Generation of mononucleate cells from post-mitotic myotubes proceeds in the absence of cell cycle progression

The remarkable regenerative ability of adult urodele amphibians depends in part of the plasticity of differentiated cells at the site of injury. Limb regeneration proceeds by formation of a mesenchymal growth zone or blastema under the wound epidermis at the end of the stump. Previous work has shown that when cultured post-mitotic newt myotubes are introduced into the blastema, they re-enter the cell cycle and undergo conversion to mononucleate cells which divide and contribute to the regenerate [11, 13]. In order to investigate the interdependence of these two aspects of plasticity, we have blocked cell cycle progression of the myotubes either by X-irradiation or by transfection of the CDK4/6 inhibitor p16. In each case, the efficacy of the block was evaluated in culture after activation of S phase re-entry by serum stimulation. The experimental myotubes were implanted into limb blastemas along with a differentially labelled control population of myotubes containing an equivalent number of nuclei. X-irradiated myotubes gave rise to mononucleate cells in the limb blastema, and the progeny were blocked in respect of S phase entry. Comparable results were obtained with the p16-expressing myotubes. We conclude that progression through S or M phase is not required for generation of mononucleate cells and suggest that such cells may arise by budding from the muscle syncytium.     

Distinctive expression of Myf5 in relation to differentiation and plasticity of newt muscle cells

Regeneration in urodele amphibians such as the newt reflects the local plasticity of differentiated cells. Newt myotubes and myofibres undergo S phase re-entry and cellularisation in the limb blastema, and we have analysed the regulation of Myf5 in relation to these events. Surprisingly, Myf5 was expressed after fusion in cultured newt myotubes and in myofibers of the adult limb, in contrast to its familiar expression in myoblasts in other vertebrates. Its expression was markedly down regulated in cultured newt myotubes after S phase re-entry induced by serum stimulation, as well as by exposure to the trisubstituted purine called myoseverin which induces cellularisation. We have attempted to relate this striking difference from other vertebrates to the requirement for multinucleate urodele muscle cells to contribute to the regeneration blastema.     

The Regenerative Plasticity of Isolated Urodele Myofibers and Its Dependence on Msx1

The conversion of multinucleate postmitotic muscle fibers to dividing mononucleate progeny cells (cellularisation) occurs during limb regeneration in salamanders, but the cellular events and molecular regulation underlying this remarkable process are not understood. The homeobox gene Msx1 has been studied as an antagonist of muscle differentiation, and its expression in cultured mouse myotubes induces about 5% of the cells to undergo cellularisation and viable fragmentation, but its relevance for the endogenous programme of salamander regeneration is unknown. We dissociated muscle fibers from the limb of larval salamanders and plated them in culture. Most of the fibers were activated by dissociation to mobilise their nuclei and undergo cellularisation or breakage into viable multinucleate fragments. This was followed by microinjection of a lineage tracer into single fibers and analysis of the labelled progeny cells, as well as by time-lapse microscopy. The fibers showing morphological plasticity selectively expressed Msx1 mRNA and protein. The uptake of morpholino antisense oligonucleotides directed to Msx1 led to a specific decrease in expression of Msx1 protein in myonuclei and marked inhibition of cellularisation and fragmentation. Myofibers of the salamander respond to dissociation by activation of an endogenous programme of cellularisation and fragmentation. Lineage tracing demonstrates that cycling mononucleate progeny cells are derived from a single myofiber. The induction of Msx1 expression is required to activate this programme. Our understanding of the regulation of plasticity in postmitotic salamander cells should inform strategies to promote regeneration in other contexts.     

