Adult Mouse Skeletal Muscle Research Articles

Fast, multiplexable and efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9

Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate genes of interest. CRISPR/Cas9-based reverse genetics is a promising method to eventually achieve this. However, such methods are sorely lacking for multi-nucleated muscle fibers, since highly efficient nuclei editing is a requisite to robustly inactive candidate genes. Here, we couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery to establish a system for highly effective somatic gene deletions in mice. Using well-characterized genes, we show that local or systemic inactivation of these genes copy the phenotype of traditional gene-knockout mouse models. Thus, this proof-of-principle study establishes a method to unravel the function of individual genes or entire signaling pathways in adult skeletal muscle fibers without the cumbersome requirement of generating knockout mice.

A High-Phosphorus Diet Moderately Alters the Lipidome and Transcriptome in the Skeletal Muscle of Adult Mice.

A high phosphorus intake has been associated with various metabolic disorders, including chronic kidney disease, cardiovascular disease, and osteoporosis. Recent studies have demonstrated the effects of dietary phosphorus on lipid and glucose metabolism. This study investigated the impact of a high-phosphorus diet on mouse skeletal muscle lipid composition and gene transcription. Adult male mice (n = 12/group) received either a diet with an adequate (0.3%) or a high (1.2%) phosphorus concentration for 6 weeks. The lipidome analysis showed that among the 17 analyzed lipid classes, the concentrations of three classes were reduced in the high phosphorus group compared to the adequate phosphorus group. These classes were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and lysophosphatidylcholine (LPC) (p < 0.05). Out of the three hundred and twenty-three individual lipid species analyzed, forty-nine showed reduced concentrations, while three showed increased concentrations in the high phosphorus group compared to the adequate phosphorus group. The muscle transcriptome analysis identified 142 up- and 222 down-regulated transcripts in the high phosphorus group compared to the adequate phosphorus group. Gene set enrichment analysis identified that genes that were up-regulated in the high phosphorus group were linked to the gene ontology terms "mitochondria" and "Notch signaling pathway", whereas genes that were down-regulated were linked to the "PI3K-AKT pathway". Overall, the effects of the high-phosphorus diet on the muscle lipidome and transcriptome were relatively modest, but consistently indicated an impact on lipid metabolism.

Targeted regulation of TAK1 counteracts dystrophinopathy in a DMD mouse model.

Muscular dystrophies make up a group of genetic neuromuscular disorders that involve severe muscle wasting. TGF-β-activated kinase 1 (TAK1) is an important signaling protein that regulates cell survival, growth, and inflammation. TAK1 has been recently found to promote myofiber growth in the skeletal muscle of adult mice. However, the role of TAK1 in muscle diseases remains poorly understood. In the present study, we have investigated how TAK1 affects the progression of dystrophic phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD). TAK1 is highly activated in the dystrophic muscle of mdx mice during the peak necrotic phase. While targeted inducible inactivation of TAK1 inhibits myofiber injury in young mdx mice, it results in reduced muscle mass and contractile function. TAK1 inactivation also causes loss of muscle mass in adult mdx mice. By contrast, forced activation of TAK1 through overexpression of TAK1 and TAB1 induces myofiber growth without having any deleterious effect on muscle histopathology. Collectively, our results suggest that TAK1 is a positive regulator of skeletal muscle mass and that targeted regulation of TAK1 can suppress myonecrosis and ameliorate disease progression in DMD.

Enzymatically dissociated muscle fibers display rapid dedifferentiation and impaired mitochondrial calcium control.

Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. By comparing adult mouse skeletal muscle fibers, isolated either by mechanical dissection or by collagenase-induced ECM digestion, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca2+ handling. RNA-sequencing showed striking differences in gene expression patterns between the two isolation methods with enzymatically dissociated fibers resembling myopathic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria following enzymatic dissociation. Repeated contractions resulted in a prolonged mitochondrial Ca2+ accumulation in enzymatically dissociated fibers, which was partially prevented by cyclophilin inhibitors. Of importance, muscle fibers of mice with severe mitochondrial myopathy show pathognomonic mitochondrial Ca2+ accumulation during repeated contractions and this accumulation was concealed with enzymatic dissociation, making this an ambiguous method in studies of native intracellular Ca2+ fluxes.

