Mouse Skeletal Muscle Development Research Articles

Opposing gene regulatory programs governing myofiber development and maturation revealed at single nucleus resolution

Skeletal muscle fibers express distinct gene programs during development and maturation, but the underlying gene regulatory networks that confer stage-specific myofiber properties remain unknown. To decipher these distinctive gene programs and how they respond to neural activity, we generated a combined multi-omic single-nucleus RNA-seq and ATAC-seq atlas of mouse skeletal muscle development at multiple stages of embryonic, fetal, and postnatal life. We found that Myogenin, Klf5, and Tead4 form a transcriptional complex that synergistically activates the expression of muscle genes in developing myofibers. During myofiber maturation, the transcription factor Maf acts as a transcriptional switch to activate the mature fast muscle gene program. In skeletal muscles of mutant mice lacking voltage-gated L-type Ca2+ channels (Cav1.1), Maf expression and myofiber maturation are impaired. These findings provide a transcriptional atlas of muscle development and reveal genetic links between myofiber formation, maturation, and contraction.

Comparative and Functional Analysis of miRNAs and mRNAs Involved in Muscle Fiber Hypertrophy of Juvenile and Adult Goats.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate several pathway intermediates and affect the skeletal muscle development in mice, pigs, sheep, and cattle. However, to date, only a small number of miRNAs have been reported in the muscle development of goats. In this report, the longissimus dorsi transcripts of one- and ten-month-old goats were analyzed by sequencing RNAs and miRNAs. The results showed that the ten-month-old Longlin goats had 327 up- and 419 down-regulated differentially expressed genes (DEGs) compared with the one-month-old. In addition, 20 co-up-regulated and 55 co-down-regulated miRNAs involved in the muscle fiber hypertrophy of goats were identified in ten-month-old Longlin and Nubian goats compared with one-month-old. Five miRNA-mRNA pairs (chi-let-7b-3p-MIRLET7A, chi-miR193b-3p-MMP14, chi-miR-355-5p-DGAT2, novel_128-LOC102178119, novel_140-SOD3) involved in the goat skeletal muscle development were identified by miRNA-mRNA negative correlation network analysis. Our results provided new insight into the functional roles of goat muscle-associated miRNAs, allowing a deeper understanding of the transformation of miRNA roles during mammalian muscle development.

Association of a variant upstream of growth differentiation factor 11 (GDF11) on carcass traits in crossbred beef cattle.

The mature peptide of growth differentiation factor 11 (GDF11) in Bos taurus breeds, shares 90% amino acid sequence similarity to myostatin (MSTN), where loss-of-function mutations result in muscular hyperplasia causing a phenotype known as double-muscling. Mutations in the MSTN coding sequence increase muscle mass and reduce fat and bone tissues, but also confer poor fertility, reduced stress tolerance, and increased calf mortality. GDF11 influences skeletal muscle development in mice, and muscular atrophy can be induced by exogenous GDF11 treatment. To date, there are no reports of GDF11's role in bovine carcass traits. To determine associations between GDF11 and carcass quality in beef cattle, bovine GDF11 was examined in crossbred Canadian beef cattle populations during finishing. Few coding variants were found in this functionally important gene, but an upstream variant c.1-1951C > T (rs136619751) with a minor allele frequency of 0.31 was identified and further genotyped in two separate populations of crossbred steers (n = 415 and 450). CC animals had lower backfat thickness, marbling percentage, and yield score than CT or TT animals (P < 0.001 and < 0.05). These data suggest a role of GDF11 in carcass quality in beef cattle and may provide a selection tool to improve carcass traits in cattle.

The histone code reader Spin1 controls skeletal muscle development

While several studies correlated increased expression of the histone code reader Spin1 with tumor formation or growth, little is known about physiological functions of the protein. We generated Spin1M5 mice with ablation of Spin1 in myoblast precursors using the Myf5-Cre deleter strain. Most Spin1M5 mice die shortly after birth displaying severe sarcomere disorganization and necrosis. Surviving Spin1M5 mice are growth-retarded and exhibit the most prominent defects in soleus, tibialis anterior, and diaphragm muscle. Transcriptome analyses of limb muscle at embryonic day (E) 15.5, E16.5, and at three weeks of age provided evidence for aberrant fetal myogenesis and identified deregulated skeletal muscle (SkM) functional networks. Determination of genome-wide chromatin occupancy in primary myoblast revealed direct Spin1 target genes and suggested that deregulated basic helix-loop-helix transcription factor networks account for developmental defects in Spin1M5 fetuses. Furthermore, correlating histological and transcriptome analyses, we show that aberrant expression of titin-associated proteins, abnormal glycogen metabolism, and neuromuscular junction defects contribute to SkM pathology in Spin1M5 mice. Together, we describe the first example of a histone code reader controlling SkM development in mice, which hints at Spin1 as a potential player in human SkM disease.

