factors MyoD and Myogenin, but not Myf5, which suggests that Six1 acts on divergent genetic networks in the embryo and in the adult. Moreover, we demonstrate that Six1 regulates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway during regeneration via direct control of Dusp6 transcription. Muscles lacking Dusp6 were able to regenerate properly but showed a marked increase in SC number after regeneration. We conclude that Six1 homeoproteins act as a rheostat system to ensure proper regeneration of the tissue and replenishment of the stem cell pool during the events that follow skeletal muscle trauma.

International audience; The accessibility to stem cells from healthy or diseased individuals, and the maintenance of their potency are challenging issues for stem cell biology. Here we report the isolation of viable and functional skeletal myogenic cells from humans up to 17 days, and mice up to 14 days post mortem, much longer beyond previous reports. Muscle stem cells are enriched in post mortem tissue, suggesting a selective survival advantage compared with other cell types. Transplantation of mouse muscle and haematopoietic stem cells regenerates tissues robustly. Cellular quiescence contributes to this cell viability where cells adopt a reversible dormant state characterized by reduced metabolic activity, a prolonged lag phase before the first cell division, elevated levels of reactive oxygen species and a transcriptional status less primed for commitment. Finally, severe hypoxia, or anoxia is critical for maintaining stem cell viability and regenerative capacity. Thus, these cells provide a useful resource for studying stem cell biology.

International audience; Skeletal muscle stem cell fate in adult mice is regulated by crucial transcription factors, including the determination genes Myf5 and Myod. The precise role of Myf5 in regulating quiescent muscle stem cells has remained elusive. Here we show that most, but not all, quiescent satellite cells express Myf5 protein, but at varying levels, and that resident Myf5 heterozygous muscle stem cells are more primed for myogenic commitment compared with wild-type satellite cells. Paradoxically however, heterotypic transplantation of Myf5 heterozygous cells into regenerating muscles results in higher self-renewal capacity compared with wild-type stem cells, whereas myofibre regenerative capacity is not altered. By contrast, Pax7 haploinsufficiency does not show major modifications by transcriptome analysis. These observations provide a mechanism linking Myf5 levels to muscle stem cell heterogeneity and fate by exposing two distinct and opposing phenotypes associated with Myf5 haploinsufficiency. These findings have important implications for how stem cell fates can be modulated by crucial transcription factors while generating a pool of responsive heterogeneous cells.

Skeletal muscle fibres form by fusion of mesoderm progenitors called myoblasts. After birth, muscle fibres do not increase in number but continue to grow in size because of fusion of satellite cells, the postnatal myogenic cells, responsible for muscle growth and regeneration. Numerous studies suggest that, on transplantation, non-myogenic cells also may contribute to muscle regeneration. However, there is currently no evidence that such a contribution represents a natural developmental option of these non-myogenic cells, rather than a consequence of experimental manipulation resulting in cell fusion. Here we show that pericytes, transgenically labelled with an inducible Alkaline Phosphatase CreERT2, but not endothelial cells, fuse with developing myofibres and enter the satellite cell compartment during unperturbed postnatal development. This contribution increases significantly during acute injury or in chronically regenerating dystrophic muscle. These data show that pericytes, resident in small vessels of skeletal muscle, contribute to its growth and regeneration during postnatal life.

International audience; Distinct cell populations with regenerative capacity have been reported to contribute to myofibres after skeletal muscle injury, including non-satellite cells as well as myogenic satellite cells. However, the relative contribution of these distinct cell types to skeletal muscle repair and homeostasis and the identity of adult muscle stem cells remain unknown. We generated a model for the conditional depletion of satellite cells by expressing a human diphtheria toxin receptor under control of the murine Pax7 locus. Intramuscular injection of diphtheria toxin during muscle homeostasis, or combined with muscle injury caused by myotoxins or exercise, led to a marked loss of muscle tissue and failure to regenerate skeletal muscle. Moreover, the muscle tissue became infiltrated by inflammatory cells and adipocytes. This localised loss of satellite cells was not compensated for endogenously by other cell types, but muscle regeneration was rescued after transplantation of adult Pax7(+) satellite cells alone. These findings indicate that other cell types with regenerative potential depend on the presence of the satellite cell population, and these observations have important implications for myopathic conditions and stem cell-based therapeutic approaches.

Tissue and organ regeneration proceed in a coordinated manner to restore proper function after trauma. Vertebrate skeletal muscle has a remarkable ability to regenerate after repeated and complete destruction of the tissue, yet limited information is available on how muscle stem and progenitor cells, and other nonmuscle cells, reestablish homeostasis after the regenerative process. The genetic pathways that regulate the establishment of skeletal muscle in the embryo have been studied extensively, and many of the genes that govern muscle stem cell maintenance and commitment are redeployed during adult homeostasis and regeneration. Therefore, correlates can be made between embryonic muscle development and postnatal regeneration. However, there are some important distinctions between prenatal development and regeneration - in the context of the cells, niche, anatomy and the regulatory genes employed. The similarities and distinctions between these two scenarios are the focus of this review.