The regenerative plasticity of isolated urodele myofibers and its dependence on MSX1

The conversion of multinucleate postmitotic muscle fibers to dividing mononucleate progeny cells (cellularisation) occurs during limb regeneration in salamanders, but the cellular events and molecular regulation underlying this remarkable process are not understood. The homeobox gene Msx1 has been studied as an antagonist of muscle differentiation, and its expression in cultured mouse myotubes induces about 5% of the cells to undergo cellularisation and viable fragmentation, but its relevance for the endogenous programme of salamander regeneration is unknown. We dissociated muscle fibers from the limb of larval salamanders and plated them in culture. Most of the fibers were activated by dissociation to mobilise their nuclei and undergo cellularisation or breakage into viable multinucleate fragments. This was followed by microinjection of a lineage tracer into single fibers and analysis of the labelled progeny cells, as well as by time-lapse microscopy. The fibers showing morphological plasticity selectively expressed Msx1 mRNA and protein. The uptake of morpholino antisense oligonucleotides directed to Msx1 led to a specific decrease in expression of Msx1 protein in myonuclei and marked inhibition of cellularisation and fragmentation. Myofibers of the salamander respond to dissociation by activation of an endogenous programme of cellularisation and fragmentation. Lineage tracing demonstrates that cycling mononucleate progeny cells are derived from a single myofiber. The induction of Msx1 expression is required to activate this programme. Our understanding of the regulation of plasticity in postmitotic salamander cells should inform strategies to promote regeneration in other contexts.     

Ganglia implantation as a means of supplying neurotrophic stimulation to the newt regeneration blastema: cell-cycle effects in innervated and denervated limbs.

Regulation of blastema cell proliferation during amphibian limb regeneration is poorly understood. One unexplained phenomenon is the relatively low level of active cell cycling in the adult newt blastema compared to that of larval axolotls. In the present study, we used ganglia implantation as a means of "superinnervating" normally innervated adult newt blastemas to test whether blastema cell subpopulations are responsive to nerve augmentation. The effectiveness of implanted ganglia to provide neurotrophic stimulation was demonstrated in denervated blastemas. Blastemas implanted with 2 dorsal root ganglia and simultaneously denervated 14 days after amputation exhibited control levels of cell cycle activity 6 days later, as measured by 3H-thymidine pulse labeling. Denervated blastemas that were sham-operated or implanted with pituitary glands exhibited cell-cycle declines similar to those of denervated blastemas without implanted ganglia. Thus, 2 implanted ganglia provide neurotrophic stimulation equivalent to that of the normal nerve supply. Dorsal root ganglia implanted into normally innervated blastemas, which should effectively double neurotrophic activity to the blastema, had no effect on cell-cycle activity, innervated blastemas implanted with ganglia for 6 days exhibited pulse labeling indices similar to those of normally innervated blastemas. These data indicate that neurotrophic stimulation is not normally limiting in innervated limbs, and that some other factor, whether extrinsic or intrinsic to blastema cells, accounts for the relatively low level of active cell cycling in the adult newt blastema.     

The origin of syncytial muscle fibres in the myotomes of Xenopus laevis--a revision

During the early stages of myogenesis in X. laevis, the primary myoblasts (of mesodermal origin) differentiate simultaneously, in each myotome, into mononucleate myotubes. At later stages mesenchymal cells appear in intermyotomal fissures and then in the myotomes between myotubes and contribute to the formation ofsyncytial muscle fibres. The pathway of mesenchymals cell during myogenesis was described in X laevis by monitoring the incorporation of 3H-thymidine. 3H-thymidine was incorporated in the nuclei of mesenchymal cells in intermyotomal fissures of younger myotomes and then in those of older myotomes between the myotubes revealing the proliferation of mesenchymal cells. As expected, nuclei of differentiating mononucleate myotubes did not incorporate 3H-thymidine. At later stages of myogenesis the myotubes were found to contain two classes of nuclei: large nuclei of the primary myoblasts (of myotomal origin) and smaller nuclei originating from secondary myoblasts ofmesenchymal origin. TEM and autoradiographic analyses confirm that mulinucleate myotubes in X. laevis arise through fusion of secondary myoblasts with mononucleate myotubes.     

