Satellite Cell Expression Research Articles

Isolation, Cryosection and Immunostaining of Skeletal Muscle.

Adult skeletal muscle is maintained and repaired by resident stem cells called satellite cells, located between the plasmalemma of a muscle fiber, and the surrounding basal lamina. When needed, satellite cells are activated to form proliferative myoblasts, that then differentiate and fuse to existing muscle fibers, or fuse together to form replacement myofibers. In parallel, a proportion of satellite cells self-renew, to maintain the stem cell pool. To date, Pax7 is the marker of choice for identifying quiescent satellite cells. Co-immunostaining of skeletal muscle with Pax7 and laminin allows both identification of satellite cells, and the myofiber that they are associated with. Furthermore, satellite cells can be followed through the early stages of the myogenic program by co-immunostaining with myogenic regulatory factors such as MyoD. To test genetically modified mice for satellite cell expression, co-immunostaining can be performed for Pax7 and reporter genes such as eGFP. Here, we describe a method for identification of satellite cells in skeletal muscle sections, including muscle isolation, cryosectioning and co-immunostaining for Pax7 and laminin.

Abstract 5153: Myogenic regulatory factors and their role in embryonal rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is a pediatric sarcoma of muscle. RAS pathway activation is the dominant oncogenic driver event in fusion negative RMS, which includes the Embryonal RMS (ERMS). We have previously shown in a transgenic zebrafish model of kRASG12D induced ERMS that tumor-propagating potential is confined to molecularly defined cells that express Myf5, m-Cadherin, c-Met and additional satellite cell markers. MYF5 and MYOD1 are bHLH myogenic regulatory factors (MRFs) that orchestrate skeletal muscle differentiation during development and regeneration. Both are sufficient to reprogram human mesenchymal cells into a myogenic fate. Importantly, these same factors are highly expressed a subset of mouse and human ERMS. Given that Myf5 is highly and specifically expressed in the TPC compartment in our ERMS model, we hypothesized that Myf5 and its transcriptional targets may regulate self-renewal and growth of TPCs. To address this question, we utilized transgenic zebrafish and expressed Myf5 in differentiated ERMS cells that lack proliferative capacity and can not make tumors when transplanted into recipient fish. Induced expression of Myf5 was sufficient to confer tumor-propagating ability to differentiated populations of ERMS cells. These “Induced TPCs” proliferate and generate aggressive tumors that re-express satellite cell markers and yet retain expression of mature muscle markers. Next, we assessed if Myf5 is required for ERMS initiation in the zebrafish model. Remarkably, kRASSG12D-induced ERMS could be generated in Myf5 loss-of-function zebrafish. Moreover, Myf5-deficient ERMS could regrow following transplantation into rag2E450fs immune-compromised zebrafish; however, these tumors are histologically and molecularly distinct when compared with ERMS arising in wild-type fish. Given the redundancy of function between Myf5 and MyoD in muscle and their differential expression in satellite cells and muscle progenitors, our current hypothesis is that Myf5-deficient zebrafish ERMS likely recapitulate a molecularly-distinct class of human ERMS. This idea is consistent with the fact that MYF5 is expressed in only 50% of primary human ERMS and a small fraction of human cell lines. Importantly, knock down experiments in human ERMS cell lines confirm independent roles of either MYF5 or MYOD1 in maintenance of ERMS cell viability in vitro. Collectively, our results support a previously unappreciated role for bHLH MRFs in ERMS cell survival and intra-tumor functional heterogeneity and suggest that myogenic factor expression may define unique subtypes of ERMS. Citation Format: Ines M. Tenente, Myron Ignatius, Eleanor Chen, Madeline Hayes, David M. Langenau. Myogenic regulatory factors and their role in embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5153. doi:10.1158/1538-7445.AM2015-5153

Dominant Lethal Pathologies in Male Mice Engineered to Contain an X-Linked DUX4 Transgene

