The production of fluorescent transgenic trout to study in vitro myogenic cell differentiation

<p>Abstract</p> <p>Background</p> <p>Fish skeletal muscle growth involves the activation of a resident myogenic stem cell population, referred to as satellite cells, that can fuse with pre-existing muscle fibers or among themselves to generate a new fiber. In order to monitor the regulation of myogenic cell differentiation and fusion by various extrinsic factors, we generated transgenic trout (<it>Oncorhynchus mykiss</it>) carrying a construct containing the green fluorescent protein reporter gene driven by a fast myosin light chain 2 (MlC2f) promoter, and cultivated genetically modified myogenic cells derived from these fish.</p> <p>Results</p> <p>In transgenic trout, green fluorescence appeared in fast muscle fibers as early as the somitogenesis stage and persisted throughout life. Using an <it>in vitro </it>myogenesis system we observed that satellite cells isolated from the myotomal muscle of transgenic trout expressed GFP about 5 days post-plating as they started to fuse. GFP fluorescence persisted subsequently in myosatellite cell-derived myotubes. Using this <it>in vitro </it>myogenesis system, we showed that the rate of muscle cell differentiation was strongly dependent on temperature, one of the most important environmental factors in the muscle growth of poikilotherms.</p> <p>Conclusions</p> <p>We produced MLC2f-gfp transgenic trout that exhibited fluorescence in their fast muscle fibers. The culture of muscle cells extracted from these trout enabled the real-time monitoring of myogenic differentiation. This <it>in vitro </it>myogenesis system could have numerous applications in fish physiology to evaluate the myogenic activity of circulating growth factors, to test interfering RNA and to assess the myogenic potential of fish mesenchymal stem cells. In ecotoxicology, this system could be useful to assess the impact of environmental factors and marine pollutants on fish muscle growth.</p

Dynamic gene expression in fish muscle during recovery growth induced by a fasting-refeeding schedule

<p>Abstract</p> <p>Background</p> <p>Recovery growth is a phase of rapid growth that is triggered by adequate refeeding of animals following a period of weight loss caused by starvation. In this study, to obtain more information on the system-wide integration of recovery growth in muscle, we undertook a time-course analysis of transcript expression in trout subjected to a food deprivation-refeeding sequence. For this purpose complex targets produced from muscle of trout fasted for one month and from muscle of trout fasted for one month and then refed for 4, 7, 11 and 36 days were hybridized to cDNA microarrays containing 9023 clones.</p> <p>Results</p> <p>Significance analysis of microarrays (SAM) and temporal expression profiling led to the segregation of differentially expressed genes into four major clusters. One cluster comprising 1020 genes with high expression in muscle from fasted animals included a large set of genes involved in protein catabolism. A second cluster that included approximately 550 genes with transient induction 4 to 11 days post-refeeding was dominated by genes involved in transcription, ribosomal biogenesis, translation, chaperone activity, mitochondrial production of ATP and cell division. A third cluster that contained 480 genes that were up-regulated 7 to 36 days post-refeeding was enriched with genes involved in reticulum and Golgi dynamics and with genes indicative of myofiber and muscle remodelling such as genes encoding sarcomeric proteins and matrix compounds. Finally, a fourth cluster of 200 genes overexpressed only in 36-day refed trout muscle contained genes with function in carbohydrate metabolism and lipid biosynthesis. Remarkably, among the genes induced were several transcriptional regulators which might be important for the gene-specific transcriptional adaptations that underlie muscle recovery.</p> <p>Conclusion</p> <p>Our study is the first demonstration of a coordinated expression of functionally related genes during muscle recovery growth. Furthermore, the generation of a useful database of novel genes associated with muscle recovery growth will allow further investigations on particular genes, pathways or cellular process involved in muscle growth and regeneration.</p

Myomixer is expressed during embryonic and post-larval hyperplasia, muscle regeneration and differentiation of myoblats in rainbow trout (Oncorhynchus mykiss)

In contrast to mice or zebrafish, trout exhibits post-larval muscle growth through hypertrophy and formation of new myofibers (hyperplasia). The muscle fibers are formed by the fusion of mononucleated cells (myoblasts) regulated by several muscle-specific proteins such as Myomaker or Myomixer. In this work, we identified a unique gene encoding a Myomixer protein of 77 amino acids (aa) in the trout genome. Sequence analysis and phylogenetic tree showed moderate conservation of the overall protein sequence across teleost fish (61% of aa identity between trout and zebrafish Myomixer sequences). Nevertheless, the functionally essential motif, AxLyCxL is perfectly conserved in all studied sequences of vertebrates. Using in situ hybridization, we observed that myomixer was highly expressed in the embryonic myotome, particularly in the hyperplasic area. Moreover, myomixer remained readily expressed in white muscle of juvenile (1 and 20 g) although its expression decreased in mature fish. We also showed that myomixer is up-regulated during muscle regeneration and in vitro myoblasts differentiation. Together, these data indicate that myomixer expression is consistently associated with the formation of new myofibers during somitogenesis, post-larval growth and muscle regeneration in trout

Additional file 2: of Global gene expression in muscle from fasted/refed trout reveals up-regulation of genes promoting myofibre hypertrophy but not myofibre production

Major functional categories of cluster IIa and lists of genes that formed them (XLSX 16 kb

Glucocorticoids regulate mRNA levels for subunits of the 19 S regulatory complex of the 26 S proteasome in fast-twitch skeletal muscles.