Isolation and clonal analysis of satellite cells from chicken pectoralis muscle

Satellite cells, liberated from the breast muscle of young adult chickens by sequential treatment with collagenase and trypsin, were fractionated by Percoll density centrifugation to remove myofibril fragments and cell debris which otherwise heavily contaminate the preparation. This procedure allowed direct measurements of cell yields (1.5-4 X 10(5) cells/g tissue), plating efficiencies (27-40%) and identification of single cells in culture. In mass cultures, satellite cells gave rise to myotubes on the fifth day, and the progeny of these cells were sequentially passaged several times without losing myogenic traits. In clonal studies, over 90% of the satellite cells gave rise to large clones of which more than 99% were myogenic as demonstrated by the appearance of myotubes. The results obtained with satellite cells differ from observations made using embryonic muscle cell preparation from chicks. In the embryonic system massive formation of myotubes was observed following the third day of culture; sequential subculturing led to overgrowth of fibroblast-like cells following the first passage; and cells gave rise to both small myogenic clones (up to 16 terminally differentiated cells per clone) and non-myogenic clones in addition to large myogenic clones. We conclude that the isolated satellite cells represent a homogeneous cell population and reside in a stem cell compartment.     

Thrombin regulates S-phase re-entry by cultured newt myotubes.

BACKGROUND: Adult urodele amphibians such as the newt have remarkable regenerative ability, and a critical aspect of this is the ability of differentiated cells to re-enter the cell cycle and lose their differentiated characteristics. Unlike mammalian myotubes, cultured newt myotubes are able to enter and traverse S phase, following serum stimulation, by a pathway leading to phosphorylation of the retinoblastoma protein. The extracellular regulation of this pathway is unknown. RESULTS: Like their mammalian counterparts, newt myotubes were refractory to mitogenic growth factors such as the platelet-derived growth factor (PDGF), which act on their mononucleate precursor cells. Cultured newt myotubes were activated to enter S phase by purified thrombin in the presence of subthreshold amounts of serum. The activation proceeded by an indirect mechanism in which thrombin cleaved components in serum to generate a ligand that acted directly on the myotubes. The ligand was identified as a second activity present in preparations of crude thrombin and that was active after removal of all thrombin activity. It induced newt myotubes to enter S phase in serum-free medium, and it acted on myotubes but not on the mononucleate precursor cells. Cultured mouse myotubes were refractory to this indirect mechanism of S-phase re-entry. CONCLUSIONS: These results provide a link between reversal of differentiation and the acute events of wound healing. The urodele myotube responds to a ligand generated downstream of thrombin activation and re-enters the cell cycle. Although this ligand can be generated in mammalian sera, the mammalian myotube is unresponsive. These results provide a model at the cellular level for the difference in regenerative ability between urodeles and mammals.     

Positional identity of adult stem cells in salamander limb regeneration

Limb regeneration in larval and adult salamanders proceeds from a mound of mesenchymal stem cells called the limb blastema. The blastema gives rise just to those structures distal to its level of origin, and this property of positional identity is reset to more proximal values by treatment with retinoic acid. We have identified a cell surface protein, called Prod1/CD59, which appears to be a determinant of proximodistal identity. Prod1 is expressed in an exponential gradient in an adult limb as determined by detection of both mRNA and immunoreactive protein. Prod1 protein is up-regulated after treatment of distal blastemas with RA and this is particularly marked in cells of the dermis. These cells have previously been implicated in pattern formation during limb regeneration.     

In vivo imaging indicates muscle fiber dedifferentiation is a major contributor to the regenerating tail blastema.

During tail regeneration in urodele amphibians such as axolotls, all of the tissue types, including muscle, dermis, spinal cord, and cartilage, are regenerated. It is not known how this diversity of cell types is reformed with such precision. In particular, the number and variety of mature cell types in the remaining stump that contribute to the blastema is unclear. Using Nomarski imaging, we followed the process of regeneration in the larval axolotl tail. Combining this with in vivo fluorescent labeling of single muscle fibers, we show that mature muscle dedifferentiates. Muscle dedifferentiation occurs by the synchronous fragmentation of the multinucleate muscle fiber into mononucleate cells followed by rapid cell proliferation and the extension of cell processes. We further show that direct clipping of the muscle fiber and severe tissue damage around the fiber are both required to initiate dedifferentiation. Our observations also make it possible to estimate for the first time how many of the blastema cells arise specifically from muscle dedifferentiation. Calculations based on our data suggest that up to 29% of nondermal-derived cells in the blastema come from dedifferentiation of mature muscle fibers. Overall, these results show that endogenous multinucleate muscle fibers can dedifferentiate into mononucleate cells and contribute significantly to the blastema.     