MicroRNA expression patterns in post-natal mouse skeletal muscle development

BackgroundMiRNAs are essential regulators of skeletal muscle development and homeostasis. To date, the role and regulation of miRNAs in myogenesis have been mostly studied in tissue culture and during embryogenesis. However, little information relating to miRNA regulation during early post-natal skeletal muscle growth in mammals is available. Using a high-throughput miRNA qPCR-based array, followed by stringent statistical and bioinformatics analysis, we describe the expression pattern and putative role of 768 miRNAs in the quadriceps muscle of mice aged 2 days, 2 weeks, 4 weeks and 12 weeks.ResultsForty-six percent of all measured miRNAs were expressed in mouse quadriceps muscle during the first 12 weeks of life. We report unprecedented changes in miRNA expression levels over time. The expression of a majority of miRNAs significantly decreased with post-natal muscle maturation in vivo. MiRNA clustering identified 2 subsets of miRNAs that are potentially involved in cell proliferation and differentiation, mainly via the regulation of non-muscle specific targets.ConclusionCollective miRNA expression in mouse quadriceps muscle is subjected to substantial levels of regulation during the first 12 weeks of age. This study identified a new suite of highly conserved miRNAs that are predicted to influence early muscle development. As such it provides novel knowledge pertaining to post-natal myogenesis and muscle regeneration in mammals.

Developmental Expression of Calcium Activated Chloride Ion Channels Anoctamin 5 in Mouse Skeletal Muscle

Anoctamin 5 (Ano5), also named TMEM16E, belongs to the Anoctamin gene family. The mutations in the Ano5 gene cause Limb-girdle muscular dystrophy (LGMD) 2L type and Miyoshi muscular dystrophy (MMD3). Both patients showed sarcolemmal lesions. The studies showed that TMEM16E mRNA expressed in the somites during embryogenesis, particularly in the myotomal cells, and also in the muscle myotome-derived progenitor cells. However, no report has been done to examine Ano5 expression during mouse skeletal muscle development. In the present study, we investigated the distribution and quantification of Ano5 in the skeletal muscles of mice during their development, with the methods of immunofluorescence, Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. The results indicated that Ano5 mRNA and protein are expressed in skeletal muscle of the mouse from 1 day to 6 months, but with development and aging, the expression of Ano5 reduced gradually. Taken together, our results demonstrate that Ano5 expression level decreased throughout development and aging, and this may explain why the musculardystrophy syndrome of Ano5 mutant patients only starts during the later stage of their lives.

Stac3 Is a Novel Regulator of Skeletal Muscle Development in Mice

The goal of this study was to identify novel factors that mediate skeletal muscle development or function. We began the study by searching the gene expression databases for genes that have no known functions but are preferentially expressed in skeletal muscle. This search led to the identification of the Src homology three (SH3) domain and cysteine rich (C1) domain 3 (Stac3) gene. We experimentally confirmed that Stac3 mRNA was predominantly expressed in skeletal muscle. We determined if Stac3 plays a role in skeletal muscle development or function by generating Stac3 knockout mice. All Stac3 homozygous mutant mice were found dead at birth, were never seen move, and had a curved body and dropping forelimbs. These mice had marked abnormalities in skeletal muscles throughout the body, including central location of myonuclei, decreased number but increased cross-sectional area of myofibers, decreased number and size of myofibrils, disarrayed myofibrils, and streaming Z-lines. These phenotypes demonstrate that the Stac3 gene plays a critical role in skeletal muscle development and function in mice.

