Search

Flesh quality recovery in female rainbow trout (Oncorhynchus mykiss) after egg production. Aquaculture Europe 2019

The formation of new myofibers in vertebrates occurs by myoblast fusion and requires fusogenic activity of the muscle-specific membrane protein myomaker. Here, using in silico (BLAST) genome analyses, we show that the myomaker gene from trout includes 14 minisatellites, indicating that it has an unusual structure compared with those of other animal species. We found that the trout myomaker gene encodes a 434 –amino acid (aa) protein, in accordance with its apparent molecular mass (40 kDa) observed by immunoblotting. The first half of the trout myomaker protein (1–220 aa) is similar to the 221-aa mouse myomaker protein, whereas the second half (222–234 aa) does not correspond to any known motifs and arises from two protein extensions. The first extension (70 aa) apparently appeared with the radiation of the bony fish clade Euteleostei, whereas the second extension (up to 236 aa) is restricted to the superorder Protacanthopterygii (containing salmonids and pike) and corresponds to the insertion of minisatellites having a length of 30 nucleotides. According to gene expression analyses, trout myomaker expression is consistently associated with the formation of new myofibers during embryonic development, postlarval growth, and muscle regeneration. Using cell-mixing experiments, we observed that trout myomaker has retained the ability to drive the fusion of mouse fibroblasts with C2C12 myoblasts. Our work reveals that trout myomaker has fusogenic function despite containing two protein extensions., SCOPUS: ar.j, DecretOANoAutActif, info:eu-repo/semantics/published

Supplementary data to this article can be found online at : https://doi.org/10.1016/j.cbpb.2018.11.005; The understanding of muscle tissue formation and regeneration is essential for the development of therapeutic approaches to treat muscle diseases or loss of muscle mass and strength during ageing or cancer. One of the critical steps in muscle formation is the fusion of muscle cells to form or regenerate muscle fibres.To identify new genes controlling myoblast fusion, we performed a siRNA screen in c2c12 myoblasts. The genes identified during this screen were then studiedi n vivo by knock down in zebrafish using morpholino. We found that N-alpha-acetyl transferase 15 (Naa15) knockdown enhanced c2c12 myoblast fusion, suggesting that Naa15 negatively regulates myogenic cell fusion. We identified two Naa15 orthologous genes in the zebrafish genome: Naa15a and Naa15b. These two orthologs were expressed in the myogenic domain of the somite. Knockdown of zebrafish Naa15a and Naa15b genes induced a“U”-shaped segmentation of the myotome and alteration of myotome boundaries, resulting in the formation of abnormally long myofibres spanning adjacent somites. Taken together, these results show that Naa15 regulates myotome formation and myogenesis in fish.

The formation of the intramuscular connective tissue was investigated in rainbow trout Oncorhynchus mykiss by combining histological and in situ gene‐expression analysis. Laminin, a primary component of basement membranes, surrounded superficial slow and deep fast muscle fibres in O. mykiss as soon as the hatching stage (c. 30 days post fertilization (dpf)). In contrast, type I collagen, the primary fibrillar collagen in muscle of vertebrates, appeared at the surface of individual slow and fast muscle fibres only at c. 90 dpf and 110 dpf, respectively. The deposition of type I collagen in laminin‐rich endomysium ensheathing individual muscle fibres correlated with the late appearance of collagen type 1 α 1 chain (col1α1) expressing fibroblasts inside slow and then fast‐muscle masses. Double in situ hybridization indicated that coll1α1 expressing muscle resident fibroblasts also expressed collagen type 5 α 2 chain (col5α2) transcripts, showing that these cells are a major cellular source of fibrillar collagens within O. mykiss muscle. At c. 140 dpf, the formation of perimysium‐like structure was manifested by the increase of type I collagen deposition around bundles of myofibres concomitantly with the alignment and elongation of some collagen‐expressing fibroblasts. Overall, this study shows that the formation of O. mykiss intramuscular connective tissue network is completed only in aged fry when fibroblast‐like cells expressing type I and V collagens arise inside of the growing myotome.

BackgroundThe dramatic increase in myotomal muscle mass in post-hatching fish is related to their ability to lastingly produce new muscle fibres, a process termed hyperplasia. The molecular and cellular mechanisms underlying fish muscle hyperplasia largely remain unknown. In this study, we aimed to characterize intrinsic properties of myogenic cells originating from fish hyperplasic muscle. For this purpose, we compared in situ proliferation, in vitro cell behavior and transcriptomic profile of myogenic precursors originating from hyperplasic muscle of juvenile trout (JT) and from non-hyperplasic muscle of fasted juvenile trout (FJT) and adult trout (AT).ResultsFor the first time, we showed that myogenic precursors proliferate in hyperplasic muscle from JT as shown by in vivo BrdU labeling. This proliferative rate was very low in AT and FJT muscle. Transcriptiomic analysis revealed that myogenic cells from FJT and AT displayed close expression profiles with only 64 differentially expressed genes (BH corrected p-val < 0.001). In contrast, 2623 differentially expressed genes were found between myogenic cells from JT and from both FJT and AT. Functional categories related to translation, mitochondrial activity, cell cycle, and myogenic differentiation were inferred from genes up regulated in JT compared to AT and FJT myogenic cells. Conversely, Notch signaling pathway, that signs cell quiescence, was inferred from genes down regulated in JT compared to FJT and AT. In line with our transcriptomic data, in vitro JT myogenic precursors displayed higher proliferation and differentiation capacities than FJT and AT myogenic precursors.ConclusionsThe transcriptomic analysis and examination of cell behavior converge to support the view that myogenic cells extracted from hyperplastic muscle of juvenile trout are intrinsically more potent to form myofibres than myogenic cells extracted from non-hyperplasic muscle. The generation of gene expression profiles in myogenic cell extracted from muscle of juvenile trout may yield insights into the molecular and cellular mechanisms controlling hyperplasia and provides a useful list of potential molecular markers of hyperplasia.

