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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.

International audience; In fish rearing industry, sexual maturation results in loss of profit or at least in a delay to valorize spawning fish. Indeed, many edible fish display poor fillet quality after sexual maturation and as such cannot be processed immediately after spawning. So, a post-spawning rearing period may allow the recovering of an acceptable fillet quality. So far, flesh quality restoration after spawning has received little attention. In the present study, the evolution of technological and organoleptic qualities was investigated in diploid autumnal strain female rainbow trout (24 months of age) after spawning. Immediately after spawning, nine groups of trout (n =25) from the same cohort were placed separately into circular tanks and fed ad libitum. Fish were then sampled at 0, 1, 2, 4, 8, 13, 16, 24, and 33 weeks after spawning (PS0, PS1, PS2, PS4, PS8, PS13, PS16, PS24, and PS33). Immature (no egg produced) female trout from the same cohort were also sampled as controls at the beginning (C0) and at the end (C33) of the experiment. Immediately after spawning, PS0 trout showed a significantly lower raw fillet yield than control (C0). Furthermore, raw fillet from these trout was less colored (lower redness a*) and presented higher lightness L* value than raw fillet from C0 trout while their fillet mechanical resistance was similar. Raw fillet yield increased after the 16th week post-spawning. The fillet lightness steadily decreased from the 4th week to the 24th week post-spawning and did not change afterwards, whereas fillet redness increased from the 8th week to the 24th week after spawning. Fillet mechanical resistance declined progressively after spawning with a significant change from the 13th week. Concerning the smoked fillet, the smoking yield of PS0 trout was significantly lower than that of C0 trout. Thereafter the smoking yield of post-spawning trout slowly increased until reaching a significantly higher value at the 24th week. The evolution of smoked fillet color and mechanical resistance after spawning was similar to that of raw fillet. At the end of the experiment, most quality parameters of PS33 trout fillet were similar to those of C33 trout. We discuss the post-spawning quality parameters recovery in three sequential phases. On the whole, our study reports that the technological and organoleptic properties of the flesh were recovered in female trout 24 weeks after spawning (≈1400 ◦C.day).

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 Compensatory growth is a phase of rapid growth, greater than the growth rate of control animals, that occurs after a period of growth-stunting conditions. Fish show a capacity for compensatory growth after alleviation of dietary restriction, but the underlying cellular mechanisms are unknown. To learn more about the contribution of genes regulating hypertrophy (an increase in muscle fibre size) and hyperplasia (the generation of new muscle fibres) in the compensatory muscle growth response in fish, we used high-density microarray analysis to investigate the global gene expression in muscle of trout during a fasting-refeeding schedule and in muscle of control-fed trout displaying normal growth. Results The compensatory muscle growth signature, as defined by genes up-regulated in muscles of refed trout compared with control-fed trout, showed enrichment in functional categories related to protein biosynthesis and maturation, such as RNA processing, ribonucleoprotein complex biogenesis, ribosome biogenesis, translation and protein folding. This signature was also enriched in chromatin-remodelling factors of the protein arginine N-methyl transferase family. Unexpectedly, functional categories related to cell division and DNA replication were not inferred from the molecular signature of compensatory muscle growth, and this signature contained virtually none of the genes previously reported to be up-regulated in hyperplastic growth zones of the late trout embryo myotome and to potentially be involved in production of new myofibres, notably genes encoding myogenic regulatory factors, transmembrane receptors essential for myoblast fusion or myofibrillar proteins predominant in nascent myofibres. Conclusion Genes promoting myofibre growth, but not myofibre formation, were up-regulated in muscles of refed trout compared with continually fed trout. This suggests that a compensatory muscle growth response, resulting from the stimulation of hypertrophy but not the stimulation of hyperplasia, occurs in trout after refeeding. The generation of a large set of genes up-regulated in muscle of refed trout may yield insights into the molecular and cellular mechanisms controlling skeletal muscle mass in teleost and serve as a useful list of potential molecular markers of muscle growth in fish. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3837-9) contains supplementary material, which is available to authorized users.

