大鼠酪氨酸羟化酶基因在肌母细胞中稳定表达

用电穿孔法将大鼠酪氨酸羟化酶（Ｔｙｒｏｓｉｎｅｈｙｄｒｏｘｙｌａｓｅ,ＴＨ）基因转染大鼠Ｌ－６ＴＧ成肌细胞株,经ＰＣＲ检测、免疫组织化学和荧光组织化学检测证明,ＴＨ基因能在细胞内稳定整合和表达,并在辅因子存在时将酪氨酸转化为多巴．移植于大鼠纹状体后可成活并表达ＴＨ。     

Transplanted wing and leg imaginal discs in Drosophila melanogaster demonstrate interactions between epidermis and myoblasts in muscle formation

 Adult muscle development in Drosophila is intimately associated with the development of the nervous system and epidermis. During metamorphosis, myoblasts from the wing imaginal disc reach target sites on the developing pupal epidermis and begin the formation of multinucleate myofibres of the dorsal thorax. The paths taken by pupal myoblasts could be specified by the nervous system and/or the epidermis. Using genetically marked donor pupal wing and leg discs transplanted onto pupal hosts, we have generated animals that have ectopic wings or legs and have examined the formation of adult muscle types. We show that thoracic myoblasts migrate over both host and donor epidermis when the transplant site on the host is thoracic. However, when the transplant site is on the abdomen, thoracic myoblasts do not migrate over abdominal epidermis. Our results show that the epidermis plays an important role in determining the migration pattern of myoblasts. Since muscles are multinucleate cells that form by the fusion of myoblasts, one way in which their molecular characteristics could be achieved is by some myoblasts acting as ”founders”. These myoblasts could influence the pattern of gene expression of those nuclei that fuse with them. We have examined, again using disc transplant experiments, if myoblasts on discs have the capacity to express fibre-specific genes as distinct from this property being conferred by other extra-discs myoblasts. Our results demonstrate that disc-associated myoblasts can indeed fuse with each other to express fibre-specific genes. We synthesize the results presented here with those from earlier experiments to suggest a mechanism for muscle patterning in the adult thorax. Received: 8 January 1995 / Accepted in revised form: 22 January 1996     

Heparan Sulfate 6-O-endosulfatases (Sulfs) Coordinate the Wnt Signaling Pathways to Regulate Myoblast Fusion during Skeletal Muscle Regeneration

Skeletal muscle regeneration is mediated by satellite cells (SCs). Upon injury, SCs undergo self-renewal, proliferation, and differentiation into myoblasts followed by myoblast fusion to form new myofibers. We previously showed that the heparan sulfate (HS) 6-O-endosulfatases (Sulf1 and -2) repress FGF signaling to induce SC differentiation during muscle regeneration. Here, we identify a novel role of Sulfs in myoblast fusion using a skeletal muscle-specific Sulf double null (Sulf^SK-DN) mouse. Regenerating Sulf^SK-DN muscles exhibit reduced canonical Wnt signaling and elevated non-canonical Wnt signaling. In addition, we show that Sulfs are required to repress non-canonical Wnt signaling to promote myoblast fusion. Notably, skeletal muscle-relevant non-canonical Wnt ligands lack HS binding capacity, suggesting that Sulfs indirectly repress this pathway. Mechanistically, we show that Sulfs reduce the canonical Wnt-HS binding and regulate colocalization of the co-receptor LRP5 with caveolin3. Therefore, Sulfs may increase the bioavailability of canonical Wnts for Frizzled receptor and LRP5/6 interaction in lipid raft, which may in turn antagonize non-canonical Wnt signaling. Furthermore, changes in subcellular distribution of active focal adhesion kinase (FAK) are associated with the fusion defect of Sulf-deficient myoblasts and upon non-canonical Wnt treatment. Together, our findings uncover a critical role of Sulfs in myoblast fusion by promoting antagonizing canonical Wnt signaling activities against the noncanonical Wnt pathway during skeletal muscle regeneration.     

