Hedgehog can drive terminal differentiation of amniote slow skeletal muscle

Background

Secreted Hedgehog (Hh) signalling molecules have profound influences on many developing and regenerating tissues. Yet in most vertebrate tissues it is unclear which Hh-responses are the direct result of Hh action on a particular cell type because Hhs frequently elicit secondary signals. In developing skeletal muscle, Hhs promote slow myogenesis in zebrafish and are involved in specification of medial muscle cells in amniote somites. However, the extent to which non-myogenic cells, myoblasts or differentiating myocytes are direct or indirect targets of Hh signalling is not known.

Results

We show that Sonic hedgehog (Shh) can act directly on cultured C2 myoblasts, driving Gli1 expression, myogenin up-regulation and terminal differentiation, even in the presence of growth factors that normally prevent differentiation. Distinct myoblasts respond differently to Shh: in some slow myosin expression is increased, whereas in others Shh simply enhances terminal differentiation. Exposure of chick wing bud cells to Shh in culture increases numbers of both muscle and non-muscle cells, yet simultaneously enhances differentiation of myoblasts. The small proportion of differentiated muscle cells expressing definitive slow myosin can be doubled by Shh. Shh over-expression in chick limb bud reduces muscle mass at early developmental stages while inducing ectopic slow muscle fibre formation. Abundant later-differentiating fibres, however, do not express extra slow myosin. Conversely, Hh loss of function in the limb bud, caused by implanting hybridoma cells expressing a functionally blocking anti-Hh antibody, reduces early slow muscle formation and differentiation, but does not prevent later slow myogenesis. Analysis of Hh knockout mice indicates that Shh promotes early somitic slow myogenesis.

Conclusions

Taken together, the data show that Hh can have direct pro-differentiative effects on myoblasts and that early-developing muscle requires Hh for normal differentiation and slow myosin expression. We propose a simple model of how direct and indirect effects of Hh regulate early limb myogenesis.

     

Community effect triggers terminal differentiation of myogenic cells derived from muscle satellite cells by quenching Smad signaling

A high concentration of bone morphogenetic proteins (BMPs) stimulates myogenic progenitor cells to undergo heterotopic osteogenic differentiation. However, the physiological role of the Smad signaling pathway during terminal muscle differentiation has not been resolved. We report here that Smad1/5/8 was phosphorylated and activated in undifferentiated growing mouse myogenic progenitor Ric10 cells without exposure to any exogenous BMPs. The amount of phosphorylated Smad1/5/8 was severely reduced during precocious myogenic differentiation under the high cell density culture condition even in growth medium supplemented with a high concentration of serum. Inhibition of the Smad signaling pathway by dorsomorphin, an inhibitor of Smad activation, or noggin, a specific antagonist of BMP, induced precocious terminal differentiation of myogenic progenitor cells in a cell density-dependent fashion even in growth medium. In addition, Smad1/5/8 was transiently activated in proliferating myogenic progenitor cells during muscle regeneration in rats. The present results indicate that the Smad signaling pathway is involved in a critical switch between growth and differentiation of myogenic progenitor cells both in vitro and in vivo. Furthermore, precocious cell density-dependent myogenic differentiation suggests that a community effect triggers the terminal muscle differentiation of myogenic cells by quenching the Smad signaling.     

The development of fibre types in grafts of a slow tonic avian muscle, the dorsocutaneous latissimus dorsi

The dorsocutaneous (DLD) and anterior (ALD) latissimus dorsii are both homogeneous slow tonic muscles. Autografts of mature DLD were attached onto the ALD of chickens to study regeneration of slow tonic muscle fibres innervated exclusively by slow tonic nerves. Fifty-three grafts were examined from 3 to 231 days after implantation for myosin ATPase, and for heavy chains of fast myosin. New muscle fibres in grafts were initially type 1 (slow) or type 2 (fast twitch). Tonic type 3 fibres were slow to differentiate and were not seen within 59 days. From 105 days many fibres were type 3A and type 1 were no longer apparent. However, type 2 fibres persisted and appeared to be present instead of type 3B fibres even after 8 months.     

