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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. [ABSTRACT FROM AUTHOR]

Presents information on a study which analyzed a key regulatory region upstream of the Myf5 gene. Materials and methods; Summary of transgenic results; Distinction between fore- and hindlimbs.

Presents information on the somite, which is the distinct feature of vertebrate mesodermal segmentation. Information on gastrulation; Definition of periodicity and somite segmentation in the vertebrate embryo; Details on the signaling molecules which have been identified and implicated in the patterning of divergent embryonic structures.

Investigates the roles of Pax-3 and Myf-5 genes in programming skeletal myogenesis. Capabilities of Pax-3; Analysis of double homozygous mutant embryos; Examination of fetuses in whole mount and sections; Roles of Pax-5 and Myf-5 in programming myogenesis; Monitoring of MyoD protein accumulation.

Satellite cells are crucial for maintaining muscle homeostasis and for regeneration following injury. In this issue, reveal that muscle satellite cells are a heterogeneous mixture of stem cells and committed myogenic progenitors. They show that asymmetric division of stem cells in the satellite cell niche is a mechanism for generating these two populations. [Copyright &y& Elsevier]

Stem cells can be maintained through symmetric cell divisions (SCDs) and asymmetric cell divisions (ACDs). How and when these divisions occur in vivo in vertebrates is poorly understood. Here, we developed a clonogenic cell tracing method that demonstrates the asymmetric distribution of transcription factors along with old and new DNA in mouse muscle stem cells during skeletal muscle regeneration. Combining single-cell tracking and artificial niches ex vivo , we show how cells switch from ACDs to SCDs, suggesting that they are not engaged in an obligate mode of cell division. Further, we generated SNAP-tagged histone H3-reporter mice and find that, unlike fly germline stem cells, differential fate outcomes are associated with a symmetric distribution of the H3.1 and H3.3 histone variants in mouse muscle stem cells. This versatile and efficient H3-SNAP labeling system will allow an investigation of mechanisms underlying the maintenance of epigenomic identity and plasticity in a variety of tissues. • Muscle stem cells divide symmetrically and asymmetrically in vivo • Muscle stem cells can switch from asymmetric to symmetric cell division ex vivo • Histone H3-SNAP reporters allow turnover measurements in vivo • H3.1 and H3.3 are symmetrically distributed during muscle stem cells divisions Using SNAP-tagged histone H3-reporter mice and clonogenic tracing, Evano et al. show that muscle stem cells can perform symmetric and asymmetric cell divisions (SCDs; ACDs) in vivo , and switch from ACDs to SCDs ex vivo , with symmetric inheritance of H3.1 and H3.3. [ABSTRACT FROM AUTHOR]

Ageing is a multi‐factorial condition that results in a gradual decline in tissue and organ function. Systemic, local and intrinsic factors play major roles in this process that also results in a decline in stem cell number and function. In this issue of The EMBO Journal, Li et al (2019) show that a subpopulation of mouse muscle stem cells is depleted in aged mice through loss of niche‐derived granulocyte colony‐stimulating factor (G‐CSF). [ABSTRACT FROM AUTHOR]