Background Excessive accumulation of adipose tissue in cultured fish is an outstanding problem in aquaculture. To understand the development of adiposity, it is crucial to identify the genes which expression is associated with adipogenic differentiation. Therefore, the transcriptomic profile at different time points (days 3, 8, 15 and 21) along primary culture development of rainbow trout preadipocytes has been investigated using an Agilent trout oligo microarray. Results Our analysis identified 4026 genes differentially expressed (fold-change >3) that were divided into two major clusters corresponding to the main phases observed during the preadipocyte culture: proliferation and differentiation. Proliferation cluster comprised 1028 genes up-regulated from days 3 to 8 of culture meanwhile the differentiation cluster was characterized by 2140 induced genes from days 15 to 21. Proliferation was characterized by enrichment in genes involved in basic cellular and metabolic processes (transcription, ribosome biogenesis, translation and protein folding), cellular remodelling and autophagy. In addition, the implication of the eicosanoid signalling pathway was highlighted during this phase. On the other hand, the terminal differentiation phase was enriched with genes involved in energy production, lipid and carbohydrate metabolism. Moreover, during this phase an enrichment in genes involved in the formation of the lipid droplets was evidenced as well as the activation of the thyroid-receptor/retinoic X receptor (TR/RXR) and the peroxisome proliferator activated receptors (PPARs) signalling pathways. The whole adipogenic process was driven by a coordinated activation of transcription factors and epigenetic modulators. Conclusions Overall, our study demonstrates the coordinated expression of functionally related genes during proliferation and differentiation of rainbow trout adipocyte cells. Furthermore, the information generated will allow future investigations of specific genes involved in particular stages of fish adipogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3728-0) contains supplementary material, which is available to authorized users.

Background Muscle fibre hyperplasia stops in most fish when they reach approximately 50 % of their maximum body length. However, new small-diameter muscle fibres can be produced de novo in aged fish after muscle injury. Given that virtually nothing is known regarding the transcriptional mechanisms that regulate regenerative myogenesis in adult fish, we explored the temporal changes in gene expression during trout muscle regeneration following mechanical crushing. Then, we compared the gene transcription profiles of regenerating muscle with the previously reported gene expression signature associated with muscle fibre hyperplasia. Results Using an Agilent-based microarray platform we conducted a time-course analysis of transcript expression in 29 month-old trout muscle before injury (time 0) and at the site of injury 1, 8, 16 and 30 days after lesions were made. We identified more than 7000 unique differentially expressed transcripts that segregated into four major clusters with distinct temporal profiles and functional categories. Functional categories related to response to wounding, response to oxidative stress, inflammatory processes and angiogenesis were inferred from the early up-regulated genes, while functions related to cell proliferation, extracellular matrix remodelling, muscle development and myofibrillogenesis were inferred from genes up-regulated 30 days post-lesion, when new small myofibres were visible at the site of injury. Remarkably, a large set of genes previously reported to be up-regulated in hyperplastic muscle growth areas was also found to be overexpressed at 30 days post-lesion, indicating that many features of the transcriptional program underlying muscle hyperplasia are reactivated when new myofibres are transiently produced during fish muscle regeneration. Conclusion The results of the present study demonstrate a coordinated expression of functionally related genes during muscle regeneration in fish. Furthermore, this study generated a useful list of novel genes associated with muscle regeneration that will allow further investigations on the genes, pathways or biological processes involved in muscle growth and regeneration in vertebrates. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3160-x) contains supplementary material, which is available to authorized users.

The dramatic increase in myotomal muscle mass in teleosts appears to be related to their sustained ability to produce new fibres in the growing myotomal muscle. To describe muscle fibre input dynamics in trout (Oncorhynchus mykiss), we generated a stable transgenic line carrying green fluorescent protein (GFP) cDNA driven by the myogenin promoter. In this myog:GFP transgenic line, muscle cell recruitment is revealed by the appearance of fluorescent, small, nascent muscle fibres. The myog:GFP transgenic line displayed fibre formation patterns in the developing trout and showed that the production of new fluorescent myofibres (muscle hyperplasia) is prevalent in the juvenile stage but progressively decreases to eventually cease at approximately 18 months post-fertilisation. However, fluorescent, nascent myofibres were formed de novo in injured muscle of aged trout, indicating that the inhibition of myofibre formation associated with trout ageing cannot be attributed to the lack of recruitable myogenic cells but rather to changes in the myogenic cell microenvironment. Additionally, the myog:GFP transgenic line demonstrated that myofibre production continues to persist during starvation.

An in situ screen for genes expressed in the skeletal muscle of eyed-stage trout embryos led to the identification of a transcript encoding a polypeptide related to CILP1, a secreted glycoprotein present in the extracellular matrix. In situ hybridisation in developing trout embryos revealed that CILP1 expression was initially detected in fast muscle progenitors of the early somite. Later, CILP1 expression was down-regulated medio-laterally in differentiating fast muscle cells, to become finally restricted to the undifferentiated muscle progenitors forming the dermomyotome-like epithelium at the surface of the embryonic myotome. At the completion of somitogenesis, CILP1 expression was concentrated in the myoseptal/tendon cells that develop between adjacent myotomes but was excluded from the skeletogenic cells of the vertebral axis to which the most medial myoseptal/tendon cells attach. Overall, our work shows that muscle cells and myoseptal/tendon cells contribute dynamically and cooperatively to the production of CILP1 during ontogeny of the trout musculoskeletal system.