Facioscapulohumeral muscular dystrophy (FSHD) isan enigmatic disease associated with epigenetic alterations in the subtelomeric heterochromatin of the D4Z4 macrosatellite repeat. Each repeat unit encodes DUX4, a gene that is normally silent in most tissues. Besides muscular loss, most patients suffer retinal vascular telangiectasias. To generate an animal model, we introduced a doxycycline-inducible transgene encoding DUX4 and 3' genomic DNA into a euchromatic region of the mouse X chromosome. Without induction, DUX4 RNA was expressed at low levels in many tissues and animals displayed a variety of unexpected dominant leaky phenotypes, including male-specific lethality. Remarkably, rare live-born males expressed DUX4 RNA in the retina and presented a retinal vascular telangiectasia. By using doxycycline to induce DUX4 expression in satellite cells, we observed impaired myogenesis invitro and invivo. This mouse model, which shows pathologies due to FSHD-related D4Z4 sequences, is likely to be useful for testing anti-DUX4 therapies in FSHD.

Myomaker is essential for muscle regeneration.

Regeneration of injured adult skeletal muscle involves fusion of activated satellite cells to form new myofibers. Myomaker is a muscle-specific membrane protein required for fusion of embryonic myoblasts, but its potential involvement in adult muscle regeneration has not been explored. We show that myogenic basic helix-loop-helix (bHLH) transcription factors induce myomaker expression in satellite cells during acute and chronic muscle regeneration. Moreover, genetic deletion of myomaker in adult satellite cells completely abolishes muscle regeneration, resulting in severe muscle destruction after injury. Myomaker is the only muscle-specific protein known to be absolutely essential for fusion of embryonic and adult myoblasts.

Developmental stage determines efficiency of gene transfer to muscle satellite cells by in utero delivery of adeno-associated virus vector serotype 2/9

Efficient gene transfer to muscle stem cells (satellite cells) has not been achieved despite broad transduction of skeletal muscle by systemically administered adeno-associated virus serotype 2/9 (AAV-9) in mice. We hypothesized that cellular migration during fetal development would make satellite cells accessible for gene transfer following in utero intravascular injection. We injected AAV-9 encoding green fluorescent protein (GFP) marker gene into the vascular space of mice ranging in ages from post-coital day 12 (E12) to postnatal day 1 (P1). Satellite cell transduction was examined using: immunohistochemistry and confocal microscopy, satellite cell migration assay, myofiber isolation and FACS analysis. GFP positive myofibers were detected in all mature skeletal muscle groups and up to 100% of the myofibers were transduced. We saw gestational variation in cardiac and skeletal muscle expression. E16 injection resulted in 27.7 ± 10.0% expression in satellite cells, which coincides with the timing of satellite cell migration, and poor satellite cell expression before and after satellite cell migration (E12 and P1). Our results demonstrate that efficient gene expression is achieved in differentiated myofibers and satellite cells after injection of AAV-9 in utero. These findings support the potential of prenatal gene transfer for muscle based treatment strategies.

Six1 Regulates MyoD Expression in Adult Muscle Progenitor Cells

Quiescent satellite cells are myogenic progenitors that enable regeneration of skeletal muscle. One of the early events of satellite cell activation following myotrauma is the induction of the myogenic regulatory factor MyoD, which eventually induces terminal differentiation and muscle function gene expression. The purpose of this study was to elucidate the mechanism by which MyoD is induced during activation of satellite cells in mouse muscle undergoing regeneration. We show that Six1, a transcription factor essential for embryonic myogenesis, also regulates MyoD expression in muscle progenitor cells. Six1 knock-down by RNA interference leads to decreased expression of MyoD in myoblasts. Chromatin immunoprecipitation assays reveal that Six1 binds the Core Enhancer Region of MyoD. Further, transcriptional reporter assays demonstrate that Core Enhancer Region reporter gene activity in myoblasts and in regenerating muscle depends on the expression of Six1 and on Six1 binding sites. Finally, we provide evidence indicating that Six1 is required for the proper chromatin structure at the Core Enhancer Region, as well as for MyoD binding at its own enhancer. Together, our results reveal that MyoD expression in satellite cells depends on Six1, supporting the idea that Six1 plays an important role in adult myogenesis, in addition to its role in embryonic muscle formation.