Sonic hedgehog inhibits the terminal differentiation of limb myoblasts committed to the slow muscle lineage

The proliferation, differentiation, and fusion of a small number of myogenic precursor cells must be precisely regulated during development to ensure the proper size, organization, and function of the limb musculature. We have examined the role of Sonic hedgehog (Shh) in these processes by both augmentation and inhibition of the Shh-mediated signaling pathway. Our data show that Shh regulates muscle development by repressing the terminal differentiation of early myogenic precursor cells and does not function as a myoblast mitogen. Shh function in hypaxial muscle appears to be spatially restricted to the early myoblast population within the ventral muscles of the posterior region of the limb. Furthermore, Shh appears to act as a permissive, rather than an inductive, signal for slow MyHC expression in myoblasts. Our data thus provide the foundation for a new hypothesis for Shh function in hypaxial skeletal muscle development.     

Controlled differentiation of myoblast cells into fast and slow muscle fibers

Skeletal muscles are classified into fast and slow muscles, which are characterized by the expression of fast-type myosin heavy chains (fMyHCs) or slow-type myosin heavy chains (sMyHCs), respectively. However, the mechanism of subtype determination during muscle fiber regeneration is unclear. We have analyzed whether the type of muscle is determined in the myoblast cells or is controlled by the environment in which the muscle fibers are formed from myoblast cells. When myoblast cells from 7-day-old chick embryo were cultured and formed into muscle fibers, more than half of the fibers produced only fMyHCs, and the remaining fibers produced both fMyHCs and sMyHCs. However, when myoblast cells were cultured in medium supplemented with a small amount of slow muscle extract, the expression of sMyHCs in muscle fibers increased, whereas the expression of fMyHCs increased in the group supplemented with fast muscle extract compared with the control group. The same results were obtained when cloned mouse myoblast cells (C₂C₁₂ cells) were cultured and formed into muscle fibers. The data presented here thus show that the subtype differentiation of muscle fiber is controlled by the environment in which the muscle fiber forms. This work was funded by the Sasakawa Scientific Research Grant of the Japan Science Society.     

In vitro differentiation in the absence of nerve of avian myoblasts derived from slow and fast muscle rudiments

Slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles of 9-day-old quail embryos were cultured in vitro without neurons for 1 to 12 weeks. Several differences could be observed between ALD- and PLD-derived cells. PLD cultures proliferated less rapidly than ALD cultures. ALD-derived muscle fibres exhibited wide Z lines, numerous mitochondria, and a poorly developed sarcotubular system, while PLD-derived muscle fibres exhibited narrow Z lines, few mitochondria, and an abundant sarcotubular system. Staining for myofibrillar ATPase revealed that all well-differentiated ALD-derived muscle fibres were of the beta' type, while PLD-derived fibres were of beta and beta R types. These results show that myoblasts from slow and fast muscle rudiments can express in vitro some of the characteristic features of slow and fast muscle fibres, independently of motor innervation.     

Regeneration of a tonic avian muscle: transformation to a twitch morphology.

The tonic anterior latissimus dorsi muscle of the pigeon was excised, minced into fine pieces, replaced into its original bed, and allowed to regenerate for periods up to 37 weeks. Although regeneration was asynchronous, regeneration patterns of the muscle fibers suggested the following sequence of fiber development: undifferentiated to tonic to twitch. Fiber types were identified on the basis of Z-line morphology and qualitative development and organization of the sarcotubular system as demonstrated by electron microscopy. Histochemical demonstration of myofibrillar adenosine triphosphatase and succinic dehydrogenase activities corroborated the morphological evidence, suggesting a transformation to a twitch morphology. In addition to the transformation to the twitch morphology, other alterations were observed in these regenerating fibers. Among these were large numbers of closely-packed 60-nm-diameter tubules, thought to be derived from the sarcoplasmic reticulum; mitochondria with intermembraneous dense material; and Z-line streaming. The transformation of the muscle fibers from tonic to twitch morphology is discussed in terms of alterations in nerve impulse activity to the regenerating muscle.     