Background In humans, mutations in the SEPN1 gene, encoding selenoprotein N (SelN), are involved in early onset recessive neuromuscular disorders, referred to as SEPN1-related-myopathies. The mechanisms behind these pathologies are poorly understood since the function of SelN remains elusive. However, previous results obtained in humans and more recently in zebrafish pointed to a potential role for SelN during embryogenesis. Using qRT-PCR, Western blot and whole mount in situ hybridization, we characterized in detail the spatio-temporal expression pattern of the murine Sepn1 gene during development, focusing particularly on skeletal muscles. Results In whole embryos, Sepn1 transcripts were detected as early as E5.5, with expression levels peaking at E12.5, and then strongly decreasing until birth. In isolated tissues, only mild transcriptional variations were observed during development, whereas a striking reduction of the protein expression was detected during the perinatal period. Furthermore, we demonstrated that Sepn1 is expressed early in somites and restricted to the myotome, the sub-ectodermal mesenchyme and the dorsal root ganglia at mid-gestation stages. Interestingly, Sepn1 deficiency did not alter somitogenesis in embryos, suggesting that SelN is dispensable for these processes in mouse. Conclusion We characterized for the first time the expression pattern of Sepn1 during mammalian embryogenesis and we demonstrated that its differential expression is most likely dependent on major post-transcriptional regulations. Overall, our data strongly suggest a potential role for selenoprotein N from mid-gestation stages to the perinatal period. Interestingly, its specific expression pattern could be related to the current hypothesis that selenoprotein N may regulate the activity of the ryanodine receptors.

Stem cells are present in all tissues and organs, and are crucial for normal regulated growth. How the pool size of stem cells and their progeny is regulated to establish the tissue prenatally, then maintain it throughout life, is a key question in biology and medicine. The ability to precisely locate stem and progenitors requires defining lineage progression from stem to differentiated cells, assessing the mode of cell expansion and self-renewal and identifying markers to assess the different cell states within the lineage. We have shown that during lineage progression from a quiescent adult muscle satellite cell to a differentiated myofibre, both symmetric and asymmetric divisions take place. Furthermore, we provide evidence that a sub-population of label retaining satellite cells co-segregate template DNA strands to one daughter cell. These findings provide a means of identifying presumed stem and progenitor cells within the lineage. In addition, asymmetric segregation of template DNA and the cytoplasmic protein Numb provides a landmark to define cell behaviour as self-renewal and differentiation decisions are being executed.

Skeletal muscle fibers form in overlapping, but distinct phases that depend on the generation of temporally different lineages of myogenic cells. During primary myogenesis (E10.5-E12.5 in the mouse), embryonic myoblasts fuse homotypically to generate primary fibers, whereas during later development (E14.5-E17.5), fetal myoblasts differentiate into secondary fibers. How these myogenic waves are regulated remains largely unknown. Studies have been hampered by the lack of markers which would distinguish embryonic from fetal myoblast populations. We show here that the homeobox gene Arx is strongly expressed in differentiating embryonic muscle, downstream of myogenic basic helix-loop-helix (bHLH) genes. Its expression progressively decreases during development. When overexpressed in the C2C12 myogenic cell line, Arx enhances differentiation. Accordingly, it stimulates the transcriptional activity from the Myogenin promoter and from multimerized E-boxes when co-expressed with MyoD and Mef2C in CH310T1/2. Furthermore, Arx co-immunoprecipitates with Mef2C, suggesting that it participates in the transcriptional regulatory network acting in embryonic muscle. Finally, embryonic myoblasts isolated from Arx-deficient embryos show a delayed differentiation in vivo together with an enhanced clonogenic capacity in vitro. We propose here that Arx acts as a novel positive regulator of embryonic myogenesis by synergizing with Mef2C and MyoD and by establishing an activating loop with Myogenin.

Development and maintenance of an abundant tissue such as skeletal muscle poses several challenges. Curiously, not all skeletal muscle stem cells are born alike, since diverse genetic pathways can specify their birth. Stem and progenitor cells that establish the tissue during development, those that maintain its homeostasis, as well as participate in its regeneration have generated considerable interest. The ability to distinguish stem cells from more committed progenitors throughout prenatal and postnatal life has guided researchers to identify stem cell properties and characterise their niche. These properties include markers that influence cell behaviour and mode of division during normal development, after trauma and cell transplantations. This review addresses these issues from a developmental perspective.