Oxygen enhances fusion of cultured chick embryo myoblasts

Fusion of mononucleate myoblasts to form multinucleated myotubes increases when skeletal muscle cells are grown in progressively higher oxygen concentrations (5%, 20%, and 40% oxygen). At four days of growth fusion of myoblasts (as expressed by the percent of all muscle nuclei that are located in myotubes) is 57 ± 2% in 5% oxygen, 68 ± 1% in 20% oxygen, and 78 ± 2% in 40% oxygen (P<0.001). However, at a concentration of 40%, oxygen depresses the rate of cell division and thereby affects the number of myoblasts available for fusion. Thus, oxygen concentration significantly modifies growth of skeletal muscle in vitro. Its net effect on myotube formation results from the interaction of its separate effects to enhance cell fusion and to depress cell proliferation.     

Stimulation in cell culture of mesenchymal cells of newt limb blastemas by EDGF I or II (basic or acidic FGF)

After amputation of a newt limb, a blastema forms on the amputation plane and later differentiates to regenerate all the missing parts of the limb. Proliferation of blastema cells is under the control of severed nerves which deliver a 'neurotrophic factor' (NTF) of unknown nature. In order to characterize this factor we use a primary culture of blastema mesenchymal cells; changes in mitotic index after 48-h colchicine treatment indicate mitogenic activity of potential growth substances. These cells, which are stimulated by nerve extracts (mitotic index X 6), were tested with two purified growth factors extracted from bovine retina or brain (EDGF I = basic FGF and EDGF II = acidic FGF). We show that these two growth factors stimulate proliferation of blastema cell cultures in a dose-dependent manner. Maximal stimulation was obtained at 3 pM for EDGF I (mitotic index X 5.7) or 300 pM for EDGF II (mitotic index X 4.9). So it appears that these two growth factors have a mitogenic activity on blastema mesenchymal cells similar to that obtained with nerve extracts. The fact that two different growth factors can stimulate these cells raises the question of whether both are present in NTF and/or whether there are receptors to both EDGF I and EDGF II on mesenchymal cell membranes.     

Immunohistochemical localization of a proto-cadherin fat tumour-suppressor homolog in the regenerating tail of lizard suggests a role in apical growth control

The present immunohistochemical and western blotting study evaluates the localization of a proto-cadherin which gene is overexpressed in the regenerating blastema of the lizard Podarcis muralis. Bioinformatic analysis suggests that the antibody recognizes FAT1/2 proteins. Western blot indicates a main band around 50 kDa, a likely fragment derived from the original membrane-bound large protein. Immunofluorescence shows main labelling in differentiating wound keratinocytes, lower in ependyma, mesenchyme and extracellular matrix of the blastema. The apical epidermal peg contains keratinocytes with labelled peripheral cytoplasm, as confirmed using ultrastructural immunogold that also reveals most labelling located along the cell surface of mesenchymal cells. Myoblasts and differentiating myotubes of regenerating muscles are less intensely labelled. The regenerating cartilaginous tube contains sparse labelled chondroblasts, especially in external and internal perichondria. In regenerating scales, differentiating beta-cells appear immunofluorescent mainly along the cell perimeter. In more differentiated muscle, cartilage and connective tissues of the new tail, the labelling lowers or disappears. The observations indicate that FAT1/2 proto-cadherins are present in the apical blastema where an intense remodelling takes place for the growth of the new tail but where also a tight control of cell division and migration is active and may regulate potential tumorigenic process.     

Detection of myosin heavy chain mRNA during myogenesis in tissue culture by in vitro and in situ hybridization

A cDNA copy of purified chick embryonic skeletal myosin heavy chain mRNA (MHC mRNA) distinguished between myogenic and nonmyogenic cells compared by in vitro and in situ hybridization. The majority of cells in replicating mononucleate myogenic cell cultures contained no detectable MHC mRNA. Among the earliest cells to contain MHC mRNA were cells engaged in mitosis. A relatively large amount of MHC mRNA was found in postmitotic monucleate cells and myotubes, and we observed nucleolar localization of MHC mRNA in these cells.     