The trunk muscle in fish is organized as longitudinal series of myomeres which are separated by sheets of connective tissue called myoseptum to which myofibers attach. In this study we show in the trout that the myoseptum separating two somites is initially acellular and composed of matricial components such as fibronectin, laminin and collagen I. However, myoseptal cells forming a continuum with skeletogenic cells surrounding axial structures are observed between adjacent myotomes after the completion of somitogenesis. The myoseptal cells do not express myogenic markers such as Pax3, Pax7 and myogenin but express several tendon-associated collagens including col1a1, col5a2 and col12a1 and angiopoietin-like 7, which is a secreted molecule involved in matrix remodelling. Using col1a1 as a marker gene, we observed in developing trout embryo an initial labelling in disseminating cells ventral to the myotome. Later, labelled cells were found more dorsally encircling the notochord or invading the intermyotomal space. This opens the possibility that the sclerotome gives rise not only to skeletogenic mesenchymal cells, as previously reported, but also to myoseptal cells. We furthermore show that myoseptal cells differ from skeletogenic cells found around the notochord by the specific expression of Scleraxis, a distinctive marker of tendon cells in amniotes. In conclusion, the location, the molecular signature and the possible sclerotomal origin of the myoseptal cells suggest that the fish myoseptal cells are homologous to the axial tenocytes in amniotes.

Myomaker (also called Tmem8c), a new membrane activator of myocyte fusion was recently discovered in mice. Using whole mount in situ hybridization on zebrafish embryos at different stages of embryonic development, we show that myomaker is transiently expressed in fast myocytes forming the bulk of zebrafish myotome. Zebrafish embryos injected with morpholino targeted against myomaker were alive after yolk resorption and appeared morphologically normal, but they were unable to swim, even under effect of a tactile stimulation. Confocal observations showed a marked phenotype characterized by the persistence of mononucleated muscle cells in the fast myotome at developmental stages where these cells normally fuse to form multinucleated myotubes. This indicates that myomaker is essential for myocyte fusion in zebrafish. Thus, there is an evolutionary conservation of myomaker expression and function among Teleostomi.

In the last decade, myostatin (MSTN), a member of the TGFβ superfamily, has emerged as a strong inhibitor of muscle growth in mammals. In fish many studies reveal a strong conservation of mstn gene organization, sequence, and protein structures. Because of ancient genome duplication, teleostei may have retained two copies of mstn genes and even up to four copies in salmonids due to additional genome duplication event. In sharp contrast to mammals, the different fish mstn orthologs are widely expressed with a tissue-specific expression pattern. Quantification of mstn mRNA in fish under different physiological conditions, demonstrates that endogenous expression of mstn paralogs is rarely related to fish muscle growth rate. In addition, attempts to inhibit MSTN activity did not consistently enhance muscle growth as in mammals. In vitro, MSTN stimulates myotube atrophy and inhibits proliferation but not differentiation of myogenic cells as in mammals. In conclusion, given the strong mstn expression non-muscle tissues of fish, we propose a new hypothesis stating that fish MSTN functions as a general inhibitors of cell proliferation and cell growth to control tissue mass but is not specialized into a strong muscle regulator.

Background A unique feature of fish is that new muscle fibres continue to be produced throughout much of the life cycle; a process termed muscle hyperplasia. In trout, this process begins in the late embryo stage and occurs in both a discrete, continuous layer at the surface of the primary myotome (stratified hyperplasia) and between existing muscle fibres throughout the myotome (mosaic hyperplasia). In post-larval stages, muscle hyperplasia is only of the mosaic type and persists until 40% of the maximum body length is reached. To characterise the genetic basis of myotube neoformation in trout, we combined laser capture microdissection and microarray analysis to compare the transcriptome of hyperplastic regions of the late embryo myotome with that of adult myotomal muscle, which displays only limited hyperplasia. Results Gene expression was analysed using Agilent trout oligo microarrays. Our analysis identified more than 6800 transcripts that were significantly up-regulated in the superficial hyperplastic zones of the late embryonic myotome compared to adult myotomal muscle. In addition to Pax3, Pax7 and the fundamental myogenic basic helix-loop-helix regulators, we identified a large set of up-regulated transcriptional factors, including Myc paralogs, members of Hes family and many homeobox-containing transcriptional regulators. Other cell-autonomous regulators overexpressed in hyperplastic zones included a large set of cell surface proteins belonging to the Ig superfamily. Among the secreted molecules found to be overexpressed in hyperplastic areas, we noted growth factors as well as signalling molecules. A novel finding in our study is that many genes that regulate planar cell polarity (PCP) were overexpressed in superficial hyperplastic zones, suggesting that the PCP pathway is involved in the oriented elongation of the neofibres. Conclusion The results obtained in this study provide a valuable resource for further analysis of novel genes potentially involved in hyperplastic muscle growth in fish. Ultimately, this study could yield insights into particular genes, pathways or cellular processes that may stimulate muscle regeneration in other vertebrates.