Gene regulatory networks that act upstream of skeletal muscle fate determinants are distinct in different anatomical locations. Despite recent efforts, a clear understanding of the cascade of events underlying the emergence and maintenance of the stem cell pool in specific muscle groups remains unresolved and debated. Here, we invalidated Pitx2 with multiple Cre-driver mice prenatally, postnatally, and during lineage progression. We showed that this gene becomes progressively dispensable for specification and maintenance of the muscle stem (MuSC) cell pool in extraocular muscles (EOMs) despite being, together with Myf5, a major upstream regulator during early development. Moreover, constitutive inactivation of Pax7 postnatally led to a greater loss of MuSCs in the EOMs compared to the limb. Thus, we propose a relay between Pitx2, Myf5 and Pax7 for EOM stem cell maintenance. We demonstrate also that MuSCs in the EOMs adopt a quiescent state earlier that those in limb muscles and do not spontaneously proliferate in the adult, yet EOMs have a significantly higher content of Pax7+ MuSCs per area pre- and post-natally. Finally, while limb MuSCs proliferate in the mdx mouse model for Duchenne muscular dystrophy, significantly less MuSCs were present in the EOMs of the mdx mouse model compared to controls, and they were not proliferative. Overall, our study provides a comprehensive in vivo characterisation of MuSC heterogeneity along the body axis and brings further insights into the unusual sparing of EOMs during muscular dystrophy. Author summary: Skeletal myogenesis serves as a paradigm to study the mechanisms controlling stem cell commitment, proliferation, and differentiation. Understanding these mechanisms is crucial for addressing their dysregulation in various diseases and ageing. Although skeletal muscle is found throughout the body, differences in embryological origins, regenerative potential, and susceptibility to myopathies exist between head and trunk muscles. Notably, extraocular muscles (EOMs) demonstrate unique resilience in conditions like Duchenne muscular dystrophy. Most research on muscle stem cell (MuSC) biology has focused on limb muscles given their accessibility for experimentation. While the transcription factor Pitx2 plays a key role in EOM formation during embryonic development, the mechanisms governing the emergence and maintenance of MuSCs at this location remain unclear. Here, we demonstrate that MuSCs in the EOMs enter a quiescent state earlier than those in limb muscles and do not spontaneously proliferate in adulthood, challenging previous assumptions. Additionally, through genetic analyses, we elucidate the interplay between Pitx2, the MuSC marker Pax7, and the myogenic factor Myf5 in regulating extraocular MuSCs, thus refining our understanding of the genetic cascades that govern craniofacial myogenesis. [ABSTRACT FROM AUTHOR]

Investigations on developmental and regenerative myogenesis have led to major advances in decrypting stem cell properties and potential, as well as their interactions within the evolving niche. As a consequence, regenerative myogenesis has provided a forum to investigate intrinsic regulators of stem cell properties as well as extrinsic factors, including stromal cells, during normal growth and following injury and disease. Here we review some of the latest advances in the field that have exposed fundamental processes including regulation of stress following trauma and ageing, senescence, DNA damage control and modes of symmetric and asymmetric cell divisions. Recent studies have begun to explore the nature of the niche that is distinct in different muscle groups, and that is altered from prenatal to postnatal stages, and during ageing. We also discuss heterogeneities among muscle stem cells and how distinct properties within the quiescent and proliferating cell states might impact on homoeostasis and regeneration. Interestingly, cellular quiescence, which was thought to be a passive cell state, is regulated by multiple mechanisms, many of which are deregulated in various contexts including ageing. These and other factors including metabolic activity and genetic background can impact on the efficiency of muscle regeneration. [ABSTRACT FROM AUTHOR]

After publication of this article [1], the authors noted that the legends for supplementary files Figures S3 and S4 were truncated in the production process, therefore lacking some information concerning these Figures. The complete legends are included in this Correction. The authors apologize for any inconvenience that this might have caused. [ABSTRACT FROM AUTHOR]

In this article, the author focuses on an extracellular protein Cripto which is an essential protein for the development of early vertebrate. He mentions that the protein is responsible for skeletal muscle regeneration and it also provides signals for satellite cell determination by antagonizing myostatin. He also informs that Cripto is helpful in revealing the signals for a stem cell lineage decision that is a key factor for skeletal muscle development and regenerative medicine.

Skeletal muscle stem cells (MuSCs) are recognised as functionally heterogeneous. Cranial MuSCs are reported to have greater proliferative and regenerative capacity when compared with those in the limb. A comprehensive understanding of the mechanisms underlying this functional heterogeneity is lacking. Here, we have used clonal analysis, live imaging and single cell transcriptomic analysis to identify crucial features that distinguish extraocular muscle (EOM) from limb muscle stem cell populations. A MyogeninntdTom reporter showed that the increased proliferation capacity of EOM MuSCs correlates with deferred differentiation and lower expression of the myogenic commitment gene Myod. Unexpectedly, EOM MuSCs activated in vitro expressed a large array of extracellular matrix components typical of mesenchymal non-muscle cells. Computational analysis underscored a distinct co-regulatory module, which is absent in limb MuSCs, as driver of these features. The EOM transcription factor network, with Foxc1 as key player, appears to be hardwired to EOM identity as it persists during growth, disease and in vitro after several passages. Our findings shed light on how high-performing MuSCs regulate myogenic commitment by remodelling their local environment and adopting properties not generally associated with myogenic cells. [ABSTRACT FROM AUTHOR]