GATA-binding Protein 4 (GATA-4) and T-cell Acute Leukemia 1 (TAL1) Regulate Myogenic Differentiation and Erythropoietin Response via Cross-talk with Sirtuin1 (Sirt1)

Erythropoietin (EPO), the cytokine required for erythrocyte production, contributes to muscle progenitor cell proliferation and delay myogenic differentiation. However, the underlying mechanism is not yet fully understood. Here, we report that EPO changes the skeletal myogenic regulatory factor expression program and delays differentiation via induction of GATA-4 and the basic helix-loop-helix TAL1 and that knockdown of both factors promotes differentiation. EPO increases the Sirt1 level, a NAD(+)-dependent deacetylase, and also induces the NAD(+)/NADH ratio that further increases Sirt1 activity. Sirt1 knockdown reduced GATA-4 and TAL1 expression, impaired EPO effect on delayed myogenic differentiation, and the Sirt1 knockdown effect was abrogated when combined with overexpression of GATA-4 or TAL1. GATA-4 interacts with Sirt1 and targets Sirt1 to the myogenin promoter and represses myogenin expression, whereas TAL1 inhibits myogenin expression by decreasing MyoD binding to and activation of the myogenin promoter. Sirt1 was found to bind to the GATA-4 promoter to directly regulate GATA-4 expression and GATA-4 binds to the TAL1 promoter to regulate TAL1 expression positively. These data suggest that GATA-4, TAL1, and Sirt1 cross-talk each other to regulate myogenic differentiation and mediate EPO activity during myogenic differentiation with Sirt1 playing a role upstream of GATA-4 and TAL1. Taken together, our findings reveal a novel role for GATA-4 and TAL1 to affect skeletal myogenic differentiation and EPO response via cross-talk with Sirt1.     

Trpc1 ion channel modulates phosphatidylinositol 3-kinase/Akt pathway during myoblast differentiation and muscle regeneration

We previously showed in vitro that calcium entry through Trpc1 ion channels regulates myoblast migration and differentiation. In the present work, we used primary cell cultures and isolated muscles from Trpc1(-/-) and Trpc1(+/+) murine model to investigate the role of Trpc1 in myoblast differentiation and in muscle regeneration. In these models, we studied regeneration consecutive to cardiotoxin-induced muscle injury and observed a significant hypotrophy and a delayed regeneration in Trpc1(-/-) muscles consisting in smaller fiber size and increased proportion of centrally nucleated fibers. This was accompanied by a decreased expression of myogenic factors such as MyoD, Myf5, and myogenin and of one of their targets, the developmental MHC (MHCd). Consequently, muscle tension was systematically lower in muscles from Trpc1(-/-) mice. Importantly, the PI3K/Akt/mTOR/p70S6K pathway, which plays a crucial role in muscle growth and regeneration, was down-regulated in regenerating Trpc1(-/-) muscles. Indeed, phosphorylation of both Akt and p70S6K proteins was decreased as well as the activation of PI3K, the main upstream regulator of the Akt. This effect was independent of insulin-like growth factor expression. Akt phosphorylation also was reduced in Trpc1(-/-) primary myoblasts and in control myoblasts differentiated in the absence of extracellular Ca(2+) or pretreated with EGTA-AM or wortmannin, suggesting that the entry of Ca(2+) through Trpc1 channels enhanced the activity of PI3K. Our results emphasize the involvement of Trpc1 channels in skeletal muscle development in vitro and in vivo, and identify a Ca(2+)-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during myoblast differentiation and muscle regeneration.     

Regulated expression of FLRT genes implies a functional role in the regulation of FGF signalling during mouse development

Within the mammalian genome, there are many multimember gene families that encode membrane proteins with extracellular leucine rich repeats which are thought to act as cell adhesion or signalling molecules. We previously showed that the members of the NLRR gene family are expressed in a developmentally restricted manner in the mouse with NLRR-1 being expressed in the developing myotome. The FLRT gene family shows a similar genomic layout and predicted protein secondary structure to the NLRRs so we analysed expression of the three FLRT genes during mouse development. FLRTs are glycosylated membrane proteins expressed at the cell surface which localise in a homophilic manner to cell-cell contacts expressing the focal adhesion marker vinculin. Each member of the FLRT family has a distinct, highly regulated expression pattern, as was seen for the NLRR family. FLRT3 has a provocative expression pattern during somite development being expressed in regions of the somite where muscle precursor cells migrate from the dermomyotome and move into the myotome, and later in myotomal precursors destined to migrate towards their final destination, for example, those that form the ventral body wall. FLRT3 is also expressed at the midbrain/hindbrain boundary and in the apical ectodermal ridge, regions where FGF signalling is known to be important, suggesting that the role for FLRT3 in FGF signalling identified in Xenopus is conserved in mammals. FLRT1 is expressed at brain compartmental boundaries and FLRT2 is expressed in a subset of the sclerotome, adjacent to the region that forms the syndetome, suggesting that interaction with FGF signalling may be a general property of FLRT proteins. We confirmed this by showing that all FLRTs can interact with FGFR1 and FLRTs can be induced by the activation of FGF signalling by FGF-2. We conclude that FLRT proteins act as regulators of FGF signalling, being induced by the signal and then able to interact with the signalling receptor, in many tissues during mouse embryogenesis. This process may, in part, be dependent on homophilic intercellular interactions between FLRT molecules.     