Session : Bases biologiques de la qualité et génétique/génomique; Recent technological advances in biology allow the collection of large sets of data which permit a precise description of the phenotypes. To process those data, computers require a standardized language to which the users (zootechnician, geneticists, physiologists, biochemists, ethologists, producers...) will be able to refer. The construction of this language is based on ontologies. INRA in collaboration with USDA in the USA have set up a program called ATOL (for "Animal Trait Ontology for Livestock") aiming to define and organize the phenotypical characters of livestock species. Integrating societal concerns, the objective is to describe the traits underlying livestock production efficiency: welfare and adaptation, nutrition and feed efficiency, fertility and reproductive efficiency, mammary gland and milk production, growth and meat production. At present, 229 traits concern growth and meat production, of 6 major species (cattle, pig, chicken, trout, rabbit, sheep) with a good genericity.

The objective of this study was to investigate the expression of two promyogenic cell surface adhesion receptors, N- and M-cadherin, in developing trout (Oncorhynchus mykiss) somite, taking account of the recent identification of a dermomyotome-like epithelium in teleosts. In situ hybridization showed that N-cadherin was expressed throughout the paraxial mesoderm and nascent somite. As the somite matured, N-cadherin expression disappeared ventrally from the sclerotome, and then mediolaterally from the differentiating slow and fast muscle cells of the embryonic myotome, to become finally restricted to the undifferentiated myogenic precursors forming the dermomyotome-like epithelium that surrounds the embryonic myotome. By contrast, M-cadherin, which was transcribed in the differentiating embryonic myotome, was never expressed in the dermomyotome-like epithelium. In late-stage trout embryos, M-cadherin transcript was only detected at the periphery of the expanding myotome, where muscle cells stemming from the N-cadherin positive dermomyotome-like epithelium differentiate. Collectively, our results support the view that, in trout embryo, N-cadherin is associated with muscle cell immaturity while M-cadherin is associated with muscle cell maturation and differentiation and this during the two successive phases of myogenesis. J. Exp. Zool. (Mol. Dev. Evol.) 318:71-77, 2012. (C) 2011 Wiley Periodicals, Inc.

International audience; Rainbow trout, Oncorhynchus mykiss, is an important aquaculture species worldwide and, in addition to being of commercial interest, it is also a research model organism of considerable scientific importance. Because of the lack of a whole genome sequence in that species, transcriptomic analyses of this species have often been hindered. Using next-generation sequencing (NGS) technologies, we sought to fill these informational gaps. Here, using Roche 454-Titanium technology, we provide new tissue-specific cDNA repertoires from several rainbow trout tissues. Non-normalized cDNA libraries were constructed from testis, ovary, brain and gill rainbow trout tissue samples, and these different libraries were sequenced in 10 separate half-runs of 454-Titanium. Overall, we produced a total of 3 million quality sequences with an average size of 328 bp, representing more than 1 Gb of expressed sequence information. These sequences have been combined with all publicly available rainbow trout sequences, resulting in a total of 242,187 clusters of putative transcript groups and 22,373 singletons. To identify the predominantly expressed genes in different tissues of interest, we developed a Digital Differential Display (DDD) approach. This approach allowed us to characterize the genes that are predominantly expressed within each tissue of interest. Of these genes, some were already known to be tissue-specific, thereby validating our approach. Many others, however, were novel candidates, demonstrating the usefulness of our strategy and of such tissue-specific resources. This new sequence information, acquired using NGS 454-Titanium technology, deeply enriched our current knowledge of the expressed genes in rainbow trout through the identification of an increased number of tissue-specific sequences. This identification allowed a precise cDNA tissue repertoire to be characterized in several important rainbow trout tissues. The rainbow trout contig browser can be accessed at the following publicly available web site (http://www.sigenae.org/).

absent

The dermomyotome is a transient epithelial sheet that forms from the dorsal aspect of the somite. The dermomyotome gives rise to a variety of tissues, most importantly myotomal muscle and dermis. Despite the central importance of the dermomyotome in the development of amniotes, the question of its existence in lower vertebrates has been lastingly eluded. The combination of single-cell lineage tracing and gene expression analysis has recently led to the identification in fish of a somitic sub-domain that exhibits structural and functional features of the amniote dermomyotome.

Sox proteins form a family of transcription factors characterized by the presence of a DNA-binding domain called the high-mobility-group domain (HMG). The presence of a large number of potential Sox5 binding sites in the trout promoter of Pax7, a gene which has emerged as an important regulator of neural and somite development, prompted us to clone trout Sox5 and to examine its expression pattern in the developing trout embryo. Using whole mount in situ hybridisation, we show here that the Sox5 transcript is first expressed before segmentation in the whole presomitic mesoderm. In newly formed somites, Sox5 labelling was observed in myogenic progenitor cells of the posterior and anterior walls. As the somite matured rostrocaudally, Sox5 expression disappeared from the differentiating embryonic myotome, deep in the somite, to become restricted to the undifferentiated myogenic precursors forming the dermomyotome-like epithelium at the surface of the embryonic myotome. Sox5 was also expressed in the developing nervous system and in pectoral fin buds. On the whole, this work suggests a hitherto unappreciated role for Sox5 in regulating myogenic cells destined to muscle formation and growth.

Chapitre 5 Croissance squelettique et musculaire; absent

International audience; Recent research has significantly broadened our understanding of how the teleost somite is patterned to achieve embryonic and postembryonic myogenesis. Medial (adaxial) cells and posterior cells of the early epithelial somite generate embryonic superficial slow and deep fast muscle fibers, respectively, whereas anterior somitic cells move laterally to form an external cell layer of undifferentiated Pax7-positive myogenic precursors surrounding the embryonic myotome. In late embryo and in larvae, some of the cells contained in the external cell layer incorporate into the myotome and differentiate into new muscle fibers, thus contributing to medio-lateral expansion of the myotome. This supports the suggestion that the teleost external cell layer is homologous to the amniote dermomyotome. Some of the signalling molecules that promote lateral movement or regulate the myogenic differentiation of external cell precursors have been identified and include stromal cell-derived factor 1 (Sdf1), hedgehog proteins, and fibroblast growth factor 8 (Fgf8). Recent studies have shed light on gene activations that underlie the differentiation and maturation of slow and fast muscle fibers, pointing out that both adaxially derived embryonic slow fibers and slow fibers formed during the myotome expansion of larvae initially and transiently bear features of the fast fiber phenotype.

