Self-renewal and expansion of single transplanted muscle stem cells

Adult muscle satellite cells have a principal role in postnatal skeletal muscle growth and regeneration. Satellite cells reside as quiescent cells underneath the basal lamina that surrounds muscle fibres and respond to damage by giving rise to transient amplifying cells (progenitors) and myoblasts that fuse with myofibres. Recent experiments showed that, in contrast to cultured myoblasts, satellite cells freshly isolated or satellite cells derived from the transplantation of one intact myofibre contribute robustly to muscle repair. However, because satellite cells are known to be heterogeneous, clonal analysis is required to demonstrate stem cell function. Here we show that when a single luciferase-expressing muscle stem cell is transplanted into the muscle of mice it is capable of extensive proliferation, contributes to muscle fibres, and Pax7(+)luciferase(+) mononucleated cells can be readily re-isolated, providing evidence of muscle stem cell self-renewal. In addition, we show using in vivo bioluminescence imaging that the dynamics of muscle stem cell behaviour during muscle repair can be followed in a manner not possible using traditional retrospective histological analyses. By imaging luciferase activity, real-time quantitative and kinetic analyses show that donor-derived muscle stem cells proliferate and engraft rapidly after injection until homeostasis is reached. On injury, donor-derived mononucleated cells generate massive waves of cell proliferation. Together, these results show that the progeny of a single luciferase-expressing muscle stem cell can both self-renew and differentiate after transplantation in mice, providing new evidence at the clonal level that self-renewal is an autonomous property of a single adult muscle stem cell.     

Synthesis of fast myosin induced by fast ectopic innervation of rat soleus muscle is restricted to the ectopic endplate region

Skeletal muscle fibres, long multinucleated cells, arise by fusion of mononucleated myoblasts to form a myotube that matures into the adult fibre. The two major types of mature fibre, fast and slow fibres, differ physiologically in their rate of isotonic shortening. At the molecular level these type-specific physiological properties are ascribed to different isoforms of myosin, a major protein involved in shortening. Differentiation of fast and slow fibres seems to be under the control of motoneurones, and mature fibres are innervated by only one motoneurone. When rat soleus muscle (SOL, a slow muscle) is dually innervated with a fast nerve, it acquires some properties of a fast muscle, that is, low sensitivity to caffeine and high glycogen content. We report here that in dually innervated soleus muscle the foreign fast nerve induces synthesis of fast isoforms of myosin, but only in the segment of the muscle fibre that is close to the foreign endplate. The localized influence of the nerve endplates suggest that factors controlling the phenotypic expression of the muscle fibre have a short range of activity.     

Fibre type composition of single motor units during synapse elimination in neonatal rat soleus muscle

Skeletal motor neurones innervate the specialized 'types' of fibres comprising most mammalian muscles in a characteristic fashion: each motor neurone forms a 'motor unit' by innervating a set of fibres all of the same type. Because the type expression of adult muscle fibres is plastic and apparently controlled by their innervation, each motor neurone is thought to impose a common type differentiation on all the fibres in its motor unit. However, the situation in developing muscles cannot be this simple. Muscle fibres in neonates receive synaptic input from several motor neurones and achieve the adult, single innervation only after a period of 'synapse elimination. Despite this polyneuronal innervation, differentiated fibre types are present in neonatal muscles. This means either that the motor neurones polyneuronally innervate fibres in a random fashion and type expression is not determined by innervation or that the polyneuronal innervation is ordered in such a way that each fibre could receive unambiguous instructions for type differentiation. We have investigated these possibilities here by determining the fibre type composition of motor units in neonatal rat soleus muscle. We find that even during the time of polyneuronal innervation each motor neurone confines its innervation to largely one of two fibre types present in the muscle. Therefore, some mechanism during early development segregates the synapses of two groups of soleus motor neurones onto two separate populations of soleus muscle fibres.     

Acetylcholine receptor-aggregating factor is similar to molecules concentrated at neuromuscular junctions

The basal lamina in the synaptic cleft of the vertebrate skeletal neuromuscular junction contains molecules that direct the formation of synaptic specializations in regenerating axons and muscle fibres. We have undertaken a series of experiments aimed at identifying and characterizing the molecules responsible for the formation of one of these specializations, the aggregates of acetylcholine receptors (AChRs) in the muscle fibre plasma membrane. We began by preparing an insoluble, basal lamina-containing fraction from Torpedo californica electric organ, a tissue which has a far higher concentration of cholinergic synapses than muscle, and showing that this fraction caused AChRs on cultured chick myotubes to aggregate. A critical step is learning whether or not the electric organ factor is similar to the receptor-aggregating molecule in the basal lamina at the neuromuscular junction. The importance of this problem is emphasized by reports that clearly non-physiological agents, such as positively charged latex beads, can cause AChR aggregation on cultured muscle cells. We have already shown that Torpedo muscle contains an AChR-aggregating factor similar to that of electric organ, although in much lower amounts. Here we demonstrate, using monoclonal antibodies, that the AChR-aggregating factor in our extracts of electric organ is, in fact, antigenically related to molecules concentrated in the synaptic cleft at the neuromuscular junction.     

Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts

An important corollary to the recent advances in our understanding of the primary cause of Duchenne muscular dystrophy, is the validation of genuine genetic homologues as animal models of the disease in which potential therapies can be tested. The persistent skeletal muscle necrosis that characterizes human Duchenne muscular dystrophy is also seen in the mdx mouse and is, in both, a consequence of a deficiency of dystrophin, probably within the muscle fibres themselves. As injected muscle precursor cells of one genotype can fuse with host muscle fibres of a different genotype and express the donor genes, we decided to test grafts of normal muscle precursor cells to see if they could induce synthesis of dystrophin in innately dystrophin-deficient mdx muscle fibres. We show that injected normal muscle precursor cells can fuse with pre-existing or regenerating mdx muscle fibres to render many of these fibres dystrophin-positive and so to partially or wholly rescue them from their biochemical defect.     

Enhanced myogenesis in NCAM-transfected mouse myoblasts

The fusion of mononucleate precursor myoblasts to form the multinucleated skeletal muscle fibre is proceeded by a series of complex cell-cell interactions but the cell-surface molecules involved in these events have not been characterized. During myogenesis in vivo and in vitro, expression of the neural cell adhesion molecule (NCAM) undergoes an isoform transition that precisely correlates with terminal myoblast differentiation and myotube formation. Altered processing of RNA results in the replacement of the transmembrane NCAM (relative molecular mass, 145,000 (145K) in proliferating myoblasts by a predominant 125K NCAM form linked to glycosyl phosphatidylinositol in myotubes. We now report that mouse myoblasts transfected to constitutively express the human muscle-specific 125K glycosylphosphatidylinositol-linked NCAM isoform more readily fuse to form myotubes. This suggests that NCAM plays a part in myoblast fusion and that the isoform switch may promote this function.     

A laminin-like adhesive protein concentrated in the synaptic cleft of the neuromuscular junction

A striking example of topographic specificity in synapse formation is the preferential reinnervation of original synaptic sites on denervated muscle fibres by regenerating motor axons. This specificity is mediated by the basal lamina of the synaptic cleft. A glycoprotein, s-laminin, has now been identified that is selectively associated with synaptic basal lamina and is recognized by motoneurons. Molecular cloning reveals that s-laminin is a novel homologue of laminin, a potent promoter of neurite outgrowth.     

Generation of chick skeletal muscle cells in groups of 16 from stem cells

The commonly accepted hypothesis explaining the control of skeletal muscle differentiation is that all myogenic precursor cells are equivalent and that they differentiate into post-mitotic muscle cells in response to exogenous signals, specifically low mitogen concentrations. Large clones derived from vertebrate myogenic cells, however, consist both of cycling precursors and of terminally differentiated, post-mitotic muscle cells. Here, we count the total number of cells and the number of terminally differentiated cells (or nuclei, in fused cells) in large myogenic clones. The number of terminally differentiated cells per clone was usually equal to or just below a multiple of 16. This finding is not expected from a model postulating a homogeneous population of muscle precursor cells. Rather, our results suggest that a self-renewing stem cell exists in the skeletal muscle lineage. This cell can generate committed precursors which then give rise to cohorts of 16 terminally differentiated muscle cells. This model of myogenesis provides a simple explanation for the protracted and asynchronous nature of muscle differentiation in vertebrate embryogenesis.     

Aggregates of acetylcholinesterase induced by acetylcholine receptor-aggregating factor

Basal lamina-rich extracts of Torpedo californica electric organ contain a factor that causes acetylcholine receptors (AChRs) on cultured myotubes to aggregate into patches. Our previous studies have indicated that the active component of these extracts is similar to the molecules in the basal lamina which direct the aggregation of AChRs in the muscle fibre plasma membrane at regenerating neuromuscular junctions in vivo. Because it can be obtained in large amounts and assayed in controlled conditions in cell culture, the AChR-aggregating factor from electric organ may be especially useful for examining in detail how the postsynaptic apparatus of regenerating muscle is assembled. Here we demonstrate that the electric organ factor causes not only the formation of AChR aggregates on cultured myotubes, but also the formation of patches of acetylcholinesterase (AChE). This finding, together with the observation that basal lamina directs the formation of both AChR and AChE aggregates at regenerating neuromuscular junctions in vivo, leads us to hypothesize that a single component of the synaptic basal lamina causes the formation of both these synaptic specializations on regenerating myofibres.     