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.

SUMMARYThe external cell layer that surrounds the fish primary myotome provides the myogenic precursors necessary for muscle growth, suggesting that this epithelium is equivalent to the amniote dermomyotome. In this study we report the identification of a trout orthologue of the dermal marker Dermo-1, and show that trout somitic external cells, which are all potentially myogenic as indicated by the transcription of Pax7 gene,express Dermo-1. This finding and our previous observation that external cells express collagen I show that these cells have dermis-related characteristics in addition to exhibiting myogenic features. In an effort to identify novel genes expressed in the external cell epithelium we performed an in situ hybridisation screen and found both collectin sub-family member 12,a transmembrane C-type lectin, and Seraf, an EGF-like repeat autocrine factor. In situ hybridisation of staged trout embryos revealed that the expression of Dermo-1, collectin sub-family member 12 and Seraf within the external cell layer epithelium was preceded by a complex temporal and spatial expression pattern in the early somite.

Les qualités des carcasses et des viandes/chairs des espèces d’intérêt agronomique (mammifères, espèces aviaires et poissons) dépendent en grande partie des caractéristiques et des proportions relatives des différents compartiments tissulaires, principalement les fibres et le tissu conjonctif musculaires et les tissus adipeux. Dans cette revue, nous décrivons la mise en place de ces trois compartiments au cours du développement embryonnaire et foetal, ainsi que leur croissance après la naissance ou l’éclosion. Les fibres musculaires et le tissu conjonctif des muscles du tronc sont issus de domaines embryonnaires distincts de ceux à l’origine des muscles des membres et de la tête. L’origine embryonnaire des adipocytes blancs reste à établir, mais est vraisemblablement multiple et dépendante de la localisation anatomique des tissus adipeux. La croissance post-embryonnaire des muscles et des tissus adipeux implique la prolifération puis la différenciation de cellules progénitrices non-embryonnaires. Le nombre total de fibres musculaires est fixé aux deux tiers ou aux quatre cinquièmes de la gestation chez le bovin et le porc respectivement, et dès la naissance chez les volailles alors qu’il augmente encore après l’éclosion chez certains poissons. Le nombre d’adipocytes augmente durant la croissance foetale et dans une moindre mesure après la naissance. Des interactions entre tissus au cours de la croissance, auparavant suggérées, sont maintenant démontrées par l’identification de protéines sécrétées par les cellules musculaires et adipeuses qui participent au dialogue entre tissus., The qualities of carcasses and products of agronomic species depend largely on characteristics and relative proportions of the different tissue components, mainly muscle fibers, muscular connective tissue and adipose tissues. This review is devoted to the prenatal and neonatal development of these three components. The embryonic origin of myofibers and connective tissue from trunk muscle differs from that of limb and head muscles. The embryonic origin of white adipocytes remains to be established and may depend on the anatomical location of adipose tissues. The post-embryonic growth of muscles and adipose tissues involves the proliferation and then the differentiation of fetal or adult progenitors. The total number of muscle fibers is fixed at around 66% (bovine) or 80% (pig) of gestation or at birth in poultry, while it increases after hatching in some fishes. The number of adipocytes increases predominantly during fetal growth and to a lesser extent after birth. Interactions between tissues during growth, formerly suggested, have now been demonstrated by the identification of proteins secreted by muscle and adipose cells that participate in the cross-talk between tissues.

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.