Conserved and divergent functions of Nfix in skeletal muscle development during vertebrate evolution

During mouse skeletal muscle development, the Nfix gene has a pivotal role in regulating fetal-specific transcription. Zebrafish and mice share related programs for muscle development, although zebrafish develops at a much faster rate. In fact, although mouse fetal muscle fibers form after 15 days of development, in fish secondary muscle fibers form by 48 hours post-fertilization in a process that until now has been poorly characterized mechanically. In this work, we studied the zebrafish ortholog Nfix (nfixa) and its role in the proper switch to the secondary myogenic wave. This allowed us to highlight evolutionarily conserved and divergent functions of Nfix. In fact, the knock down of nfixa in zebrafish blocks secondary myogenesis, as in mouse, but also alters primary slow muscle fiber formation. Moreover, whereas Nfix mutant mice are motile, nfixa knockdown zebrafish display impaired motility that probably depends upon disruption of the sarcoplasmic reticulum. We conclude that, during vertebrate evolution, the transcription factor Nfix lost some specific functions, probably as a consequence of the different environment in which teleosts and mammals develop.

G-CSF influences mouse skeletal muscle development and regeneration by stimulating myoblast proliferation

G-CSF influences mouse skeletal muscle development and regeneration by stimulating myoblast proliferation

G-CSF influences mouse skeletal muscle development and regeneration by stimulating myoblast proliferation

After skeletal muscle injury, neutrophils, monocytes, and macrophages infiltrate the damaged area; this is followed by rapid proliferation of myoblasts derived from muscle stem cells (also called satellite cells). Although it is known that inflammation triggers skeletal muscle regeneration, the underlying molecular mechanisms remain incompletely understood. In this study, we show that granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is expressed in developing somites. G-CSFR and G-CSF were expressed in myoblasts of mouse embryos during the midgestational stage but not in mature myocytes. Furthermore, G-CSFR was specifically but transiently expressed in regenerating myocytes present in injured adult mouse skeletal muscle. Neutralization of endogenous G-CSF with a blocking antibody impaired the regeneration process, whereas exogenous G-CSF supported muscle regeneration by promoting the proliferation of regenerating myoblasts. Furthermore, muscle regeneration was markedly impaired in G-CSFR-knockout mice. These findings indicate that G-CSF is crucial for skeletal myocyte development and regeneration and demonstrate the importance of inflammation-mediated induction of muscle regeneration.

Expression and alternative splicing of N‐RAP during mouse skeletal muscle development

N-RAP alternative splicing and protein localization were studied in developing skeletal muscle tissue from pre- and postnatal mice and in fusing primary myotubes in culture. Messages encoding N-RAP-s and N-RAP-c, the predominant isoforms of N-RAP detected in adult skeletal muscle and heart, respectively, were present in a 5:1 ratio in skeletal muscle isolated from E16.5 embryos. N-RAP-s mRNA levels increased three-fold over the first 3 weeks of postnatal development, while N-RAP-c mRNA levels remained low. N-RAP alternative splicing during myotube differentiation in culture was similar to the pattern observed in embryonic and neonatal muscle, with N-RAP-s expression increasing and N-RAP-c mRNA levels remaining low. In both developing skeletal muscle and cultured myotubes, N-RAP protein was primarily associated with developing myofibrillar structures containing alpha-actinin, but was not present in mature myofibrils. The results establish that N-RAP-s is the predominant spliced form of N-RAP present throughout skeletal muscle development.

MIR-206 regulates connexin43 expression during skeletal muscle development

Skeletal myoblast fusion in vitro requires the expression of connexin43 (Cx43) gap junction channels. However, gap junctions are rapidly downregulated after the initiation of myoblast fusion in vitro and in vivo. In this study we show that this downregulation is accomplished by two related microRNAs, miR-206 and miR-1, that inhibit the expression of Cx43 protein during myoblast differentiation without altering Cx43 mRNA levels. Cx43 mRNA contains two binding sites for miR-206/miR-1 in its 3′-untranslated region, both of which are required for efficient downregulation. While it has been demonstrated before that miR-1 is involved in myogenesis, in this work we show that miR-206 is also upregulated during perinatal skeletal muscle development in mice in vivo and that both miR-1 and miR-206 downregulate Cx43 expression during myoblast fusion in vitro. Proper development of singly innervated muscle fibers requires muscle contraction and NMJ terminal selection and it is hypothesized that prolonged electrical coupling via gap junctions may be detrimental to this process. This work details the mechanism by which initial downregulation of Cx43 occurs during myogenesis and highlights the tight control mechanisms that are utilized for the regulation of gap junctions during differentiation and development.