Circulating levels of glucocorticoids are increased in many traumatic and muscle-wasting conditions that include insulin-dependent diabetes, acidosis, infection, and starvation. On the basis of indirect findings, it appeared that these catabolic hormones are required to stimulate Ub (ubiquitin)-proteasome-dependent proteolysis in skeletal muscles in such conditions. The present studies were performed to provide conclusive evidence for an activation of Ub-proteasome-dependent proteolysis after glucocorticoid treatment. In atrophying fast-twitch muscles from rats treated with dexamethasone for 6 days, compared with pair-fed controls, we found (i) increased MG132-inhibitable proteasome-dependent proteolysis, (ii) an enhanced rate of substrate ubiquitination, (iii) increased chymotrypsin-like proteasomal activity of the proteasome, and (iv) a co-ordinate increase in the mRNA expression of several ATPase (S4, S6, S7 and S8) and non-ATPase (S1, S5a and S14) subunits of the 19 S regulatory complex, which regulates the peptidase and the proteolytic activities of the 26 S proteasome. These studies provide conclusive evidence that glucocorticoids activate Ub-proteasome-dependent proteolysis and the first in vivo evidence for a hormonal regulation of the expression of subunits of the 19 S complex. The results suggest that adaptations in gene expression of regulatory subunits of the 19 S complex by glucocorticoids are crucial in the regulation of the 26 S muscle proteasome

Collagen I is concentrated at the surface of the somites.

<p>(<b>A and B</b>) Frontal sections through the trunk of a 13 dpf trout embryo. (<b>A</b>) Posterior tail. Collagen I immunofluorescence localises to the anterior and posterior edges and at the lateral surface of the dermomyotome. (<b>B</b>) Anterior tail. Collagen I immunofluorescence is present along the space separating adjacent somites. der: dermomyotome. Scale bars in A and B, 15 μm.</p

Ubiquitin-proteasome-dependent proteolysis in skeletal muscle

3 tables 2 graph.International audienceThe ubiquitin-proteasome proteolytic pathway has recently been reported to be of major importance in the breakdown of skeletal muscle proteins. The first step in this pathway is the covalent attachment of polyubiquitin chains to the targeted protein. Polyubiquitylated proteins are then recognized and degraded by the 26S proteasome complex. In this review, we critically analyse recent findings in the regulation of this pathway, both in animal models of muscle wasting and in some human diseases. The identification of regulatory steps of ubiquitin conjugation to protein substrates and/or of the proteolytic activities of the proteasome should lead to new concepts that can be used to manipulate muscle protein mass. Such concepts are essential for the development of anti-cachectic therapies for many clinical situations

Comparison of the early axial musculoskeletal system between amniote and fish.

<p>Schematic frontal sections through trunk region of amniote (<b>A</b>) and fish embryos (<b>B</b>). (<b>A</b>) Amniote embryo. The sclerotome that derives from an important portion of the somite gives rise to chondroprogenitors surrounding axial structures (notochord and neural tube). Scleraxis expressing tendon progenitors originating from the dorsolateral edge of the early sclerotome lie between adjacent myotomes (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091876#pone.0091876-Charvet2" target="_blank">[13]</a>). (<b>B</b>) Fish embryo (17dpf). The sclerotome that represents a reduced somite derivative gives rise to a limited number of chondroprogenitors that express osteoblast-specific factor2/periostin. Adjacent myotomes are separated by Scleraxis-expressing myoseptal cells. A dermomyotome (white and red vertical lines) at the surface of the myotome persists late during fish development and expresses high levels of <i>col1a1</i>, <i>col5a2</i> and <i>col12a1</i>.</p

Myoseptal cells express genes involved in extracellular matrix production and remodelling.

<p>(<b>A</b>–<b>D</b>) Frontal sections of eyed stage (17 dpf) embryos. Expression of (<b>A</b>) <i>col1a1</i>, (<b>B</b>) <i>col5a2</i>, (<b>C</b>) <i>col12a1</i> and (<b>D</b>) Angiopoietin-7 like. mc: myoseptal cell; sc: skeletogenic cells; m: myotome; n: notochord. Scale bars, 20 μm.</p

Apparent movement of <i>col1a1</i> expressing cells suggests a sclerotomal origin of myoseptal cells.

<p>(<b>A</b>) Transverse section of a 13 dpf embryo. <i>Col1a1</i> staining is present in the ventrally located sclerotome cells (arrow). (<b>B</b>) Transverse section of a 14 dpf embryon. Labelled cells have migrated dorsally to surround the notochord (arrow). (<b>C</b>–<b>E</b>) Frontal section of trout embryo at the level of the notochord. (<b>C</b>) 14 dpf embryo. Labelled cells surround the notochord. (<b>D</b>) 15 dpf embryo. Some labelled cells occupy the medial aspect of the intermyotomal space. (<b>E</b>) 16 dpf embryo. The medial-lateral extent of the intermyotomal space contains labeled cells. (<b>C</b>′–<b>E</b>′) Merged images showing <i>col1a1</i> labelling and Hoechst nuclear staining, white arrows indicate the intermyotomal space. n: notochord; m: myotome. Scale bars in A and B, 50 μm; C, C′, D, D′, E and E′, 30 μm.</p