Skeletal muscles are composed of different fiber types, largely defined by differential expression of protein isoforms involved in myofibrillogenesis or metabolism. To learn more about the gene activations that underlie the differentiation and the diversification of embryonic fish myotomal fibers, we investigated the developmental expression of 25 muscle genes in trout embryos by in situ hybridization of muscle-specific transcripts. The earliest event of muscle differentiation, at approximately the 25-somite stage, was the expression of a variety of muscle-specific genes, including slow-twitch and fast-twitch muscle isoforms. The activation of these muscle genes started in the deep somitic domain, where the slow muscle precursors (the adaxial cells) were initially located, and progressively spread laterally throughout the width of the myotome. This mediolateral progression of gene expression was coordinated with the lateral migration of slow adaxial cells, which specifically expressed the slow myosin light chain 1 and the SLIM1/FHL1 genes. Subsequently, the fast and slow skeletal muscle isoforms precociously expressed in the course of the mediolateral wave of muscle gene activation became down-regulated in the superficial slow fibers and the deep fast fibers, respectively. Finally, several muscle-specific genes, including troponins, a slow myosin-binding protein C, tropomodulins, and parvalbumin started their transcription only in late embryos. Taken together, these findings show in fish embryos that a common myogenic program is triggered in a mediolateral progression in all muscle cells. The acquisition of the slow phenotype involves the additional activation of several slow-specific genes in migrating adaxial muscle cells. These events are followed by sequential gene activations and repressions in fast and slow muscle cells.

Chez les espèces d’intérêt agronomique, les caractéristiques des fibres musculaires jouent un rôle important dans le déterminisme de la qualité organoleptique de la viande. Ce dossier fait le point des connaissances concernant la caractérisation des fibres musculaires et l’acquisition de leurs propriétés (contractiles et métaboliques) dans les principales espèces productrices de viande (bovins, porcs, volailles) et chez les poissons. La mise en place des fibres musculaires, qui débute très tôt au cours de la vie fœtale, implique des vagues successives de plusieurs générations de cellules myogéniques qui sont à l’origine des différents types de fibres. Le nombre total de fibres reste stable après la naissance chez le bovin, le porc et les volailles. En revanche, chez certains poissons, il augmente encore au cours de la vie postnatale. Chez le bovin, les principales étapes de la différenciation contractile et métabolique des fibres ont lieu durant le dernier tiers de la vie fœtale. En revanche, l’acquisition de ces propriétés se fait principalement pendant les deux semaines qui suivent la naissance chez le porc ou la volaille. Ainsi, si les principes généraux du développement musculaire sont comparables dans les différentes espèces, ils se déroulent selon une cinétique différente en relation avec la maturité de chaque espèce à la naissance. Toutefois, dans toutes les espèces, les propriétés des fibres sont caractérisées par une large plasticité tout au long de la vie des animaux, ce qui laisse la possibilité de les modifier par le biais des facteurs d’élevage.

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.

SUMMARY Myostatin (GDF8) has been shown to be a major genetic determinant of skeletal muscle growth in mammals. In this study, we report the cloning of two trout cDNAs that encode two distinct myostatin-related proteins. The presence in this fish species of two myostatin genes (Tmyostatin 1 and Tmyostatin 2) probably results from the recent tetraploïdisation of the salmonid genome. A comparative reverse-transcriptase-linked polymerase chain reaction assay revealed that Tmyostatin 1 mRNA was present ubiquitously in trout tissues, while Tmyostatin 2 mRNA expression was restricted to muscle and brain. In developing muscle, Tmyostatin 1 expression was observed in eyed-stage embryos well before hatching, whereas Tmyostatin 2 was expressed only in free-swimming larvae. In myotomal muscle from adult animals, Tmyostatin 1 mRNA accumulation was similar in both slow- and fast-twitch fibres, and its concentration did not change during the muscle wasting associated with sexual maturation. In contrast, Tmyostatin 2 mRNA accumulated predominantly in slow-twitch fibres, and its concentration decreased dramatically in wasting muscles from maturing animals. This work shows that two distinct myostatin genes are present in the trout genome. Furthermore, it indicates that these two trout myostatin genes (i) exhibit a distinct expression pattern in muscle and non-muscle tissues and (ii) are not upregulated during the muscle wasting that accompanies sexual maturation.