Why animals have different muscle fibre types

Animals have different muscle fibre types: slow fibres with a low maximum velocity of shortening (Vmax) and fast fibres with a high Vmax. An advantage conferred by the use of different fibre types during locomotion has been proposed solely on the basis of their in vitro properties. Isolated muscle experiments show that force generation, mechanical power production and efficiency are all functions of V/Vmax, where V is the velocity of muscle shortening. But it is not known whether animals actually use the different fibres at shortening velocities that are optimal for mechanical power production and efficiency. Here we compare the V of muscle fibres during locomotion with their Vmax. This comparison shows that during slow locomotion, the slow fibres shorten at a velocity that gives peak mechanical power and efficiency and the fast fibres shorten at their optimal velocity when powering maximal movements. Our results also show that maximal movements are impossible without fast fibres because the slow ones cannot shorten rapidly enough.     

Neurons derived from radial glial cells establish radial units in neocortex

The neocortex of the adult brain consists of neurons and glia that are generated by precursor cells of the embryonic ventricular zone. In general, glia are generated after neurons during development, but radial glia are an exception to this rule. Radial glia are generated before neurogenesis and guide neuronal migration. Radial glia are mitotically active throughout neurogenesis, and disappear or become astrocytes when neuronal migration is complete. Although the lineage relationships of cortical neurons and glia have been explored, the clonal relationship of radial glia to other cortical cells remains unknown. It has been suggested that radial glia may be neuronal precursors, but this has not been demonstrated in vivo. We have used a retroviral vector encoding enhanced green fluorescent protein to label precursor cells in vivo and have examined clones 1-3 days later using morphological, immunohistochemical and electrophysiological techniques. Here we show that clones consist of mitotic radial glia and postmitotic neurons, and that neurons migrate along clonally related radial glia. Time-lapse images show that proliferative radial glia generate neurons. Our results support the concept that a lineage relationship between neurons and proliferative radial glia may underlie the radial organization of neocortex.     

Fast and slow myosin within single skeletal muscle fibres of adult rabbits.

There is good evidence for the coexistence of different myosin types both in developing muscles and in Purkinje cells from adult chicken hearts. In skeletal muscle fibres of adult animals, however, coexistence of fast (FM) and slow (SM) myosin has only been demonstrated after long-term electrical stimulation. The term 'promiscuity' has recently been coined to describe the coexistence of different myosin isoenzymes within a single fibre. Using novel, refined immunological methods we demonstrate here the presence of both FM and SM within single fibres of the musculus tibialis anterior of adult rabbits. Essentially identical results were also obtained with other muscles. Our findings imply that the genes coding for FM and SM can be expressed simultaneously within the same cell throughout an animal's entire life, and not only during development or after artificial electrical stimulation.     

Cell lineage analysis reveals multipotency of some avian neural crest cells

A major question in developmental biology is how precursor cells give rise to diverse sets of differentiated cell types. In most systems, it remains unclear whether the precursors can form many or all cell types (multipotent or totipotent), or only a single cell type (predetermined). The question of cell lineage is central to the neural crest because it gives rise to numerous and diverse derivatives including peripheral neurons, glial and Schwann cells, pigment cells, and cartilage. Although the sets of derivatives arising from different populations of neural crest cells have been well-documented, relatively little is known about the developmental potentials of individual neural crest cells. We have iontophoretically microinjected the vital dye, lysinated rhodamine dextran (LRD) into individual dorsal neural tube cells to mark unambiguously their descendants. Many of the resulting labelled clones consisted of multiple cell types, as judged by both their location and morphology. Cells as diverse as sensory neurons, presumptive pigment cells, ganglionic supportive cells, adrenomedullary cells and neural tube cells were found within individual clones. Our results indicate that at least some neural crest cells are multipotent before their departure from the neural tube.     

Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice

In vertebrates, skeletal muscle is a model for the acquisition of cell fate from stem cells. Two determination factors of the basic helix-loop-helix myogenic regulatory factor (MRF) family, Myf5 and Myod, are thought to direct this transition because double-mutant mice totally lack skeletal muscle fibres and myoblasts. In the absence of these factors, progenitor cells remain multipotent and can change their fate. Gene targeting studies have revealed hierarchical relationships between these and the other MRF genes, Mrf4 and myogenin, where the latter are regarded as differentiation genes. Here we show, using an allelic series of three Myf5 mutants that differentially affect the expression of the genetically linked Mrf4 gene, that skeletal muscle is present in the new Myf5:Myod double-null mice only when Mrf4 expression is not compromised. This finding contradicts the widely held view that myogenic identity is conferred solely by Myf5 and Myod, and identifies Mrf4 as a determination gene. We revise the epistatic relationship of the MRFs, in which both Myf5 and Mrf4 act upstream of Myod to direct embryonic multipotent cells into the myogenic lineage.     