International audience; NLRR-1 (neuronal leucine-rich repeat-l) is a transmembrane protein that functions as a cell adhesion molecule regulating morphogenesis. A previous study in the mouse reported that the somitic expression of NLRR-1 is restricted to the dorsal lip of the dermomyotome that gives rise to the epaxial muscle. In this study, we report the expression of a NLRR-1 gene in the trout-developing somite. Whole mount in situ hybridization showed that NLRR-1 transcript accumulated in a rostro-caudal wave in the adaxial slow muscle cells, which are initially found deep in the somite, immediately adjacent to the notochord. No labelling was observed in the segmental plate from which somites form. As somites mature along an anteroposterior axis, the NLRR-1-positive adaxial cells exhibited an apparent migration radially to the lateral surface of the myotome where they ultimately form the peripheral slow muscle fibres. These observations show that a NLRR-1 gene is expressed in a subpopulation of myogenic cells of the trout embryo, but the anatomical location and the fate of this subpopulation are distinct from those of the NLRR-1 positive myogenic cells in amniotes. NLRR-l was also transcribed in distinct areas of the developing nervous system including the telencephalon, the optic tectum, the cerebellum, the neural tube, the retina, and the branchial arches.

Background 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. Results 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. Conclusion 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.

International audience; In fish, the skeletal muscle of the trunk and the tail derives from the somites which form in the paraxial mesoderm in a rostro-caudal sequence. The development of the fish myotome begins with the onset of myogenic regulatory factors expression and continues with the formation of a distinct superficial layer of slow muscle fibres that covers a bulk of fast muscle fibres located in the deep portion of the myotome. Muscle fibres of the slow-twitch lineage originate in fish embryos from adaxial cells, a distinct subpopulation of the paraxial mesoderm that flanks the notochord. During the early maturation of the somite these adaxial cells migrate away from the notochord towards the lateral part of the somite where they form the superficial slow fibres. Lateral presomitic cells that remain deep in the myotome differentiate into fast muscle fibres. Morphogens of the hedgehog family secreted by the notochord have a pivotal role in inducing the slow-twitch lineage. In late embryos, additional fibres are added from discrete germinal zones situated at the ventral and dorsal extremes of the developing myotome. This regionalised process has been termed "stratified hyperplasia." In fish which grow to a large final size this is followed by a mosaic hyperplastic process that leads to the formation of new fibres throughout the whole myotome. Current knowledge about the endocrine and autocrine factors that potentially regulate the proliferation and the differentiation of muscle cells within the embryonic and larval fish myotome is reviewed.

International audience

SUMMARYMuscle differentiation is inhibited by members of the Id family that block the transcriptional effect of myogenic bHLH regulators by forming inactive heterodimers with them. Also, Id proteins promote cell proliferation by interacting with key regulators of the cell cycle. In order to determine the role of Id-encoding genes during fish development and especially in early myogenesis, we examined the expression patterns of Id1, Id2 and two nonallelic Id6 (Id6a and Id6b)-encoding genes in developing trout embryos. These four Id paralogs were found to exhibit discrete expression in the developing nervous system and in the eye rudiment. During the segmentation process, Id6a, Id6b and Id1 were expressed in the tail bud, the paraxial mesoderm and the ventral and dorsal domains of neoformed somites. As the somite matured in a rostrocaudal progression, the labelling for Id1 transcripts rapidly faded whereas labelling for Id6 transcripts was found to persist until at least the completion of segmentation. By contrast, Id2 transcripts were visualised transiently only in dorsal domains of neoformed somites and strongly accumulated in the pronephros. The preferential localisation of Id6a, Id6b, Id1 and Id2 transcripts within ventral and/or dorsal extremes of the developing somites, suggests that these areas, which were the last ones to express muscle-specific genes, contain dividing cells involved in somite expansion.