Importance of satellite cells in the strength recovery after eccentric contraction-induced muscle injury

The purpose of this study was to determine if the elimination of satellite cell proliferation using gamma-irradiation would inhibit normal force recovery after eccentric contraction-induced muscle injury. Adult female ICR mice were implanted with a stimulating nerve cuff on the common peroneal nerve and assigned to one of four groups: 1) irradiation- and eccentric contraction-induced injury, 2) eccentric contraction-induced injury only, 3) irradiation only, and 4) no intervention. Anterior crural muscles were irradiated with a dose of 2,500 rad and injured with 150 in vivo maximal eccentric contractions. Maximal isometric torque was determined weekly through 35 days postinjury. Immediately after injury, maximal isometric torque was reduced by approximately 50% and had returned to normal by 28 days postinjury in the nonirradiated injured mice. However, torque production of irradiated injured animals did not recover fully and was 25% less than that of injured nonirradiated mice 35 days postinjury. These data suggest that satellite cell proliferation is required for approximately half of the force recovery after eccentric contraction-induced injury.     

Six family genes control the proliferation and differentiation of muscle satellite cells

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4^+/−Six5^−/− mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.     

Desmin is present in proliferating rat muscle satellite cells but not in bovine muscle satellite cells.

The presence of desmin was characterized in cultured rat and bovine satellite cells and its potential usefulness as a marker for identifying satellite cells in vitro was evaluated. In primary cultures, positive immunohistochemical staining for desmin and skeletal muscle myosin was observed in rat and bovine myotubes. A small number of mononucleated cells (20% of rat satellite cells and 5% of bovine satellite cells) were myosin-positive, indicative of post-mitotic differentiated myocytes. In bovine satellite cell cultures 13% of the mononucleated cells were desmin-positive, while 84% of the mononucleated cells in rat satellite cell cultures were desmin-positive. Rat satellite cell mass cultures and bovine satellite cell clonal density cultures were pulsed with 3H-thymidine, and autoradiographic data revealed that greater than 94% of dividing rat cells were desmin-positive, suggesting that desmin is synthesized in proliferating rat satellite cells. However, no desmin was seen in cells that incorporated labeled thymidine in bovine satellite cell clones. Analysis of clonal density cultures revealed that only 14% of the mononucleated cells in bovine satellite cell colonies were desmin-positive, whereas 98% of the cells in rat satellite cell colonies were desmin-positive. Fibroblast colonies from both species were desmin-negative. In order to further examine the relationship between satellite cell differentiation and desmin expression, 5-bromo-2'-deoxyuridine (BrdU) was added to culture medium at the time of plating to inhibit differentiation. Fusion was inhibited in rat and bovine cultures, and cells continued to divide. Very few desmin-positive cells were found in bovine cultures, but greater than 90% of the cells in rat cultures stained positive for desmin. The presence of desmin and sarcomeric myosin was also evaluated in regenerating rat tibialis anterior five days after bupivacaine injection. In regenerating areas of the muscle many desmin-positive cells were present, and only a few cells stained positive for skeletal muscle myosin. Application of desmin staining to rat satellite cell growth assays indicated that rat satellite cells cultured in serum-containing medium were contaminated with fibroblasts at levels that ranged from approximately 5% in 24 hr cultures to 15% in mature cultures. In defined medium 4 day cultures contain approximately 95% to 98% desmin-positive satellite cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Release of hepatocyte growth factor from mechanically stretched skeletal muscle satellite cells and role of pH and nitric oxide

Application of mechanical stretch to cultured adult rat muscle satellite cells results in release of hepatocyte growth factor (HGF) and accelerated entry into the cell cycle. Stretch activation of cultured rat muscle satellite cells was observed only when medium pH was between 7.1 and 7.5, even though activation of satellite cells was accelerated by exogenous HGF over a pH range from 6.9 to 7.8. Furthermore, HGF was only released in stretched cultures when the pH of the medium was between 7.1 and 7.4. Conditioned medium from stretched satellite cell cultures stimulated activation of unstretched satellite cells, and the addition of anti-HGF neutralizing antibodies to stretch-conditioned medium inhibited the stretch activation response. Conditioned medium from satellite cells that were stretched in the presence of nitric-oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester hydrochloride did not accelerate activation of unstretched control satellite cells, and HGF was not released into the medium. Conditioned medium from unstretched cells that were treated with a nitric oxide donor, sodium nitroprusside dihydrate, was able to accelerate the activation of satellite cells in vitro, and HGF was found in the conditioned medium. Immunoblot analysis indicated that both neuronal and endothelial NOS isoforms were present in satellite cell cultures. Furthermore, assays of NOS activity in stretched satellite cell cultures demonstrated that NOS is stimulated when satellite cells are stretched in vitro. These experiments indicate that stretch triggers an intracellular cascade of events, including nitric oxide synthesis, which results in HGF release and satellite cell activation.     