PH值对应用肌球蛋白 ATP酶法进行梭内肌纤维分型的影响

探讨酸性和碱性预孵育处理，对应用肌球蛋白ATP酶法进行梭内肌纤维分型的影响。采用肌球蛋白ATP酶法。PH4．3和PH4．6预孵育液孵育后，梭内肌纤维中的核袋纤维染色呈阳性，核链纤维染色相对较浅；PH9．4预孵育后，核袋与核链纤维均呈阳性或强阳性；PH10．4预孵育后，核袋1纤维呈阴性，核袋2纤维呈阳性，核链纤维呈强阳性。结论是：用mATP酶法研究梭内肌纤维的分型时，预孵育液的PH值对分型结果有显著影响，应当说明并严格把握预孵育液的PH值。     

Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses

IN mammalian muscle, the subunit composition of the nicotinic acetylcholine receptor (AChR) and the distribution of AChRs along the fibre are developmentally regulated. In fetal muscle, AChRs are distributed over the entire fibre length whereas in adult fibres they are concentrated at the end-plate. We have used in situ hybridization techniques to measure the development of the synaptic localization of the messenger RNAs (mRNAs) encoding the alpha-subunit and the epsilon-subunit of the rat muscle AChR. The alpha-subunit is present in both fetal and adult muscle, whereas the epsilon-subunit appears postnatally and specifies the mature AChR subtype. The synaptic localization of alpha-subunit mRNA in adult fibres may arise from the selective down-regulation of constitutively expressed mRNA from extrasynaptic fibre segments. In contrast, epsilon-subunit mRNA appears locally at the site of neuromuscular contact and its accumulation at the end-plate is not dependent on the continued presence of the nerve terminal very early during synapse formation. This suggests that epsilon-subunit mRNA expression is induced locally via a signal which is restricted to the end-plate region and is dependent on the presence of the nerve only during a short period of early neuromuscular contact. Evidently, several mechanisms operate to confine AChR mRNAs to the adult end-plate region, and the levels of alpha-subunit and epsilon-subunit mRNAs depend on these mechanisms to differing degrees.     

新基因hSSB1逆转录病毒载体的构建、鉴定和稳定株的筛选

 hSSB1 (Human Single strand DNA binding protein) 是参与细胞DNA损伤应答的一个重要信号分子。根据GenBank 提供的hSSB1基因序列扩增其cDNA序列,插入到pBABE逆转录病毒载体中, 连接后的质粒转化后经过双酶切,PCR扩增及测序来鉴定pBABE-hSSB1阳性克隆。将阳性表达的pBABE-hSSB1和包装质粒转染到HEK293T细胞中,产生病毒液。将包装好的病毒感染细胞并用嘌呤霉素(puromycin)筛选稳定表达hSSB1的细胞株。重组pBABE-hSSB1质粒经双酶切,PCR扩增鉴定及DNA测序分析等方法证实克隆成功。Western blotting检测发现转染重组质粒pBABE-hSSB1的细胞株中hSSB1蛋白的表达水平高于对照组。该研究成功构建了针对hSSB1基因的逆转录病毒载体(pBABE-hSSB1),并得到了稳定高表达hSSB1的细胞株, 为深入研究其功能奠定了基础。     

Fibulin-5/DANCE is essential for elastogenesis in vivo.

The elastic fibre system has a principal role in the structure and function of various types of organs that require elasticity, such as large arteries, lung and skin. Although elastic fibres are known to be composed of microfibril proteins (for example, fibrillins and latent transforming growth factor (TGF)-beta-binding proteins) and polymerized elastin, the mechanism of their assembly and development is not well understood. Here we report that fibulin-5 (also known as DANCE), a recently discovered integrin ligand, is an essential determinant of elastic fibre organization. fibulin-5-/- mice generated by gene targeting exhibit a severely disorganized elastic fibre system throughout the body. fibulin-5-/- mice survive to adulthood, but have a tortuous aorta with loss of compliance, severe emphysema, and loose skin (cutis laxa). These tissues contain fragmented elastin without an increase of elastase activity, indicating defective development of elastic fibres. Fibulin-5 interacts directly with elastic fibres in vitro, and serves as a ligand for cell surface integrins alphavbeta3, alphavbeta5 and alpha9beta1 through its amino-terminal domain. Thus, fibulin-5 may provide anchorage of elastic fibres to cells, thereby acting to stabilize and organize elastic fibres in the skin, lung and vasculature.