CCAAT/Enhancer Binding Protein Beta is Expressed in Satellite Cells and Controls Myogenesis

Upon injury, muscle satellite cells become activated and produce skeletal muscle precursors that engage in myogenesis. We demonstrate that the transcription factor CCAAT/enhancer binding protein beta (C/EBPβ) is expressed in the satellite cells of healthy muscle. C/EBPβ expression is regulated during myogenesis such that C/EBPβ is rapidly and massively downregulated upon induction to differentiate. Furthermore, persistent expression of C/EBPβ in myoblasts potently inhibits differentiation at least in part through the inhibition of MyoD protein function and stability. As a consequence, myogenic factor expression, myosin heavy chain expression, and fusogenic activity were reduced in C/EBPβ-overexpressing cells. Using knockout models, we demonstrate that loss of Cebpb expression in satellite cells results in precocious differentiation of myoblasts in growth conditions and greater cell fusion upon differentiation. In vivo, loss of Cebpb expression in satellite cells resulted in larger muscle fiber cross-sectional area and improved repair after muscle injury. Our results support the notion that C/EBPβ inhibits myogenic differentiation and that its levels must be reduced to allow for activation of MyoD target genes and the progression of differentiation.

Satellite cell expression of myogenic regulatory factors is altered when co‐cultured with preadipocytes

Postnatal skeletal muscle hypertrophy requires activation, proliferation, and differentiation of muscle satellite cells (SCs) driven by expression of several myogenic regulatory factors (MRFs). The interaction between SCs and non‐myogenic cells within the unique microenvironment of the muscle is poorly characterized. SCs induced to differentiate in either a mono‐culture (MC) or co‐culture (CC) with preadipocytes, were isolated using laser‐assisted cell catapulting. Temporal expression of paired box transcription factor 7 (Pax7) and four MRFs; myogenic differentiation factor (MyoD), myogenin (Myf4), myogenic factor 5 and myocyte enhancement factor 2a were compared in SCs. MRF expression using TaqMan™ qRT‐PCR was performed at 0, 4, 8, 16, 24, 32, 40 and 48 hours post‐differentiation (PD). As compared to MC, differentiation of SCs in CC resulted in i) an increased Pax7 expression at 32 (p=0.05) and 48 (p=0.02) hrs PD ii) an elevated MyoD expression at 16 (p=0.05) and 48 (p=0.06), hrs PD and iii) a reduced Myf4 expression at 32 (p=0.04) and 40 (p=0.06) hrs PD. Altered MRF expression in SCs grown in CC, as compared to MC, indicates that preadipocytes can influence activation and differentiation of SCs. Furthermore, this CC is an improved model of the in vivo satellite cell niche and may aid in elucidating the complex mediation of postnatal SC activation.

Retention of Pax3 Expression in Satellite Cells of Muscle Spindles

Intrafusal fibers within muscle spindles retain features characteristic of immaturity, unlike the larger and more numerous extrafusal fibers constituting the bulk of skeletal muscle. Satellite cells (SCs), myogenic progenitors, are detected on the surfaces of both intrafusal and extrafusal fibers, but little is known of spindle SCs. We have recently demonstrated that, like their extrafusal counterparts, SCs in muscle spindles of posthatch chickens express paired box transcription factor 7 (Pax7) protein. During vertebrate embryogenesis, myogenic progenitors express both Pax7 and Pax3 proteins. In postnatal mice, Pax3 appears in rare SC subsets, whereas Pax7 is expressed by all SCs within extrafusal fibers. Here we test the hypothesis that Pax3 protein maintains localized expression within SCs of muscle spindles. Immunohistochemical techniques were used to identify SCs by their Pax7 expression within anterior latissimus dorsi muscle excised from posthatch chickens of various ages. A greater percentage of SCs express Pax3 within intrafusal than extrafusal fibers at each age, and the proportion of SCs expressing Pax3 declines with aging. This is the first study to localize Pax3 expression in posthatch avian muscle and within SCs of muscle spindles. We suggest that Pax3-positive SCs are involved in fiber maintenance.