Prenatal EGCG consumption causes obesity and perturbs glucose homeostasis in adult mice

Epigallocatechin gallate (EGCG) has a wide consumption for its health advantages. The current study investigates the effects of prenatal EGCG administration on glucose metabolism and obesity in adulthood. Pregnant C57BL/6J mice were supplemented with EGCG in drinking water (3 µg/mL) for 16 d. Abdominal obesity was observed in both male and female adult mice, which was associated with the upregulation of adipose-specific genes, including C/ebpα and Srebf1 (Srebf1 only in males), and the downregulation of genes related to lipolysis, such as Acox1, Atgl and Pdk4 (only in males) in visceral adipose tissue. Elevated fasting glucose levels and hyperinsulinemia were observed in adult males, while females exhibit lower glucose level in glucose tolerance test, which might be due to reduced glucagon levels. Though hepatic expression of the insulin receptor signaling pathway was upregulated in males and was not altered in females, prenatal treatment with EGCG downregulated the expression of this signaling pathway in the skeletal muscle of adult mice, which was further demonstrated in primary human skeletal muscle cells treated with EGCG. The methylation levels in promotor of genes related to the insulin receptor signaling were matched with their transcription in mice, while the expression of acetylated histones was downregulated in human skeletal muscle cells. These results suggest that EGCG consumption during pregnancy should be a risk factor for the disruption of glucose homeostasis in adulthood.

Lipid-independent activation of a muscle-specific PKCα splicing variant.

Protein kinase C-α (PKCα) plays a major role in a diverse range of cellular processes. Studies to date have defined the regulatory controls and function of PKCα entirely based upon the previously annotated ubiquitously expressed prototypical isoform. From RNA-seq-based transcriptome analysis in murine heart, we identified a previously unannotated PKCα variant produced by alternative RNA splicing. This PKCα transcript variant, which we named PKCα-novel exon (PKCα-NE), contains an extra exon between exon 16 and exon 17, and is specifically detected in adult mouse cardiac and skeletal muscle, but not other tissues; it is also detected in human hearts. This transcript variant yields a PKCα isoform with additional 16 amino acids inserted in its COOH-terminal variable region. Although the canonical PKCα enzyme is a lipid-dependent kinase, in vitro kinase assays show that PKCα-NE displays a high level of basal lipid-independent catalytic activity. Our unbiased proteomic analysis identified a specific interaction between PKCα-NE and eukaryotic elongation factor-1α (eEF1A1). Studies in cardiomyocytes link PKCα-NE expression to an increase in eEF1A1 phosphorylation and elevated protein synthesis. In summary, we have identified a previously uncharacterized muscle-specific PKCα splicing variant, PKCα-NE, with distinct biochemical properties that plays a unique role in the control of the protein synthesis machinery in cardiomyocytes.NEW & NOTEWORTHY PKCα is an important signaling molecule extensively studied in many cellular processes. However, no isoforms have been reported for PKCα except one prototypic isoform. Alternative mRNA splicing of Prkca gene was detected for the first time in rodent and human cardiac tissue, which can produce a previously unknown PKCα-novel exon (NE) isoform. The biochemistry and molecular effects of PKCα-NE are markedly different from PKCα wild type, suggesting potential functional diversity of PKCα signaling in muscle.