Four SIX homeoproteins display a combinatorial expression throughout embryonic developmental myogenesis and they modulate the expression of the myogenic regulatory factors. Here, we provide a deep characterization of their role in distinct mouse developmental territories. We showed, at the hypaxial level, that the Six1:Six4 double knockout (dKO) somitic precursor cells adopt a smooth muscle fate and lose their myogenic identity. At the epaxial level, we demonstrated by the analysis of Six quadruple KO (qKO) embryos, that SIX are required for fetal myogenesis, and for the maintenance of PAX7+ progenitor cells, which differentiated prematurely and are lost by the end of fetal development in qKO embryos. Finally, we showed that Six1 and Six2 are required to establish craniofacial myogenesis by controlling the expression of Myf5. We have thus described an unknown role for SIX proteins in the control of myogenesis at different embryonic levels and refined their involvement in the genetic cascades operating at the head level and in the genesis of myogenic stem cells. Author summary: We demonstrate with double, triple and quadruple Six KO mouse embryos that specific Six combinations are required for proper myogenesis depending on the level of the mouse embryonic body axis. We show that the Six1 and Six2 genes are required for craniofacial myogenesis by controlling the engagement of unsegmented cranial paraxial mesodermal cells in the myogenic pathway. We also show that hypaxial somitic dermomyotomal cells from embryos mutant for the Six1 and Six4 genes are unable to engage in the skeletal muscle lineage. Last, we show that embryos mutant for the four Six genes expressed in the myogenic lineage exhibit a defect in self-renewal of PAX7+ stem cells present in their residual muscle masses, and that SIX proteins interact directly with several enhancer elements at the Pax7 locus to control its expression. We have thus characterized new functions of SIX proteins in the control of myogenesis at different embryonic levels and refined their involvement in the genetic cascades that govern the genesis of myogenic stem cells. [ABSTRACT FROM AUTHOR]

A major challenge in the study of living systems is understanding how tissues and organs are established, maintained during homeostasis, reconstituted following injury or deteriorated during disease. Most of the studies that interrogate in vivo cell biological properties of cell populations within tissues are obtained through static imaging approaches. However, in vertebrates, little is known about which, when, and how extracellular and intracellular signals are dynamically integrated to regulate cell behaviour and fates, due largely to technical challenges. Intravital imaging of cellular dynamics in mammalian models has exposed surprising properties that have been missed by conventional static imaging approaches. Here we highlight some selected examples of intravital imaging in mouse intestinal stem cells, hematopoietic stem cells, hair follicle stem cells, and neural stem cells in the brain, each of which have distinct features from an anatomical and niche-architecture perspective. Intravital imaging of mouse skeletal muscles is comparatively less advanced due to several technical constraints that will be discussed, yet this approach holds great promise as a complementary investigative method to validate findings obtained by static imaging, as well as a method for discovery. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Gene targeting has indicated that Myf5 and MyoD are required for myogenic determination because skeletal myoblasts and myofibers are missing in mouse embryos lacking both Myf5 and MyoD. To investigate the fate of Myf5:MyoD-deficient myogenic precursor cells during embryogenesis, we examined the sites of epaxial, hypaxial, and cephalic myogenesis at different developmental stages. In newborn mice, excessive amounts of adipose tissue were found in the place of muscles whose progenitor cells have undergone long-range migrations as mesenchymal cells. Analysis of the expression pattern of Myogenin-lacZ transgene and muscle proteins revealed that myogenic precursor cells were not able to acquire a myogenic fate in the trunk (myotome) nor at sites of MyoD induction in the limb buds. Importantly, the Myf5-dependent precursors, as defined by Myf5nlacZ-expression, deficient for both Myf5 and MyoD, were observed early in development to assume nonmuscle fates (e.g., cartilage) and, later in development, to extensively proliferate without cell death. Their fate appeared to significantly differ from the fate of MyoD-dependent precursors, as defined by 258/−2.5lacZ-expression (−20 kb enhancer of MyoD), of which a significant proportion failed to proliferate and underwent apoptosis. Taken together, these data strongly suggest that Myf5 and MyoD regulatory elements respond differentially in different compartments. [Copyright &y& Elsevier]