Satellite-cell pool size does matter: defining the myogenic potency of aging skeletal muscle

The deteriorating in vivo environment is thought to play a major role in reduced stem cell function with age. The capacity of stem cells to support tissue maintenance depends not only on their response to cues from the surrounding niche, but also on their abundance. Here, we investigate satellite cell (myogenic stem cell) pool size and its potential to participate in muscle maintenance through old age. The numbers and performance of mouse satellite cells have been analyzed using molecular markers that exclusively characterize quiescent satellite cells and their progeny as they transit through proliferation, differentiation and generation of reserve cells. The study establishes that abundance of resident satellite cells declines with age in myofibers from both fast- and slow-twitch muscles. Nevertheless, the inherent myogenic potential of satellite cells does not diminish with age. Furthermore, the aging satellite cell niche retains the capacity to support effective myogenesis upon enrichment of the mitogenic milieu with FGF. Altogether, satellite cell abundance, but not myogenic potential, deteriorates with age. This study suggests that the population of satellite cells that participate in myofiber maintenance during routine muscle utilization is not fully replenished throughout life.     

Myostatin regulation of muscle development: molecular basis, natural mutations, physiopathological aspects

Since its identification in 1997, myostatin has been considered as a novel and unique negative regulator of muscle growth, as mstn-/- mice display a dramatic and widespread increase in skeletal muscle mass. Myostatin also appears to be involved in muscle homeostasis in adults as its expression is regulated during muscle atrophy. Moreover, deletion of the myostatin gene seems to affect adipose tissue mass in addition to skeletal muscle mass. Natural myostatin gene mutations occur in cattle breeds such as Belgian Blue, exhibiting an obviously increased muscle mass, but also in humans, as has recently been demonstrated. Here we review these natural mutations and their associated phenotypes as well as the physiological influence of the alterations in myostatin expression and the physiopathological consequences of changes in myostatin expression, especially with regard to satellite cells. Interestingly, studies have demonstrated some rescue effects of myostatin in muscular pathologies such as myopathies, providing a novel pharmacological strategy for treatment. Furthermore, the myostatin pathway is now better understood thanks to in vitro studies and it consists of inhibition of myoblast progression in the cell cycle, inhibition of myoblast terminal differentiation, in both cases associated to protection from apoptosis. The molecular pathway driving the myogenic myostatin influence is currently under extensive study and many molecular partners of myostatin have been identified, suggesting novel potent muscle growth enhancers for both human and agricultural applications.     

Overexpression of Kelch domain containing-2 (mKlhdc2) inhibits differentiation and directed migration of C2C12 myoblasts

Targeted migration of muscle precursor cells to the anlagen of limb muscles is a complex process, which is only partially understood. We have used Lbx1 mutant mice, which are unable to establish correct migration paths of muscle precursor cells into the limbs to identify new genes involved in the accurate placement of myogenic cells in developing muscles. We found that mKlhdc2 (Kelch domain containing-2), a novel member of the family of Kelch domain containing proteins, is significantly downregulated in Lbx1 homozygous mutant embryos. Functional characterization of mKlhdc2 by targeted overexpression in 10T1/2 fibroblasts and C2C12 muscle cells rendered these cells unable to respond to chemoattractants such as HGF. Furthermore, C2C12 myoblasts overexpressing mKlhdc2 display altered cellular morphology and are unable to differentiate into mature myotubes. Our results suggest that a tightly controlled expression of mKlhdc2 is essential for a faithful execution of the myogenic differentiation and migration program.