Deletion of the Vitamin D Receptor in Skeletal Muscle is Associated with Improved Glucose Tolerance and Reduced Muscle Function

Skeletal muscle insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus. Epidemiological studies have demonstrated that vitamin D deficiency is associated with the presence of type 2 diabetes in humans. To probe the role of the vitamin D receptor (VDR), an intracellular and nuclear protein that mediates the bioactivity of the active vitamin D ligand, 1,25‐dihydroxyvitamin D, in skeletal muscle mediated glucose disposal, we deleted the skeletal muscle VDR in adult mice. We assessed glucose disposal, insulin concentrations and sensitivity, and skeletal muscle gene expression in Vdr knockout and wild‐type Vdr mice. Following the induction of a skeletal muscle‐specific Crerecombinase with tamoxifen in adult male Vdrfl/flmice, efficient deletion of the Vdrwas demonstrated. In skeletal muscle‐specific Vdrknockout mice, basal glucose concentrations were similar to those in wild‐type Vdrmice. In Vdrknockout mice, blood glucose concentrations decreased more rapidly following the administration of intraperitoneal glucose than in control mice expressing wild‐type Vdr (Figure 1A and Figure 1B). The AUC for glucose concentrations in Vdrfl/fl.actin iCre mice treated with tamoxifen was reduced by 20% (p=0.015) compared to the AUC of blood glucose concentrations prior to tamoxifen treatment. Pre‐and post‐treatment AUC’s for blood glucose in Vdrfl/fl.actin iCre mice treated with vehicle and in Vdrfl/flmice treated with tamoxifen were unchanged (p=0.97,0.23). Plasma insulin concentrations were identical in knockout and wild‐type Vdr mice following a bolus of glucose, demonstrating that deletion of the Vdr in skeletal muscle did not influence the release of insulin in response to glucose (one‐way ANOVA, p=0.45). Insulin sensitivity was similar in Vdrknockout and wild‐type Vdrmice. Gene expression analysis of skeletal muscle mRNA derived from Vdrknockout mice and wild‐type mice demonstrated an increase in the expression of genes associated with oxidative phosphorylation, fatty acid oxidation and adipogenesis pathways that could potentially alter glucose utilization. We conclude that selective deletion of the Vdr in the skeletal muscle of adult mice results in more efficient glucose disposal than in wild‐type Vdrmice; basal and glucose‐stimulated insulin serum concentrations are identical in knock‐out and wild type VDR mice with no changes in insulin sensitivity. The data suggest that modulation of the vitamin‐D endocrine system is unlikely to improve glucose utilization.

Supraphysiological activation of TAK1 promotes skeletal muscle growth and mitigates neurogenic atrophy

Skeletal muscle mass is regulated through coordinated activation of multiple signaling pathways. TAK1 signalosome has been found to be activated in various conditions of muscle atrophy and hypertrophy. However, the role and mechanisms by which TAK1 regulates skeletal muscle mass remain less understood. Here, we demonstrate that supraphysiological activation of TAK1 in skeletal muscle of adult mice stimulates translational machinery, protein synthesis, and myofiber growth. TAK1 causes phosphorylation of elongation initiation factor 4E (eIF4E) independent of mTOR. Inactivation of TAK1 disrupts neuromuscular junction morphology and causes deregulation of Smad signaling. Using genetic approaches, we demonstrate that TAK1 prevents excessive loss of muscle mass during denervation. TAK1 favors the nuclear translocation of Smad4 and cytoplasmic retention of Smad6. TAK1 is also required for the phosphorylation of eIF4E in denervated skeletal muscle. Collectively, our results demonstrate that TAK1 supports skeletal muscle growth and prevents neurogenic muscle atrophy in adult mice.

The IRE1/XBP1 signaling axis promotes skeletal muscle regeneration through a cell non-autonomous mechanism.