The dorsal root ganglion in Friedreich’s ataxia

Atrophy of dorsal root ganglia (DRG) and thinning of dorsal roots (DR) are hallmarks of Friedreich's ataxia (FRDA). Many previous authors also emphasized the selective vulnerability of larger neurons in DRG and thicker myelinated DR axons. This report is based on a systematic reexamination of DRG, DR and ventral roots (VR) in 19 genetically confirmed cases of FRDA by immunocytochemistry and single- and double-label immunofluorescence with antibodies to specific proteins of myelin, neurons and axons; S-100alpha as a marker of satellite and Schwann cells; laminin; and the iron-responsive proteins ferritin, mitochondrial ferritin, and ferroportin. Confocal images of axons and myelin allowed the quantitative analysis of fiber density and size, and the extent of DR and VR myelination. A novel technology, high-definition X-ray fluorescence (HDXRF) of polyethylene glycol-embedded fixed tissue, was used to "map" iron in DRG. Unfixed frozen tissue of DRG in three cases was available for the chemical assay of total iron. Proliferation of S-100alpha-positive satellite cells accompanied neuronal destruction in DRG of all FRDA cases. Double-label visualization of peripheral nerve myelin protein 22 and phosphorylated neurofilament protein confirmed the known loss of large myelinated DR fibers, but quantitative fiber counts per unit area did not change. The ratio of myelinated to neurofilament-positive fibers in DR rose significantly from 0.55 to 0.66. In VR of FRDA patients, fiber counts and degree of myelination did not differ from normal. Pooled histograms of axonal perimeters disclosed a shift to thinner fibers in DR, but also a modest excess of smaller axons in VR. Schwann cell cytoplasm in DR of FRDA was depleted while laminin reaction product remained prominent. Numerous small axons clustered around fewer Schwann cells. Ferritin in normal DRG localized to satellite cells, and proliferation of these cells in FRDA caused wide rims of reaction product about degenerating nerve cells. Mitochondrial ferritin was not detectable. Ferroportin was present in the cytoplasm of normal satellite cells and neurons, and in large axons of DR and VR. In FRDA, some DRG neurons lost their cytoplasmic ferroportin immunoreactivity, whereas the cytoplasm of satellite cells remained ferroportin positive. Ferroportin in DR axons disappeared in parallel with atrophy of large fibers. HDXRF of DRG detected regional and diffuse increases in iron fluorescence that matched ferritin expression in satellite cells. The observations support the conclusions that satellite cells and DRG neurons are affected by iron dysmetabolism; and that regeneration and inappropriate myelination of small axons in DR are characteristic of the disease.

The Effects of Inflammation on Glial Fibrillary Acidic Protein Expression in Satellite Cells of the Dorsal Root Ganglion

After undergoing L5 hemilaminectomy, chromic gut suture was placed onto the DRG and the animals were sacrificed at various time-points. The purpose of this study was to identify the effects of inflammation on satellite cells (SCs) of the dorsal root ganglion (DRG) by analyzing glial fibrillary acidic protein (GFAP) expression in of the DRG at various time points. SCs are neuroglial cells that closely interact with nerve cells of the DRG. The role of SC remains unknown GFAP expression increases in response to CNS injury. Loss of GFAP has impaired Schwann cell proliferation and delayed nerve regeneration after injury. Sixty rats underwent a left L5 hemilaminectomy. In Group I, a chromic-gut suture was place topically on the DRG (n = 30), Group II was the sham surgery group (n = 30). DRGs were harvested at 6, 24, 48, 72 hours, and 7 days after surgery. In Group III, 6 control rats were killed and their bilateral L5 DRG harvested. The harvested DRG were analyzed using light microscopy for SC immunoreactivity, using GFAP, HIS-36, TNF-alpha, IL-1alpha, IL-1beta, IL-6 monoclonal antibodies. One hundred thirty-two DRGs were harvested for analysis. Naïve controls and neurons did not express GFAP. The SC sheath expressed GFAP as early as 6 hours postchromic gut application. In Group I, GFAP expression steadily increased after chromic-gut application with 100% of SC soma and SC sheaths being GFAP positive at 7 days. The contralateral DRG demonstrated delayed GFAP expression, with 83% of SC soma and SC sheaths were GFAP positive at 7 days. In Group II, 89% of sacs expressed GFAP by 7 compared to 79% in the contralateral undisturbed DRG. Under physiologic conditions, the expression of GFAP by SCs is undetectable. As the inflammatory process develops, GFAP expression steadily increases with 100% of SCs being GFAP immunoreactive 7 days after chromic gut application. These data suggest that SCs are the primary source of GFAP in the DRG. We hypothesize that SC play an important role in the response to early inflammatory injury.