Background: Myogenin is a transcription factor that is expressed during terminal myoblast differentiation in embryonic development and adult muscle regeneration. Investigation of this cell state transition has been hampered by the lack of a sensitive reporter to dynamically track cells during differentiation. Results: Here, we report a knock-in mouse line expressing the tdTOMATO fluorescent protein from the endogenous Myogenin locus. Expression of tdTOMATO in MyogntdTom mice recapitulated endogenous Myogenin expression during embryonic muscle formation and adult regeneration and enabled the isolation of the MYOGENIN+ cell population. We also show that tdTOMATO fluorescence allows tracking of differentiating myoblasts in vitro and by intravital imaging in vivo. Lastly, we monitored by live imaging the cell division dynamics of differentiating myoblasts in vitro and showed that a fraction of the MYOGENIN+ population can undergo one round of cell division, albeit at a much lower frequency than MYOGENIN− myoblasts. Conclusions: We expect that this reporter mouse will be a valuable resource for researchers investigating skeletal muscle biology in developmental and adult contexts. [ABSTRACT FROM AUTHOR]

Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments. The extraocular muscles (EOMs) are an evolutionarily conserved group of muscles that are precisely engineered for fine displacement of the eyeball, and thus crucial for visual acuity. This study presents the first integrative blueprint for morphogenesis of the EOM functional unit, providing genetic evidence for the existence of a retinoic acid signaling module that coordinates the emergence of individual EOMs, their tendons and insertion sites. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Skeletal muscle satellite (stem) cells are quiescent in adult mice and can undergo multiple rounds of proliferation and self-renewal following muscle injury. Several labs have profiled transcripts of myogenic cells during the developmental and adult myogenesis with the aim of identifying quiescent markers. Here, we focused on the quiescent cell state and generated new transcriptome profiles that include subfractionations of adult satellite cell populations, and an artificially induced prenatal quiescent state, to identify core signatures for quiescent and proliferating. Methods: Comparison of available data offered challenges related to the inherent diversity of datasets and biological conditions. We developed a standardized workflow to homogenize the normalization, filtering, and quality control steps for the analysis of gene expression profiles allowing the identification up- and down-regulated genes and the subsequent gene set enrichment analysis. To share the analytical pipeline of this work, we developed Sherpa, an interactive Shiny server that allows multi-scale comparisons for extraction of desired gene sets from the analyzed datasets. This tool is adaptable to cell populations in other contexts and tissues. Results: A multi-scale analysis comprising eight datasets of quiescent satellite cells had 207 and 542 genes commonly up- and down-regulated, respectively. Shared up-regulated gene sets include an over-representation of the TNFα pathway via NFKβ signaling, Il6-Jak-Stat3 signaling, and the apical surface processes, while shared down-regulated gene sets exhibited an over-representation of Myc and E2F targets and genes associated to the G2M checkpoint and oxidative phosphorylation. However, virtually all datasets contained genes that are associated with activation or cell cycle entry, such as the immediate early stress response genes Fos and Jun. An empirical examination of fixed and isolated satellite cells showed that these and other genes were absent in vivo, but activated during procedural isolation of cells. Conclusions: Through the systematic comparison and individual analysis of diverse transcriptomic profiles, we identified genes that were consistently differentially expressed among the different datasets and shared underlying biological processes key to the quiescent cell state. Our findings provide impetus to define and distinguish transcripts associated with true in vivo quiescence from those that are first responding genes due to disruption of the stem cell niche. [ABSTRACT FROM AUTHOR]