Analysis of cartilage differentiation from skeletal muscle grown on bone matrix: I. Ultrastructural aspects

Previous studies have demonstrated that embryonic skeletal muscle is competent to form hyaline cartilage when cultured in vitro on demineralized bone matrix (Nogami, H., and Urist, M. R. (1970). Exp. Cell Res.63, 404–410; Nathanson, M. A., et al. (1978). Develop. Biol.64, 99–117). The present experiments were undertaken to determine the nature of the morphological alterations which attend this phenotypic transformation and to investigate the ultrastructural characteristics of the myoblasts and fibroblasts of skeletal muscle during the transformation. Nineteen-day embryonic rat limb muscles were minced and the tissue fragments explanted to bone matrix or collagen gels. The trauma of excision and mincing causes syncytial myotubes to degenerate and the nuclei of mononucleate cells to enter a heterochromatic “resting stage.” In culture, nuclei of mononucleate cells rapidly regain euchromasia. No myoblast or fibroblast cell death can be detected. On bone matrix, the entire mononucleate population transforms into fibroblast-like cells. Myoblasts are the major contributor to this population; they dissociate from the degenerate myotubes and begin to acquire endoplasmic reticulum by 24 h in vitro. The fibroblast-like morphology persists through 4 days in vitro. By 6 days in vitro some of these fibroblast-like cells acquire the phenotypic characteristics of chondrocytes, and by 10 days masses of hyaline cartilage are found. In control explants of skeletal muscle onto collagen gels, the heterochromatic nuclei of the mononucleated cells expand after 24 hr in vitro, but the mononucleated cells remain as myoblasts and fibroblasts and begin to regenerate skeletal muscle by 4 days in vitro. No cartilage forms. The results indicate that both myoblasts and fibroblasts have chondrogenic potential when grown on demineralized bone. It is tempting to conclude that the embryonic mesenchymal cells which give rise to skeletal muscle, cartilage, and other connective tissue of the limb have similar developmental potentials and that local influences, rather than separate cell lineages, account for the final pattern of differentiation.     

Proximodistal patterning during limb regeneration

Regeneration is an ability that has been observed extensively throughout metazoan phylogeny. Amongst vertebrates, the urodele amphibians stand out for their exceptional capacity to regenerate body parts such as the limb. During this process, only the missing portion of the limb is precisely replaced--amputation in the upper arm results in regeneration of the entire limb, while amputation at the wrist produces a hand. Limb regeneration occurs through the formation of a local proliferative zone called the blastema. Here, we examine how proximodistal identity is established in the blastema. Using cell marking and transplantation experiments, we show that distal identities have already been established in the earliest stages of blastemas examined. Transplantation of cells into new environments is not sufficient to respecify cell identity. However, overexpression of the CD59, a cell surface molecule previously implicated in proximodistal identity during limb regeneration, causes distal blastema cells to translocate to a more proximal location and causes defects in the patterning of the distal elements of the regenerate. We suggest a model for the limb regeneration blastema where by 4 days post-amputation the blastema is already divided into distinct growth zones; the cells of each zone are already specified to give rise to upper arm, lower arm, and hand.     

Myotomal myogenesis in Triturus vulgaris L. (Urodela) with special reference to the role of mesenchymal cells

Two stages can be distinguished in the differentiation of myotomal muscle fibres in Triturus vulgaris. In the first stage only synchronously differentiating myotomal cells are engaged; in the second stage mesenchymal cells also take part in the process. Myotomal cells (primary myoblasts) fuse to form 2-3 nucleate myotubes. Only in the caudal part of the embryo mononucleate myotubes persist. The mononucleate myotubes, like polynucleate ones, occupy the whole length of the myotome. The differentiation of myotubes is accompanied by vitellolysis. At further development stages mesenchymal cells enter the intermyotomal fissure, after which they migrate to the myotomes, between the myotubes. The cells that remain in the intermyotomal fissures retain their fibroblastic potential (they synthesise collagen). Their daughter cells adjoining the myotubes acquire myogenic abilities. Their myoblastic potential is evidenced by their ability to fuse with the myotube. Fusion of secondary myoblasts (of mesenchymal origin) with the myotube results in further growth of the myotubes. In T. vulgaris myotomal myotubes and muscle fibres developing from them are of myotomal-mesenchymal origin.     