Local Gene Transfer and Expression Following Intramuscular Administration of FGF-1 Plasmid DNA in Patients With Critical Limb Ischemia

NV1FGF is an expression plasmid encoding sp.FGF-1(21-154) currently under investigation for therapeutic angiogenesis in clinical trials. NV1FGF plasmid distribution and transgene expression following intramuscular (IM) injection in patients is unknown. The study involved six patients with chronic critical limb ischemia (CLI) planned to undergo amputation. A total dose of 0.5, 2, or 4 mg NV1FGF was administered as eight IM injections (0.006, 0.25, or 0.5 mg per injection) 3-5 days before amputation. Injected sites (30 cm(3)) were divided into equally sized smaller pieces to assess spatial distribution of NV1FGF sequences (PCR), NV1FGF mRNA (reverse transcriptase-PCR), and fibroblast growth factor-1 (FGF-1)-expressing cells (immunohistochemistry). Data indicated gene expression at all doses. The distribution area was within 5-12 cm for NV1FGF sequences containing the expression cassette, up to 5 cm for NV1FGF mRNA, and up to 3 cm for FGF-1-expressing myofibers. All FGF receptors were detected indicating robust potential for bioactivity after NV1FGF gene transfer. Circulating levels of NV1FGF sequences were shown to decrease within days after injection. Data support demonstration of plasmid-mediated gene transfer and expression in muscles from patients with CLI. FGF-1 expression was shown to be limited to injection sites, which supports the concept of multiple-site injection for therapeutic use.

Axotomy-induced up-regulation of tumor necrosis factor-alpha in the dorsal root ganglia

Objectives: Neuropathic pain is a chronic pain syndrome associated with drug, injury or disease-induced damage or destruction of sensory afferent fibers of the dorsal root ganglia (DRG). Although the exact underlying pathologic mechanisms are not known, pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) are recognized as potential modulators of peripheral and central nervous system inflammatory responses. They play a crucial role in injury and the pathologic development of chronic pain syndromes such as neuropathic pain.Methods: Twenty-four rats were divided into a naive control (n=6), sham (surgery exposing sciatic nerve, n=6), and peripheral nerve lesion group (unilateral axotomy of sciatic nerve, n=12).Results: The results of this study demonstrate a transient up-regulation of TNF-α expression within ipsi- and contralateral DRG following complete unilateral sciatic nerve axotomy as confirmed by immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR. Elevated expression of TNF-α was noted to occur within the first 7 days post-axotomy, which subsequently normalized to baseline levels by day 14. This transient up-regulation was also associated with a switch in cellular source from predominant satellite cell expression at baseline to that involving satellite cells and abundant numbers of sensory neurons.Discussion: These results support the role of TNF-α in the upstream cascade of cellular events involved in the underlying pathogenesis of neuropathic pain. Strategies targeting the early attenuation of TNF-α within the DRG during the first week post-injury may have significant clinical impact in preventing the downstream cascade of events involved in the underlying cellular pathology of neuropathic pain.

The A17 enhancer directs expression of Myf5 to muscle satellite cells but Mrf4 to myonuclei

The myogenic regulatory factors, Myf5 and Mrf4, play key roles in the specification and differentiation of skeletal muscle, respectively. Many cis-acting regulatory elements at the Mrf4/Myf5 locus have been identified, including the A17 enhancer. During development, A17 in conjunction with the Mrf4 or Myf5 promoter, directs transgene expression either to embryonic or fetal muscles. We now show that this enhancer also regulates Mrf4/Myf5 transcription in the adult. A17 linked to the Myf5 promoter drives expression in muscle satellite cells, whereas with the Mrf4 promoter, A17 directs transgene expression to myonuclei. Interestingly, expression of A17-Mrf4-nlacZ transgenes in myonuclei varies between muscles, revealing muscle autonomous transcriptional regulation. During muscle repair, satellite cells are induced to proliferate and differentiate to provide new myonuclei. A17 directs Myf5 expression in satellite cell progeny while it only drives the Mrf4 promoter after differentiation. Importantly, therefore, this promoter-specific activity directed by A17 reflects aspects of the expression profiles of the endogenous Myf5 and Mrf4 genes.