Like many poecilotherms, salmonids exhibit seasonal variations of growth rate in relation with seasonal temperatures and plasma GH level. However, temperature alters other parameters like food intake, which may directly modify the level of plasma GH. In order to determine whether temperature regulates plasma GH levels independently of nutritional status, fish were reared at 8, 12, or 16 degrees C and either fed ad libitum (fish with different food intake) to determine the global effect of temperature, or with the same ration (1.2%/body weight) to observe the temperature effect in fish with the same growth rate. Plasma insulin level was inversely proportional to the temperature (8, 12, and 16 degrees C) in fish fed ad libitum (12.1+/-0.3 ng/ml, 10.9+/-0.3 ng/ml, 9.5+/-0.4 ng/ml; P0.001) and in restricted fish (14.0+/-0.3 ng/ml, 11.3+/-0.3 ng/ml, 10.0+/-0.2 ng/ml; P0.0001), probably due to a prolonged nutrient absorption, and delayed recovery of basal insulin level at low temperature. Conversely, temperature did not affect plasma T3 level of fish fed ad libitum (2.5+/-0.2 ng/ml, 2.4+/-0.1 ng/ml, 2.5+/-0.1 ng/ml at 8, 12, and 16 degrees C) while fish fed with the same ration present less T3 at 16 degrees C than at 8 degrees C (1.83+/-0.1 ng/ml versus 1.2+/-0.1 ng/ml; P0.001) throughout the experiment; these observations indicate that different plasma T3 levels reflect the different nutritional status of the fish. The levels of GH1 and GH2 mRNA, and GH1/GH2 ratio were not different for whatever the temperature or the nutritional status. Pituitary GH content, of fish fed ad libitum did not exhibit obvious differences at 8, 12, or 16 degrees C (254+/-9 ng/g bw, 237+/-18 ng/g bw, 236+/-18 ng/g bw), while fish fed with the same ration have higher pituitary GH contents at 16 degrees C than at 8 degrees C (401+/-30 ng/g bw versus 285+/-25 ng/g bw; P0.0001). Interestingly, high temperature strongly increases plasma GH levels (2.5+/-0.3 ng/ml at 8 degrees C versus 4.8+/-0.6 ng/ml at 16 degrees C; P0.0001) to the same extent in both experiments, since at a given temperature average plasma GH was similar between fish fed ad libitum or a restricted diet. Our results, demonstrate that temperature regulates plasma GH levels specifically but not pituitary GH content, nor the levels of GH1 and GH2 mRNA. In addition no differential regulation of both GH genes was evidenced whatever the temperature.

Fish endure long periods of fasting and demonstrate an extensive capacity for rapid and complete recovery after refeeding. The underlying mechanisms through which nutrient intake activates an increase in somatic growth and especially in muscle growth is poorly understood. In this study we examined the expression profile of major muscle growth regulators in trout white muscle 4, 12, and 34 days after refeeding, using real-time quantitative RT-PCR. Mean insulin-like growth factor I (IGFI) mRNA level in muscle increased dramatically 8- and 15-fold, 4 and 12 days, respectively, after refeeding compared to fasted trout. This declined thereafter. Conversely, only a weak but gradual increase in mean insulin-like growth factor II (IGFII) mRNA level was observed during refeeding. Inversely to IGFI, mean IGF receptor Ia (IGFRIa) mRNA level declined after ingestion of food. In contrast, IGF receptor Ib (IGFRIb) mRNA level was not affected by refeeding. Mean fibroblast growth factor 2 (FGF2) mRNA level increased by 2.5-fold both 4 and 12 days after refeeding, whereas fibroblast growth factor 6 (FGF6) and myostatin mRNA levels were unchanged. Subsequent to IGFI and FGF2 gene activation, an increase in myogenin mRNA accumulation was observed at 12 days post-refeeding suggesting that an active differentiation of myogenic cells succeeds their proliferation. In conclusion, among the potential growth factors we examined in this study, IGFI and FGF2 were identified as candidate genes whose expression may contribute to muscle compensatory growth induced by refeeding.

The transcription factors of the MyoD family have essential functions in myogenic lineage determination and muscle differentiation. These myogenic regulatory factors (MRFs) activate muscle-specific transcription through binding to a DNA consensus sequence known as the E-box present in the promoter of numerous muscle genes. Four members, MyoD, myogenin, myf5 and MRF4/herculin/myf6, have been identified in higher vertebrates and have been shown to exhibit distinct but overlapping functions. Homologues of these four MRFs have also been isolated in a variety of lower vertebrates, including amphibians and fish. Differences have been observed, however, in both the expression patterns of MRFs during muscle development and the function of individual MRFs between lower and higher vertebrates. These differences reflect the variety of body muscle formation patterns among vertebrates. Furthermore, as a result of an additional polyploidy that occurred during the evolution of some amphibians and fish, MyoD, myogenin, myf5 and MRF4 may exist in lower vertebrates in two distinct copies that have evolved separately, acquiring specific roles and resulting in increased complexity of the myogenic regulatory network. Evidence is now accumulating that many of the co-factors (E12, Id, MEF2 and CRP proteins) that regulate MRF activity in mammals are also present in lower vertebrates. The inductive signals controlling the initial expression of MRFs within the developing somite of lower vertebrate proteins are currently being elucidated.

Previously we identified two nonallelic MyoD encoding genes in the rainbow trout. These two MyoD genes (TMyoD and TMyoD2) were duplicated during the tetraploidization of the salmonid genome. In this study we show that TMyoD and TMyoD2 exhibit a distinct spatiotemporal pattern of expression that defines discrete cell populations in the developing somite. TMyoD expression is first detected in the mid-gastrula on either side of the elongating embryonic shield. During the anterior-to-posterior wave of somite formation the TMyoD transcript is initially present in adaxial cells of both the presomitic mesoderm and the forming somites. A lateral extension of TMyoD expression occurs only when the myotomes acquire their characteristic chevron shape pointing rostrally. By contrast, the initial expression of TMyoD2 occurs in somites that have already formed and is limited to the posterior compartment of somites. Further, in postlarval trout we observed a differential expression of TMyoD and TMyoD2 genes in muscle fibers with differing phenotype. Collectively, these data provide evidence that the two trout MyoD encoding genes have evolved to become functionally different. A comparison of the expression patterns of the two trout MyoD genes with that of myogenin allowed us to position them in the regulatory pathway leading to muscle differentiation.