An immunofluorescent study of the distribution of fibronectin and laminin during limb regeneration in the adult newt

Fibronectin and laminin are two extracellular glycoproteins which are involved in various processes of cellular development and differentiation. The present investigation describes changes in their distribution during regeneration of the newt forelimb, as determined by indirect immunofluorescence. The distribution of fibronectin and laminin was similar in normal limb tissue components. These glycoproteins were localized in the pericellular region of the myofibers corresponding to its basement membrane; the perineurium and endoneurium of the nerves; and the basement membranes of blood vessels, skin epithelium, and dermal glands. The cytoplasm of myofibers, axons, skin epithelium, and bone matrix lacked fluorescence for both glycoproteins. After limb amputation in the regenerating blastema, extensive presence of fibronectin, but not laminin, was seen in and around the undifferentiated blastemal cells. Increased fluorescence for fibronectin was also seen during blastema growth, blastemal cell aggregation, and early stages of redifferentiation. As redifferentiation continued, staining for fibronectin slowly disappeared from the cartilage matrix and the myoblast fusion zone. Laminin was first observed around the regenerated myotubes; this was followed by the appearance of fibronectin suggesting a sequential formation of these two components of the new myotube basement membrane. In the regenerated limb, the distribution of laminin and fibronectin was similar to that seen in normal limb. Based on the distribution pattern of these glycoproteins, it is concluded that fibronectin may play an important role in blastemal cell aggregation, cell alignment, and initiation of redifferentiation. After redifferentiation, both laminin and fibronectin may be important in the determination of the architecture of the regenerated limb.     

Development of a clonal myogenic cell line with unusual biochemical properties.

The morphological, ultrastructural, biochemical and electrophysiological properties of B104-F, a clonal cell line derived from a nitrosoethylurea-induced neoplasm in a rat, were studied as a function of the growth phase of the culture. Cells in exponentially growing cultures are mononucleate and produce action potentials when stimulated electrically. Stationary phase cultures contain three types of cells: cells of the first type are mononucleate and have long processes containing microfilaments and many parallel microtubules; cells of the second type are mononucleate but contain no microtubules and few microfilaments; and cells of the third type have ultrastructural features typical of multinucleate, striated myotubes. Multinucleate cells generate action potentials with both sodium and calcium components and are depolarized by acetylcholine. The acetylcholine response is blocked by d-tubocurarine. The specific activity of creatine phosphokinase is nine times higher in stationary phase cultures than in exponentially growing ones while the myokinase specific activity is unchanged. The gamma-aminobutyric acid content of the cells is 3.5- to 26-fold higher in stationary phase than in exponentially growing cultures, depending on the degree of fusion of the culture. The properties of B104-F are discussed in relation to the properties of developing skeletal muscle and of central nervous system cell lines.     

Bcl-2 Expression Identifies an Early Stage of Myogenesis and Promotes Clonal Expansion of Muscle Cells

We show that Bcl-2 expression in skeletal muscle cells identifies an early stage of the myogenic pathway, inhibits apoptosis, and promotes clonal expansion. Bcl-2 expression was limited to a small proportion of the mononucleate cells in muscle cell cultures, ranging from ∼1–4% of neonatal and adult mouse muscle cells to ∼5–15% of the cells from the C2C12 muscle cell line. In rapidly growing cultures, some of the Bcl-2–positive cells coexpressed markers of early stages of myogenesis, including desmin, MyoD, and Myf-5. In contrast, Bcl-2 was not expressed in multinucleate myotubes or in those mononucleate myoblasts that expressed markers of middle or late stages of myogenesis, such as myogenin, muscle regulatory factor 4 (MRF4), and myosin. The small subset of Bcl-2–positive C2C12 cells appeared to resist staurosporine-induced apoptosis. Furthermore, though myogenic cells from genetically Bcl-2–null mice formed myotubes normally, the muscle colonies produced by cloned Bcl-2–null cells contained only about half as many cells as the colonies produced by cells from wild-type mice. This result suggests that, during clonal expansion from a muscle progenitor cell, the number of progeny obtained is greater when Bcl-2 is expressed.