Muscle satellite cells are resistant to cytotoxic agents, and they express several genes that confer resistance to stress, thus allowing efficient dystrophic muscle regeneration after transplantation. However, once they are activated, this capacity to resist to aggressive agents is diminished resulting in massive death of transplanted cells. Although cell immaturity represents a survival advantage, the signalling pathways involved in the control of the immature state remain to be explored. Here, we show that incubation of human myoblasts with retinoic acid impairs skeletal muscle differentiation through activation of the retinoic-acid receptor family of nuclear receptor. Conversely, pharmacologic or genetic inactivation of endogenous retinoic-acid receptors improved myoblast differentiation. Retinoic acid inhibits the expression of early and late muscle differentiation markers and enhances the expression of myogenic specification genes, such as PAX7 and PAX3. These results suggest that the retinoic-acid-signalling pathway might maintain myoblasts in an undifferentiated/immature stage. To determine the relevance of these observations, we characterised the retinoic-acid-signalling pathways in freshly isolated satellite cells in mice and in siMYOD immature human myoblasts. Our analysis reveals that the immature state of muscle progenitors is correlated with high expression of several genes of the retinoic-acid-signalling pathway both in mice and in human. Taken together, our data provide evidences for an important role of the retinoic-acid-signalling pathway in the regulation of the immature state of muscle progenitors. [ABSTRACT FROM AUTHOR]

During development, lymph node (LN) initiation is coordinated by lymphoid tissue organizer (LTo) cells that attract lymphoid tissue inducer (LTi) cells at strategic positions within the embryo. The identity and function of LTo cells during the initial attraction of LTi cells remain poorly understood. Using lineage tracing, we demonstrated that a subset of Osr1- expressing cells was mesenchymal LTo progenitors. By investigating the heterogeneity of Osr1+ cells, we uncovered distinct mesenchymal LTo signatures at diverse anatomical locations, identifying a common progenitor of mesenchymal LTos and LN-associated adipose tissue. Osr1 was essential for LN initiation, driving the commitment of mesenchymal LTo cells independent of neural retinoic acid, and for LN-associated lymphatic vasculature assembly. The combined action of chemokines CXCL13 and CCL21 was required for LN initiation. Our results redefine the role and identity of mesenchymal organizer cells and unify current views by proposing a model of cooperative cell function in LN initiation. [Display omitted] • Osr1 labels mLTo cells and progenitors for adult mesenchymal LN stromal cells • Identifies a common progenitor of mesenchymal LTo cells and LN-associated adipose tissue • Osr1 is required for mesenchymal LTo cell commitment via retinoic acid signaling • Osr1+ cells control LEC assembly, and mLTo-LEC combined action leads to LN initiation The initial step of lymph node (LN) formation relies on lymphoid tissue organizer (LTo) cells, which are poorly characterized. Vallecillo-García et al. reveal that Osr1 labels mesenchymal LTo cells and their progenitors, is essential for their commitment via retinoic acid signaling, and is required for LN-associated lymphatic vasculature assembly. [ABSTRACT FROM AUTHOR]

Summary The esophagus links the oral cavity to the stomach and facilitates the transfer of bolus. Using genetic tracing and mouse mutants, we demonstrate that esophagus striated muscles (ESMs) are not derived from somites but are of cranial origin. Tbx1 and Isl1 act as key regulators of ESMs, which we now identify as a third derivative of cardiopharyngeal mesoderm that contributes to second heart field derivatives and head muscles. Isl1 -derived ESM progenitors colonize the mouse esophagus in an anterior-posterior direction but are absent in the developing chick esophagus, thus providing evolutionary insight into the lack of ESMs in avians. Strikingly, different from other myogenic regions, in which embryonic myogenesis establishes a scaffold for fetal fiber formation, ESMs are established directly by fetal myofibers. We propose that ESM progenitors use smooth muscle as a scaffold, thereby bypassing the embryonic program. These findings have important implications in understanding esophageal dysfunctions, including dysphagia, and congenital disorders, such as DiGeorge syndrome. [ABSTRACT FROM AUTHOR]

The conserved Notch pathway functions in diverse developmental and disease-related processes, requiring mechanisms to ensure appropriate target selection and gene activation in each context. To investigate the influence of chromatin organisation and dynamics on the response to Notch signalling, we partitioned Drosophila chromatin using histone modifications and established the preferred chromatin conditions for binding of Su(H), the Notch pathway transcription factor. By manipulating activity of a co-operating factor, Lozenge/Runx, we showed that it can help facilitate these conditions. While many histone modifications were unchanged by Su(H) binding or Notch activation, we detected rapid changes in acetylation of H3K56 at Notch-regulated enhancers. This modification extended over large regions, required the histone acetyl-transferase CBP and was independent of transcription. Such rapid changes in H3K56 acetylation appear to be a conserved indicator of enhancer activation as they also occurred at the mammalian Notch-regulated Hey1 gene and at Drosophila ecdysone-regulated genes. This intriguing example of a core histone modification increasing over short timescales may therefore underpin changes in chromatin accessibility needed to promote transcription following signalling activation. [ABSTRACT FROM AUTHOR]