Skeletal muscle regeneration is regulated by coordinated activation of multiple signaling pathways. The unfolded protein response (UPR) is a major mechanism that detects and alleviates protein-folding stresses in the endoplasmic reticulum. However, the role of individual arms of the UPR in skeletal muscle regeneration remain less understood. In the present study, we demonstrate that IRE1α (also known as ERN1) and its downstream target, XBP1, are activated in skeletal muscle of mice upon injury. Myofiber-specific ablation of IRE1α or XBP1 in mice diminishes skeletal muscle regeneration that is accompanied with reduced number of satellite cells. Ex vivo cultures of myofiber explants demonstrate that ablation of IRE1α reduces the proliferative capacity of myofiber-associated satellite cells. Myofiber-specific ablation of IRE1α dampens Notch signaling and canonical NF-κB pathway in skeletal muscle of adult mice. Finally, targeted ablation of IRE1α also reduces Notch signaling, abundance of satellite cells, and skeletal muscle regeneration in the mdx mice, a model of Duchenne muscular dystrophy. Collectively, our experiments suggest that the IRE1α-mediated signaling promotes muscle regeneration through augmenting the proliferation of satellite cells in a cell non-autonomous manner.

Down-regulation of pro-necroptotic molecules blunts necroptosis during myogenesis

Cell death and differentiation are closely related at the molecular level. Differentiation of skeletal muscle cells attenuates susceptibility to apoptosis. Necroptosis has recently been recognized as a form of regulated cell death but its role in myogenesis has not been studied. This study aimed to compare the sensitivity to TNF-induced necroptosis in skeletal muscle at the undifferentiated (myoblasts) and differentiated (myotubes) stages. Surprisingly, our results showed that TNF-induced necroptosis was blunted during myoblast differentiation. Moreover, our data revealed that the key molecules involved in necroptosis, including receptor-interacting serine/threonine protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), were significantly down-regulated during myogenic differentiation, resulting in suppression of necroptosis signal transduction in differentiated myotubes. In addition, RIPK1, RIPK3, and MLKL expression levels were significantly lower in the skeletal muscle of adult mice than in newborn mice, suggesting that the susceptibility to necroptosis might be attenuated in differentiated muscle tissue. In conclusion, this study revealed that expression of key molecules involved in necroptosis is down-regulated during muscle differentiation, which results in the differentiation of muscles becoming insensitive to necroptotic cell death.

Targeting the Expression of Long Noncoding RNAs in Murine Satellite Cells from Single Myofibers.

LncRNAs have been recently implicated in the epigenetic control of muscle differentiation and their functional characterization has traditionally relied upon in vitro models of myogenic differentiation. However, the use of experimental paradigms to specifically target lncRNAs expression in muscle stem cells (MuSCs), also known as satellite cells, represents an important requisite to interrogate their function in more physiological contexts. Since isolation and culture of single myofibers preserves satellite cells within their physiological niche underneath the surrounding basal lamina, this procedure represents the optimal approach to follow satellite cell dynamics ex-vivo, such as activation from quiescence, expansion of committed progenitors, differentiation, and self-renewal. Here, we detail an optimized protocol to isolate viable single myofibers from the extensor digitorum longus (EDL) skeletal muscle of adult mice and to manipulate the expression of lncRNAs by antisense LNA GapmeRs-mediated knock-down (KD). Furthermore, we describe a method of EdU incorporation that, coupled to lncRNA KD and subsequent immunofluorescence analysis of proliferating, differentiating, and satellite cell-specific markers, permits the inference of lncRNAs function on muscle stem cells dynamics. Graphic abstract: Graphical representation of the single myofiber isolation method. Experimental workflow showing the main steps of the protocol procedure: EDL muscle harvesting from the mouse hindlimb; EDL digestion into single myofibers; transfection with antisense oligos and culture for 96h; immunofluorescence protocol and image outcome.

Drp1 knockdown induces severe muscle atrophy and remodelling, mitochondrial dysfunction, autophagy impairment and denervation.