Analysis of the spatial, temporal and tissue-specific transcription of γ-sarcoglycan gene using a transgenic mouse

To evaluate the promoter function of the 5′-flanking sequence of mouse γ-sarcoglycan ( γ-SG) gene in vivo, we generated transgenic mice harboring this sequence fused with enhanced green fluorescent protein reporter gene. The reporter expression was restricted in striated muscles and particularly strong in all myofibers in skeletal muscles. Using these mice, we examine the spatial and temporal transcriptional patterns of the γ-SG gene during mouse skeletal muscle development. The expression of basic helix loop helix transcriptional factors preceded that of the reporter. Differences between the expression of reporter and endogenous γ-SG genes in non-muscle tissues suggested the existence of additional promoter elements in the endogenous gene, and the analysis of endogenous mRNAs demonstrated the existence of a novel upstream exon and promoter active in non-muscle tissues.

Nitric oxide synthase (NOS) in mouse skeletal muscle development and differentiated myoblasts.

The neuronal isoform of nitric oxide synthase (nNOS, termed also NOS-I) is expressed in normal adult skeletal muscle, suggesting important functions for NO in muscle biology. However, the expression and subcellular localization of NOS in muscle development and myoblast differentiation are largely unknown. In the present study, NOS was immunolocalized with isoform-specific antibodies in developing muscle and in differentiated myoblast cultures (mouse C2C12) together with histochemical NADPH-dependent diaphorase activity that is blocked by specific NOS inhibitors and therefore designated as NOS-associated diaphorase activity (NOSaD). Western blot analysis revealed immunoreactive bands for NOS-I-III in lysates from perinatal and adult muscle tissue and C2C12-myotubes that comigrated with prototypical proteins. In embryonic skeletal muscle, but not in adult myofibers, diffuse cytosolic staining and lack of sarcolemmal NOSaD activity and NOS-I immunoreaction were evident. In both myoblasts and fusioned myotubes, NOSaD and NOS isoforms I-III colocalize in the cytosol. Additionally, members of the sarcolemmal dystrophin-glycoprotein complex (i.e., dystrophin, adhalin, beta1-dystroglycan) immunolocalize in the cytosol of differentiating myoblasts, whereas anti-dystrophin and anti-beta1-dystroglycan clearly delineate the sarcolemma in myotubes. Thus, expression of NOS isoforms I-III and NOSaD is cytosolic in fusion-competent myoblasts during myotube formation in vitro. Interaction of NOSaD/NOS-I with the sarcolemmal dystrophin-complex known from mature myofibers is apparently lacking in prenatal muscle development and differentiating myoblasts. Localization of NOS isoforms thus characterized in myogenic cultures may help further to investigate regulated NO formation in muscle cells in vitro.

Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development

The myogenic basic HLH transcription factor family of genes, composed of MyoD, myogenin, Myf-5, and Myf-6, are thought to regulate skeletal muscle differentiation. To understand the role of MyoD in myogenesis, we have introduced a null mutation of MyoD into the germline of mice. Surprisingly, mice lacking MyoD are viable and fertile. Histological examination of skeletal muscle failed to reveal any morphological abnormalities in these mice. Furthermore, Northern analysis revealed normal levels of skeletal muscle-specific mRNAs. Significantly, Myf-5 mRNA levels are elevated in postnatal mutant mice. Normally, Myf-5 expression becomes markedly reduced at day 12 of gestation when MyoD mRNA first appears. This suggests that Myf-5 expression is repressed by MyoD. Our results indicate that MyoD is dispensable for skeletal muscle development in mice, revealing some degree of functional redundancy in the control of the skeletal myogenic developmental program.

Actin and myosin genes are transcriptionally regulated during mouse skeletal muscle development

During primary and secondary myotube formation in utero and subsequent maturation of muscle fibers after birth there are complex changes in the pattern of contractile protein gene expression at the RNA and protein levels. In order to determine the degree of transcriptional regulation of actin and myosin genes we have carried out “nuclear run-on” experiments using nuclei prepared from the limb muscle of mice at 14.5, 15.5, 17.5, and 18.5 days in utero and at 10–12 and 12.5 days after birth. We show that transitions in the expression of these genes in vivo are regulated transcriptionally. Transcription of the sarcomeric α-actins changes from cardiac to predominantly skeletal actin over this time period; transcription of the β-actin gene is repressed. The myosin heavy chain and myosin light chain genes also undergo transcriptional transitions during muscle development. Notably, transcription from the MLC3 F promoter is activated after that of the MLC1 F promoter, which is part of the same gene. These results are discussed in the context of published RNA data.