163. The Effects of Inflammation on GFAP Expression in Satellite Cells of the Dorsal Root Ganglion

163. The Effects of Inflammation on GFAP Expression in Satellite Cells of the Dorsal Root Ganglion

Sox15 and Fhl3 transcriptionally coactivate Foxk1 and regulate myogenic progenitor cells

The regulation of myogenic progenitor cells during muscle regeneration is not clearly understood. We have previously shown that the Foxk1 gene, a member of the forkhead/winged helix family of transcription factors, is expressed in myogenic progenitor cells in adult skeletal muscle. In the present study, we utilize transgenic technology and demonstrate that the 4.6 kb upstream fragment of the Foxk1 gene directs beta-galactosidase expression to the myogenic progenitor cell population. We further establish that Sox15 directs Foxk1 expression to the myogenic progenitor cell population, as it binds to an evolutionarily conserved site and recruits Fhl3 to transcriptionally coactivate Foxk1 gene expression. Knockdown of endogenous Sox15 results in perturbed cell cycle kinetics and decreased Foxk1 expression. Furthermore, Sox15 mutant mice display perturbed skeletal muscle regeneration, due in part to decreased numbers of satellite cells and decreased Foxk1 expression. These studies demonstrate that Sox15, Fhl3 and Foxk1 function to coordinately regulate the myogenic progenitor cell population and skeletal muscle regeneration.

Alterations of M‐cadherin, neural cell adhesion molecule and β‐catenin expression in satellite cells during overload‐induced skeletal muscle hypertrophy

Neural cell adhesion molecule (NCAM) and M-cadherin are cell adhesion molecules expressed on the surface of skeletal muscle satellite cell (SC). During myogenic morphogenesis, M-cadherin participates in mediating terminal differentiation and fusion of myoblasts by forming a complex with beta-catenin and that NCAM contributes to myotube formation by fusion of myoblasts. Hypertrophy and hyperplasia of functionally overloaded skeletal muscle results from the fusion with SCs into the existing myofibres or new myofibre formation by SC-SC fusion. However, the alterations of NCAM, M-cadherin and beta-catenin expressions in SCs in response to functional overload have not been investigated. Using immunohistochemical approaches, we examined the temporal and spatial expression patterns of these factors expressed in SCs during the functional overload of skeletal muscles. Myofibres with SCs showing NCAM+/M-cadherin-, NCAM+/M-cadherin+ or NCAM-/M-cadherin+ were detected in overloaded muscles. The percentage changes of myofibres with SCs showing NCAM+/M-cadherin-, NCAM+/M-cadherin+ or NCAM-/M-cadherin+ were elevated in day-3 post-overloaded muscles, and then only the percentage changes of myofibres with SCs showing NCAM-/M-cadherin+ were significantly increased in day-7 post-overload muscles (P < 0.05). Both beta-catenin and M-cadherin were co-localized throughout quiescent, proliferation and differentiation stages of SCs. These results suggested that the expressions of NCAM, M-cadherin and beta-catenin in SCs may be controlled by distinct regulatory mechanisms during functional overload, and that interactions among NCAM, M-cadherin and beta-catenin in SCs may play important roles to contribute to overload-induced muscle hypertrophy via fusion with each other or into the existing myofibres of SCs.