Abstract: Skeletal muscle satellite cells play a critical role during muscle growth, homoeostasis and regeneration. Selective induction of the muscle determination genes Myf5, Myod and Mrf4 during prenatal development can potentially impact on the reported functional heterogeneity of adult satellite cells. Accordingly, expression of Myf5 was reported to diminish the self-renewal potential of the majority of satellite cells. In contrast, virtually all adult satellite cells showed antecedence of Myod activity. Here we examine the priming of myogenic cells by Mrf4 throughout development. Using a Cre-lox based genetic strategy and novel highly sensitive Pax7 reporter alleles compared to the ubiquitous Rosa26-based reporters, we show that all adult satellite cells, independently of their anatomical location or embryonic origin, have been primed for Mrf4 expression. Given that Mrf4 Cre and Mrf4 nlacZ are active exclusively in progenitors during embryogenesis, whereas later expression is restricted to differentiated myogenic cells, our findings suggest that adult satellite cells emerge from embryonic founder cells in which the Mrf4 locus was activated. Therefore, this level of myogenic priming by induction of Mrf4, does not compromise the potential of the founder cells to assume an upstream muscle stem cell state. We propose that embryonic myogenic cells and the majority of adult muscle stem cells form a lineage continuum. [Copyright &y& Elsevier]

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-nGFPHi cells are less primed for commitment and have a lower metabolic status and delayed first mitosis compared to Pax7-nGFPLo cells. Pax7-nGFPHi can give rise to Pax7-nGFPLo cells after serial transplantations. Proliferating Pax7-nGFPHi 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-nGFPLo cells perform random DNA segregation. Therefore, quiescent Pax7-nGFPHi cells represent a reversible dormant stem cell state, and during muscle regeneration, Pax7-nGFPHi 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. [Copyright &y& Elsevier]

Abstract: Mechanical forces are essential for normal adult bone function and repair, but the impact of prenatal muscle contractions on bone development remains to be explored in depth in mammalian model systems. In this study, we analyze skeletogenesis in two ‘muscleless’ mouse mutant models in which the formation of skeletal muscle development is disrupted; Myf5nlacZ/nlacZ:MyoD−/− and Pax3 Sp/Sp (Splotch). Ossification centers were found to be differentially affected in the muscleless limbs, with significant decreases in bone formation in the scapula, humerus, ulna and femur, but not in the tibia. In the scapula and humerus, the morphologies of ossification centers were abnormal in muscleless limbs. Histology of the humerus revealed a decreased extent of the hypertrophic zone in mutant limbs but no change in the shape of this region. The elbow joint was also found to be clearly affected with a dramatic reduction in the joint line, while no abnormalities were evident in the knee. The humeral deltoid tuberosity was significantly reduced in size in the Myf5nlacZ/nlacZ:MyoD−/− mutants while a change in shape but not in size was found in the humeral tuberosities of the Pax3 Sp/Sp mutants. We also examined skeletal development in a ‘reduced muscle’ model, the Myf5nlacZ/+:MyoD−/− mutant, in which skeletal muscle forms but with reduced muscle mass. The reduced muscle phenotype appeared to have an intermediate effect on skeletal development, with reduced bone formation in the scapula and humerus compared to controls, but not in other rudiments. In summary, we have demonstrated that skeletal development is differentially affected by the lack of skeletal muscle, with certain rudiments and joints being more severely affected than others. These findings indicate that the response of skeletal progenitor cells to biophysical stimuli may depend upon their location in the embryonic limb, implying a complex interaction between mechanical forces and location-specific regulatory factors affecting bone and joint development. [Copyright &y& Elsevier]