In the trout Oncorhynchus mykiss genome, two distinct MyoD genes (TMyoD and TMyoD2) and a single myogenin gene (Tmyogenin) have been identified. The two MyoD genes are believed to arise from a recent tetraploidization of the salmonid genome. During the anterior-to-posterior wave of somite formation, the TMyoD transcript is present initially in adaxial cells of both the presomitic mesoderm and the forming somites. A lateral extension of TMyoD labelling is observed in maturing somites when progressively they acquire the characteristic chevron shape. In contrast, the initial expression of TMyoD2 takes place in somites which have been formed already and is limited to cells of the posterior domain of the somites. Later, when all the myotomes have acquired their chevron shape, TMyoD2 transcript disappears progressively from the inner part of the myotomes until it is present only in the superficial part where slow oxidative fibres differentiate. The expression of Tmyogenin is detected first in adaxial cells of forming somites. Shortly after the formation of the somite, Tmyogenin expression extends from the adaxial cells to the posterior lateral regions of the somites and then progresses towards the anterior region. TMyoD, TMyoD2 and Tmyogenin are dynamically and differentially expressed in the developing somite suggesting that they are piaying distinet roles in the early myogenesis of trout.

FGF6, a member of the fibroblast growth factor (FGF) family, is specifically expressed in developing skeletal muscle and may participate in muscle maintenance and regeneration. Until now, no convincing evidence for the existence of an FGF6 gene in non-mammalian vertebrates has been put forward. Only a hybrid growth factor containing features characteristic of both FGF4 and FGF6 has been identified in frogs and chickens, suggesting that the step of duplication which created FGF4 and FGF6 took place with the emergence of mammals. In this study, we report the isolation and characterization of a genomic clone encoding the trout (Oncorhynchus mykiss) fibroblast growth factor 6 (TFGF6). An initial cDNA clone was generated by PCR amplification using degenerate oligo primers corresponding to a conserved region of protein found in the mouse and human homologs. The screening of a genomic library with the cloned PCR product led to the isolation of a clone composed of three exons encoding a putative protein of 206 amino acids which exhibits a potential signal peptide and shows 64.6 and 63.6% similarity with mouse and human FGF6, respectively (77% over the carboxy two-thirds of the protein) and only 46.5% similarity with mouse and human FGF4 (62% over the carboxy two-thirds of the protein). The splice position of the three exons was found to be analogous to the human and mouse FGF6 and the start translation site of TFGF6 was preceded by a long stretch of nucleotides that is highly and specifically conserved in mammalian FGF6. Furthermore, a comparative reverse transcriptase-linked PCR assay revealed that the expression pattern of TFGF6 is close to that of mammals, TFGF6 transcripts being present in muscle (fast-twitch and to a lesser extent slow-twitch fibers), heart, testis and brain. Interestingly, the prolonged phase of muscle fiber hyperplasia which occurs in trout is accompanied by the lasting expression of TFGF6 up to the adult stage suggesting that TFGF6 may participate in the continuous generation of muscle fibers within the myotomal musculature of post larval animals.

Members of the cysteine-rich protein (CRP) define a subclass of LIM-only proteins implicated mainly in muscle differentiation. Until now, very little is known concerning the expression of CRP encoding genes during vertebrate development. We describe here the isolation of a trout (Oncorhynchus mykiss) gene encoding a cysteine-rich protein (TCRP) and the pattern of its mRNA accumulation during embryogenesis, focusing on somitogenesis. TCRP encodes a putative protein with two LIM domains linked to a short glycine-rich region that displays 86%, 76%, 67% identity with chicken CRP2, CRP1 and MLP/CRP3 proteins, respectively. Whole-mount in situ hybridisation showed that TCRP transcript is first detected just before somitogenesis in the paraxial mesoderm, while it is absent in the axial structures. During somitogenesis, the expression of TCRP was observed caudally in the elongating presomitic mesoderm and in the last formed somites. The labelling for TCRP was found to fade as the somites mature. At the end of the somitogenesis, TCRP transcripts accumulation was restricted to pronephros and bronchial arches.

The dramatic increase in myotomal muscle mass in fish is related to their unique ability to lastingly produce new muscle fibres, a process termed hyperplasia. In this study, we aimed to characterize intrinsic properties of myogenic cells originating from hyperplasic fish muscle. For this purpose, we compared in situ proliferation, in vitro cell behavior and transcriptomes (RNAs and miRNAs) of myogenic precursors from hyperplasic muscle of juvenile trout (JT) and from non-hyperplastic muscle of fasted juvenile trout (FJT) and adult trout (AT). We showed an higher proliferation rate of trout satellite cells in vivo in hyperplastic muscle compared to non-hyperplastic muscle. Functional analysis of transcriptomics data showed, among the over-expressed genes in JT myogenic cells compared to AT and FJT, an enrichment in genes related to mitochondrial activity, cell cycle and myogenic differentiation.We found also an enrichment in genes involved in Notch signaling pathway, indicating a repression of quiescency markers in JT myogenic cells compared to AT and FJT. Moreover, the miRnome analysis also revealed overexpressed miRNAs in JT whose targets may regulate the proliferation and the differentiation of myogenic precursors. In line with our results, JT myogenic precursors displayed in vitro higher proliferation and differentiation capacities than FJT and AT myogenic precursors. On the whole, our results converge to support the view that myogenic cells extracted from hyperplastic muscle of juvenile trout are intrinsically more potent to form myofibres than myogenic cells extracte; La remarquable capacité de croissance musculaire des poissons est liée notamment à leur capacité à produire durablement de nouvelles fibres (hyperplasie). Nous avons cherché à caractériser au niveau cellulaire et moléculaire les propriétés intrinsèques des précurseurs myogéniques de muscle hyperplasique de la truite. Ainsi, nous avons comparé la prolifération in situ, le comportement in vitro, et les transcriptomes (ARN messagers et microARNs) de précurseurs myogéniques de muscle hyperplasique de truites juvéniles (JT) et de muscles non-hyperplasiques de truites juvéniles soumises à un jeûne prolongé (FJT) ou de truites adultes (AT). Nous avons observé un fort taux de prolifération des cellules satellites au sein du muscle hyperplasique comparé au muscle non-hyperplasique.L’analyse du transcriptome montre que les gènes surexprimés dans les précurseurs du muscle hyperplasique (JT) sont principalement impliqués dans l'activité mitochondriale, le cycle cellulaire et la différenciation myogénique. L’analyse du miRnome a mis en évidence des miRNA surexprimés chez les JT dont les cibles potentielles régulent la prolifération ou la différenciation. En accord avec ces résultats, les précurseurs myogéniques JT présentent in vitro des capacités de prolifération et de différenciation plus élevées que les précurseurs myogéniques FJT et AT. L’ensemble de nos résultats montrent que les précurseurs myogéniques extraits de muscle en croissance hyperplasique présentent, de par les expressions géniques dont ils sont le siège et leur comportement in vitro, de plus grandes capacités intrins