The maintenance of optimal mitochondrial content and function is critical for muscle health. Mitochondrial dynamics play key roles in mitochondrial quality control; however, the exact role that mitochondrial fission plays in skeletal muscle health remains unclear. Here we report knocking down Drp1 (a protein regulating mitochondrial fission) for 4months in adult mouse skeletal muscle resulted in severe muscle atrophy (40-50%). Drp1 knockdown also led to a reduction in ADP-stimulated respiration, an increase in markers of impaired autophagy and increased muscle regeneration, denervation, fibrosis and oxidative stress. Our data indicate that Drp1 is crucial for the maintenance of normal mitochondrial function and that Drp1 depletion severely impairs muscle health. Mitochondria play central roles in skeletal muscle physiology, including energy supply, regulation of energy-sensitive signalling pathways, reactive oxygen species production/signalling, calcium homeostasis and the regulation of apoptosis. The maintenance of optimal mitochondrial content and function is therefore critical for muscle cells. Mitochondria are now well known as highly dynamic organelles, able to change their morphology through fusion and fission processes. Solid experimental evidence indicates that mitochondrial dynamics play key roles in mitochondrial quality control, and alteration in the expression of proteins regulating mitochondrial dynamics have been reported in many conditions associated with muscle atrophy and wasting. However, the exact role that mitochondrial fission plays in skeletal muscle health remains unclear. To address this issue, we investigated the impact of Drp1 (a protein regulating mitochondrial fission) knockdown, introduced via intramuscular injection of adeno-associated virus (AAV) on adult mouse skeletal muscle. Knocking down Drp1 for 4months resulted in very severe muscle atrophy (40-50%). Drp1 knockdown also led to a reduction in ADP-stimulated respiration and increases in markers of muscle regeneration, denervation, fibrosis, oxidative stress and impaired autophagy. Our findings indicate that Drp1 is essential for the maintenance of normal mitochondrial function and that Drp1 suppression severely impairs muscle health.

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

SUN-670 P300 and CBP Are Necessary for Skeletal Muscle Insulin-Stimulated Glucose Uptake

Introduction: Akt is a critical mediator of insulin-stimulated glucose uptake in skeletal muscle. The acetyltransferases, E1A binding protein p300 (p300) and cAMP response element-binding protein binding protein (CBP) are phosphorylated and activated by Akt, and p300/CBP can acetylate and inactivate Akt, thus giving rise to a possible Akt-p300/CBP axis. Our objective was to determine the importance of p300 and CBP to skeletal muscle insulin sensitivity. Methods: We used Cre-LoxP methodology to generate mice with a tamoxifen-inducible, conditional knock out of Ep300 and/or Crebbp in skeletal muscle. At 13-15 weeks of age, the knockout was induced via oral gavage of tamoxifen and oral glucose tolerance, ex vivo skeletal muscle insulin sensitivity, and microarray and proteomics analysis were done. Results: Loss of both p300 and CBP in adult mouse skeletal muscle rapidly and severely impairs whole body glucose tolerance and skeletal muscle insulin sensitivity. Furthermore, giving back a single allele of either p300 or CBP rescues both phenotypes. Moreover, the severe insulin resistance in the p300/CBP double knockout mice is accompanied by significant changes in both mRNA and protein expression of transcript/protein networks critical for insulin signaling, GLUT4 trafficking, and metabolism. Lastly, in human skeletal muscle samples, p300 and CBP protein levels correlate significantly and negatively with markers of insulin resistance. Conclusions: p300 and CBP are jointly required for maintaining whole body glucose tolerance and insulin sensitivity in skeletal muscle.

Mitochondrial Organization is Severly Modified in Skeletal Muscles of Septin 7 Knockdown Animals

Components of the cytoskeleton have essential role in the organization and appropriate distribution of ion channels and proteins involved in calcium homeostasis, signal transduction, and contractile function of striated muscle. Our goal was to identify the role of septin 7, as extra-sarcomeric cytoskeletal protein in the organization and regeneration of the skeletal muscle. As knocking out septin 7 is lethal at the embryonic stage, a tissue specific, tamoxifen-induced Cre-Lox system was used to downregulate (KD) the expression of septin 7 in adult mouse skeletal muscle.

Isolation, Purification and Characterization of Exosomes fromFibroblast Cultures of Skeletal Muscle.