Abstract: The myogenic determination genes Myf5, Myod and Mrf4 direct skeletal muscle cell fate prenatally. In adult myogenesis, Myod has been shown to regulate myoblast differentiation, however, our understanding of satellite cell regulation is incomplete since the roles of Myf5 and Mrf4 had not been clearly defined. Here we examine the function of Myf5 and Mrf4 in the adult using recently generated alleles. Mrf4 is not expressed in normal or Myf5 null satellite cells and myoblasts, therefore excluding a role for this determination gene in adult muscle progenitors. Skeletal muscles of adult Myf5 null mice exhibit a subtle progressive myopathy. Crucially, adult Myf5 null mice exhibit perturbed muscle regeneration with a significant increase in muscle fibre hypertrophy, delayed differentiation, adipocyte accumulation, and fibrosis after freeze-injury. Satellite cell numbers are not significantly altered in Myf5 null animals and they show a modest impaired proliferation under some conditions in vitro. Mice double mutant for Myf5 and Dystrophin were more severely affected than single mutants, with enhanced necrosis and regeneration. Therefore, we show that Myf5 is a regulator of regenerative myogenesis and homeostasis, with functions distinct from those of Myod and Mrf4. [Copyright &y& Elsevier]

Abstract: Skeletal muscle development occurs asynchronously and it has been proposed to be dependent upon the generation of temporally distinct populations of myogenic cells. This long-held hypothesis has not been tested directly due to the inability to isolate and analyze purified populations of myoblasts derived from specific stages of prenatal development. Using a mouse strain with the GFP reporter gene targeted into the Myf5 locus, a cell-sorting method was developed for isolating embryonic and fetal myoblasts. The two types of myoblasts show an intrinsic difference in fusion ability, proliferation, differentiation and response to TGFβ, TPA and BMP-4 in vitro. Microarray and quantitative PCR were used to identify differentially expressed genes both before and after differentiation, thus allowing a precise phenotypic analysis of the two populations. Embryonic and fetal myoblasts differ in the expression of a number of transcription factors and surface molecules, which may control different developmental programs. For example, only embryonic myoblasts express a Hox code along the antero-posterior axis, indicating that they possess direct positional information. Taken together, the data presented here demonstrate that embryonic and fetal myoblasts represent intrinsically different myogenic lineages and provide important information for the understanding of the molecular mechanisms governing skeletal muscle development. [Copyright &y& Elsevier]

The growth and repair of skeletal muscle after birth depends on satellite cells that are characterized by the expression of Pax7. We show that Pax3, the paralogue of Pax7, is also present in both quiescent and activated satellite cells in many skeletal muscles. Dominant-negative forms of both Pax3 and -7 repress MyoD, but do not interfere with the expression of the other myogenic determination factor, Myf5, which, together with Pax3/7, regulates the myogenic differentiation of these cells. In Pax7 mutants, satellite cells are progressively lost in both Pax3-expressing and -nonexpressing muscles. We show that this is caused by satellite cell death, with effects on the cell cycle. Manipulation of the dominant-negative forms of these factors in satellite cell cultures demonstrates that Pax3 cannot replace the antiapoptotic function of Pax7. These findings underline the importance of cell survival in controlling the stem cell populations of adult tissues and demonstrate a role for upstream factors in this context. [ABSTRACT FROM AUTHOR]

The myogenic regulatory factors, Mrf4 and Myf5, play a key role in skeletal muscle formation. An enhancer trap approach, devised to isolate positive-acting elements from a 200-kb YAC covering the mouse Mrf4–Myf5 locus in a C2 myoblast assay, yielded an enhancer, A17, which mapped at −8 kb 5′ of Mrf4 and −17 kb 5′ of Myf5. An E-box bound by complexes containing the USF transcription factor is critical for enhancer activity. In transgenic mice, A17 gave two distinct and mutually exclusive expression profiles before birth, which correspond to two phases of Mrf4 transcription. Linked to the Tk or Mrf4 minimal promoters, the nlacZ reporter was expressed either in embryonic myotomes, or later in fetal muscle, with the majority of Mrf4 lines showing embryonic expression. When linked to the Myf5 minimal promoter, only fetal muscle expression was detected. These observations identify A17 as a sequence that targets sites of myogenesis in vivo and raise questions about the mutually exclusive modes of expression and possible promoter/enhancer interactions at the Mrf4–Myf5 locus. [Copyright &y& Elsevier]