In Vivo and In Vitro Dysferlin Expression in Human Muscle Satellite Cells

Dysferlin is a protein of the sarcolemma that is mutated in patients with limb girdle muscular dystrophy 2B, Miyoshi myopathy, and distal anterior myopathy. It has been implicated in muscle signaling and sarcolemma repair. To further understand its functional role we studied dysferlin expression in satellite cells (SCs) in normal and pathological human muscle biopsies, as well as in primary cultures of human skeletal muscle. Using immunohistochemistry we detected dysferlin-positive (Dysf+) SCs. Double staining with c-met+, a total SC marker, showed that the number of Dysf+ SCs ranged from 33.7% +/- 4.4% in normal muscle to 68.0% +/- 6.2% in pathological muscles, whereas double staining with MyoD/Dysf showed that all activated SC (MyoD+) were also Dysf+. These results indicate that dysferlin is upregulated in activated SCs. In vitro, immunohistochemistry, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and real-time PCR showed that both dysferlin mRNA and protein expression were higher in multinucleated myotubes than in the myoblast stage (p < 0.05). Furthermore, experiments of inhibition of myoblast fusion with amiloride, a type T calcium channel antagonist, showed that dysferlin levels were lower in treated than in non-treated cultures (p < 0.001), demonstrating that dysferlin expression reached peak levels upon differentiation into myotubes. These results and the in vivo findings of dysferlin expression when SCs are activated confirm the involvement of dysferlin in human muscle regeneration/repair and its possible role in fusion events during muscle development.

Myf5 expression in satellite cells and spindles in adult muscle is controlled by separate genetic elements

The myogenic regulatory factor Myf5 is integral to the initiation and control of skeletal muscle formation. In adult muscle, Myf5 is expressed in satellite cells, stem cells of mature muscle, but not in the myonuclei that sustain the myofibre. Using the Myf5 nlacZ/+ mouse, we now show that Myf5 is also constitutively expressed in muscle spindles-stretch-sensitive mechanoreceptors, while muscle denervation induces extensive reactivation of the Myf5 gene in myonuclei. To identify the elements involved in the regulation of Myf5 in adult muscle, we analysed reporter gene expression in a transgenic bacterial artificial chromosome (BAC) deletion series of the Mrf4/Myf5 locus. A BAC carrying 140 kb upstream of the Myf5 transcription start site was sufficient to drive all aspects of Myf5 expression in adult muscle. In contrast, BACs carrying 88 and 59 kb upstream were unable to drive consistent expression in satellite cells, although expression in muscle spindles and reactivation of the locus in myonuclei were retained. Therefore, as during development, multiple enhancers are required to generate the full expression pattern of Myf5 in the adult. Together, these observations show that elements controlling adult Myf5 expression are genetically separable and possibly distinct from those that control Myf5 during development. These studies are a first step towards identifying cognate transcription factors involved in muscle stem cell regulation.

Metastasis-associated S100A4 (Mts1) protein is expressed in subpopulations of sensory and autonomic neurons and in Schwann cells of the adult rat.

S100A4 (Mts1) is a member of a family of calcium-binding proteins of the EF-hand type, which are widely expressed in the nervous system, where they appear to be involved in the regulation of neuron survival, plasticity, and response to injury or disease. S100A4 has previously been demonstrated in astrocytes of the white matter and rostral migratory stream of the adult rat. After injury, S100A4 is markedly up-regulated in affected central nervous white matter areas as well as in the periventricular area and rostral migratory stream. Here, we show that S100A4 is expressed in a subpopulation of dorsal root, trigeminal, geniculate, and nodose ganglion cells; in a subpopulation of postganglionic sympathetic and parasympathetic neurons; in chromaffin cells of the adrenal medulla; and in satellite and Schwann cells. In dorsal root ganglia, S100A4-positive cells appear to constitute a subpopulation of small ganglion neurons, a few of which coexpressed calcitonin gene-related peptide (CGRP) and Griffonia simplicifolia agglutinin (GSA) isolectin B4 (B4). S100A4 protein appears to be transported from dorsal root ganglia to the spinal cord, where it is deposited in the tract of Lissauer. After peripheral nerve or dorsal root injury, a few S100A4-positive cells coexpress CGRP, GSA, or galanin. Peripheral nerve or dorsal root injury induces a marked up-regulation of S100A4 expression in satellite cells in the ganglion and in Schwann cells at the injury site and in the distal stump. This pattern of distribution partially overlaps that of the previously studied S100B and S100A6 proteins, indicating a possible functional cooperation between these proteins. The presence of S100A4 in sensory neurons, including their processes in the central nervous system, suggests that S100A4 is involved in propagation of sensory impulses in specific fiber types.