Exosomes are dynamic nanovesicles secreted by virtually all cells and are present in all biological fluids. Given their highly heterogeneous content exosomes have been implicated in many physiological and pathological processes that they exert by influencing cell-cell and cell-ECM communication. In recent years an increasing number of methods have been established for the purification and characterization of exosomes. These include ultracentrifugation, ultrafiltration, size exclusion chromatography, immune capture and precipitation using a proprietary polymer. Here, we provide a protocol based on differential ultracentrifugation and sucrose density gradients tailored for the isolation of crude and ultra-pure exosomes from primary fibroblast cultures derived from adult mouse skeletal muscle. This protocol can be adapted and modified for the isolation and characterization of exosomes from a variety of tissues and bodily fluids.

Equivalent L-type channel (CaV1.1) function in adult female and male mouse skeletal muscle fibers

Loss of total muscle force during aging has both atrophic and non-atrophic components. The former deficit is a direct consequence of reduced muscle mass while the latter has been attributed to a depression of excitation-contraction (EC) coupling. It is well established that age-onset reductions in sex hormone production regulate the atrophic component in both males and females. However, it is unknown whether the non-atrophic component is influenced by sex hormones. Since the non-atrophic component has been linked mechanistically to reduced expression of the skeletal muscle L-type Ca2+ channel (CaV1.1), we recorded L-type Ca2+ currents, gating charge movements and depolarization-induced changes in myoplasmic Ca2+ from flexor digitorum brevis (FDB) fibers of naïve and gonadectomized mice of both sexes. Our first set of experiments sought to identify any basal differences in EC coupling or L-type Ca2+ flux between the sexes; no detectable differences in any of the aforementioned parameters were observed between FDB harvested from either naïve males or females. In the latter segments of the study, ovariectomy (OVX) and orchiectomy (ORX) models were used to assess the possible influence of sex hormones on EC coupling and/or L-type Ca2+ flux. In these experiments, FDB fibers harvested from OVX and ORX mice both showed no differences in L-type Ca2+ current, gating charge movement or depolarization-induced changes in Ca2+ release from the sarcoplasmic reticulum. Taken together, our results indicate L-type Ca2+ channel function and EC coupling are: 1) equivalent between the sexes, and 2) not significantly regulated by sex hormones. Since recent NIH review guidelines mandate the consideration of sex differences as a criterion for review, our work indicates the suitability of either sex for the study of the fundamental mechanisms of EC coupling. Thus, our findings may accelerate the research process by conserving animals, labor and financial resources.

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants.

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Sema3a-Nrp1 Signaling Mediates Fast-Twitch Myofiber Specificity of Tw2+ Cells.

We previously identified a unique population of interstitial muscle progenitors, marked by expression of the Twist2 transcription factor, which fuses specifically to type IIb/x fast-twitch myofibers. Tw2+ progenitors are distinct from satellite cells, a muscle progenitor that expresses Pax7 and contributes to all myofiber types. Through RNA sequencing and immunofluorescence, we identify the membrane receptor, Nrp1, as a marker of Tw2+ cells but not Pax7+ cells. We also found that Sema3a, a chemorepellent ligand for Nrp1, is expressed by type I and IIa myofibers but not IIb myofibers. Using stripe migration assays, chimeric cell-cell fusion assays, and a Sema3a transgenic mouse model, we identify Sema3a-Nrp1 signaling as a major mechanism for Tw2+ cell fiber-type specificity. Our findings reveal an extracellular signaling mechanism whereby a cell-surface receptor for a chemorepellent confers specificity of intercellular fusion of a specific muscle progenitor with its target tissue.

Correction: Single cell analysis of adult mouse skeletal muscle stem cells in homeostatic and regenerative conditions (doi: 10.1242/dev.174177).

There was an error published in Development (2019) 146, [dev174177][1] ([doi:10.1242/dev.174177][2]). On p. 6, the definition of TA was given incorrectly. Corrected: Tibialis anterior (TA) and extensor digitorum longus (EDL) muscles of two 3-month-old C56BL/6J mice were injured with the myotoxic