Ultrastructural duality of extrafusal fibers in a slow (tonic) skeletal muscle

Summary Ultrastructural and stereological assessment of the mature avian anterior latissimus dorsi (ALD) muscle showed that it contains two kinds of extrafusal fibers. This fine structural dichotomy of fiber types in the ALD correlated well with their previously reported histochemical duality. Distinct differences occur in sarcomere banding, myofibrillar area, sarcotubular and mitochondrial density, and in morphology of motor-nerve terminals. Both myofiber types in this muscle were interpreted as representing varieties of slow or tonic muscle fibers.Both fibers contain myofibrils that, despite differences in cross-sectional area, were large, irregular, and ribbon-shaped, typical of the Felderstruktur appearance of true slow fibers. Whereas the majority of fibers (type-1) are devoid of well-defined M-bands, the minor fiber population (type-2) exhibit prominent M-bands in the center of each sarcomere. In addition, type-1 tonic fibers contain a significantly lower mitochondrial and sarcotubular volume than the tonic fibers of type-2. While both fiber types exhibit motor-nerve terminals that are small, smooth and punctate in appearance, those on the type2 fibers often had a number of shallow postjunctional folds. Whether or not these two classes of extrafusal fiber in this muscle represent two separate and distinct types of motor units remains to be determined functionally.Supported by grants from the Medical Research Council and the Muscular Dystrophy Association of Canada. The author gratefully acknowledges the excellent technical assistance of Susan L. Shinn     

Activation of muscle satellite cells in single-fiber cultures.

Satellite stem cell activation is the process by which quiescent precursor cells resident on muscle fibers are recruited to cycle and move. Two processes are reported to affect satellite cell activation. In vivo, nitric oxide (NO) produced by NO synthase in fibers (NOS-Imu) promotes activation. In cell cultures, hepatocyte growth factor (HGF) is the major activating factor isolated from crushed muscle extract (CME). In this study we hypothesized that distinct and possibly related events were mediated by NO and HGF during activation. Intact fibers were cultured in the presence of bromodeoxyuridine (BrdU) to label DNA synthesis over 48 h. Experiments were designed to test the effects of CME, HGF, a NOS substrate L-arginine, and the NOS inhibitor L-NAME on activation, determined as the number of BrdU-positive satellite cells per fiber. Activation was increased significantly by CME, HGF, and L-arginine. L-Arginine increased activation in a dose-response manner. CME-induced activation was reduced significantly by NOS inhibition. Exposure to marcaine (10 min) caused reversible membrane damage without hypercontraction, as shown by characterizing the sarcolemmal integrity. The resulting decrease in satellite cell activation could be overcome by exogenous HGF. Results support the hypothesis that NO is involved in recruiting to cycle those satellite cells resident on fibers. Separate assessments of resident and free muscle cells showed that HGF and NO also participate in mobilizing satellite cells. Since HGF counteracted NOS inhibition and marcaine-induced membrane damage, data suggest that NO may mediate early steps in activation and precede HGF-mediated events.     

Depot-specific gene expression profiles during differentiation and transdifferentiation of bovine muscle satellite cells, and differentiation of preadipocytes

We report a systematic study of gene expression during myogenesis and transdifferentiation in four bovine muscle tissues and of adipogenesis in three bovine fat tissues using DNA microarray analysis. One hundred hybridizations were performed and 7245 genes of known and unknown function were identified as being differentially expressed. Supervised hierarchical cluster analysis of gene expression patterns revealed the tissue specificity of genes. A close relationship in global gene expression observed for adipocyte-like cells derived from muscle and adipocytes derived from intramuscular fat suggests a common origin for these cells. The role of transthyretin in myogenesis is a novel finding. Different genes were highly induced during the transdifferentiation of myogenic satellite cells and in the adipogenesis of preadipocytes, indicating the involvement of different molecular mechanisms in these processes. Induction of CD36 and FABP4 expression in adipocyte-like cells and adipocytes may share a common pathway.