The dramatic increase in myotomal muscle mass in fish is related to their unique ability to lastingly produce new muscle fibres, a process termed hyperplasia. In this study, we aimed to characterize intrinsic properties of myogenic cells originating from hyperplasic fish muscle. For this purpose, we compared in situ proliferation, in vitro cell behavior and transcriptomes (RNAs and miRNAs) of myogenic precursors from hyperplasic muscle of juvenile trout (JT) and from non-hyperplastic muscle of fasted juvenile trout (FJT) and adult trout (AT). We showed an higher proliferation rate of trout satellite cells in vivo in hyperplastic muscle compared to non-hyperplastic muscle. Functional categories related to mitochondrial activity, cell cycle and myogenic differentiation were inferred from genes up regulated in JT myogenic cells compared to AT and FJT myogenic cells. We found also an enrichment in genes involved in Notch signaling pathway, indicating a repression of quiescency markers in JT myogenic cells compared to AT and FJT. Moreover, the miRnome analysis also revealed overexpressed miRNAs in JT whose targets may regulate the proliferation and the differentiation of myogenic precursors. In line with our results, JT myogenic precursors displayed in vitro higher proliferation and differentiation capacities than FJT and AT myogenic precursors. On the whole, our results converge to support the view that myogenic cells extracted from hyperplastic muscle of juvenile trout are intrinsically more potent to form myofibres than myogenic cells extracted from non-hyperplasic muscle.; La remarquable capacité de croissance musculaire des poissons est liée notamment à leur capacité à produire durablement de nouvelles fibres (hyperplasie). Dans cette étude, nous avons cherché à caractériser au niveau cellulaire et moléculaire les propriétés intrinsèques, des cellules myogéniques issues du muscle hyperplasique du poisson. Dans ce but, nous avons comparé la prolifération in situ, le comportement in vitro, et les transcriptomes (ARN messagers et microARNs) de précurseurs myogéniques musculaires extraits de muscle hyperplasique de truites juvéniles (JT) et de muscles non-hyperplasiques de truites juvéniles soumises à un jeun prolongé (FJT) ou de truites adultes (AT). Nous avons observé un fort taux de prolifération des cellules satellites au sein du muscle hyperplasique comparé au muscle non-hyperplasique. Des catégories fonctionnelles liées à l'activité mitochondriale, au cycle cellulaire et à la différenciation myogénique ont été déduites des gènes surexprimés dans les précurseurs JT par rapport aux précurseurs AT et FJT. En outre, nous avons identifié des gènes impliqués dans la voie Notch sous exprimés chez les JT indiquant une répression des marqueurs de quiescence. L’analyse du miRnome à permis également de mettre en évidence des miRNA surexprimés chez les JT dont les cibles peuvent réguler la prolifération ou la différenciation des précurseurs myogéniques. En accord avec ces résultats, les précurseurs myogéniques JT présentaient in vitro des capacités de prolifération et de différenciation plus élevées que les précurseurs myogéniques FJT et AT. L’ensemble de nos résultats montrent que les précurseurs myogéniques extraits de muscle en croissance hyperplasique présentent de plus grandes capacités intrinsèques à former des myofibres comparés aux précurseurs myogéniques extraits de muscle non hyperplasique.

Diplôme : Dr. d'Universite; La musculature squelettique des poissons est composée d'un muscle lent périphérique et d'un muscle rapide profond. Les fibres lentes dérivent de précurseurs somitiques adaxiaux migrant en périphérie du myotome et les précurseurs somitiques latéraux se différencient sur place en fibres rapides. Une modalité particulière de la croissance musculaire des poissons est la formation continue de nouvelles fibres, recrutées en périphérie du myotome chez la larve (hyperplasie stratifiée) et dans tout le myotome chez les poissons de grande taille finale (hyperplasie mosaïque). Nos travaux visaient à décrire chez la truite les régulations transcriptionnelles sous-tendant la différenciation contractile et métabolique des fibres musculaires mises en place au cours du développement, par l'examen systématique par hybridation in situ des patrons d'expression de marqueurs musculaires. Nous montrons que la différenciation des fibres musculaires chez le poisson inclue un programme myogénique commun, observé transitoirement dans toutes les fibres embryonnaires et dans les néofibres lentes formées par hyperplasie stratifiée. Par ailleurs, nous montrons que l'IGF1 et le FGF2 sont de bons candidats impliqués dans le maintien de l'hyperplasie chez le poisson.