SUMMARYThe axial muscle of most teleost species consists of a deep bulk of fast-contracting white fibres and a superficial strip of slow-contracting red fibres. To investigate the embryological development of fast and slow muscle in trout embryos, we carried out single and double in situ hybridisation with fast and slow myosin heavy chain (MyHC)-isoform-specific riboprobes. This showed that the slow-MyHC-positive cells originate in a region of the somite close to the notochord. As the somite matures in a rostrocaudal progression, the slow-MyHC-positive cells appear to migrate radially away from the notochord to the lateral surface of the myotome, where they form the superficial strip of slow muscle. Surprisingly, the expression pattern of the fast MyHC showed that the differentiation of fast muscle commences in the medial domain of the somite before the differentiation and migration of the slow muscle precursors. Later, as the differentiation of fast muscle progressively spreads from the inside to the outside of the myotome, slow-MyHC-expressing cells become visible medially. Our observations that the initial differentiation of fast muscle takes place in proximity to axial structures and occurs before the differentiation and migration of slow muscle progenitors are not in accord with the pattern of muscle formation in teleosts previously described in the zebrafish Danio rerio, which is often used as the model organism in fishes.

Helix-loop-helix (HLH) proteins related to the inhibitor of DNA binding/differentiation (Id) serve as general antagonists of cell differentiation. They lack a basic DNA-binding domain and are thought to function in a dominant negative manner by sequestering basic HLH (bHLH) transcription factors that are involved in cell determination and differentiation. Four Id-encoding genes have been shown in mammals, they have a distinct pattern of expression suggesting different functions for each member in different cell lineage. In this study we describe the identification and cloning of two trout cDNAs which encode helix-loop-helix proteins showing a high degree of similarity with mammalian Id family members. One cDNA encodes a trout putative Id1 protein (TId1) that is 63% identical to the human Id1 protein over the entire length and 78% identical within the HLH region. The other cDNA encodes a trout putative Id2 protein (TId2) that shows 82% identity to the human Id2 protein and only one change that is conservative over the HLH region. In the 3′ untranslated region, TId2 mRNA exhibits 16 nucleotides upstream from the AATAAA site, a palindromic sequence similar to the cytoplasmic polyadenylation element (CPE) which is also present in Id2 and Id3 mRNAs from mammals and in XIdx/XIdI mRNA from Xenopus. In the fish, TIdl and TId2 are expressed in a tissue-specific manner, with slightly different patterns. During myogenesis, TId1 and TId2 are highly expressed in the myotomal musculature of fish embryos and of early alevins but are down-regulated in that of late alevins. In muscle from juveniles and adults, TId1 and TId2 transcripts are abundant in the slow oxidative fibers while they are absent in the fast glycolytic fibers. This expression pattern suggests that Id genes play a role in the regulation of muscle fiber pheno-type in addition to controlling early myogenesis. On the whole. the identification of two HLH-Id encoding genes in a major taxonomic group like teleosts, suggests an early divergence of Id genes in vertebrate evolution. The observation that Id transcripts are present selectively in the slow muscle reveals that their expression is more complicated than previously appreciated.

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.

We report the cloning of a new trout myogenic cDNA which encodes helix-loop-helix protein homologous to the myogenic factor myogenin. Northern analyses indicate that trout myogenin (Tmyogenin) transcripts accumulate in large amounts in the myotomal masculature of embryos and frys. In adults, transcripts concentrate within the thin lateral layer of red (slow oxydative) muscle fibres. They are present only in low amounts in white (fast glycolytic) muscle fibres which constitute the major part of the trunk musculature. Using an in vitro myogenesis system, we observed that the trout myogenin encoding gene is not activated until myosatellite cells fuse to generate multinucleated myotubes, indicating that Tmyogenin lies downstream of muscle determination factors. All these observations show that in a major taxinomic group like teleosts, a gene with homology to myogenin exists. Its activation during myogenesis suggests that it acts as a major developmental regulator of muscle differentiation.

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.

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/).

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.

Background 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 (Oncorhynchus mykiss) 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. Results In transgenic trout, green fluorescence appeared in fast muscle fibers as early as the somitogenesis stage and persisted throughout life. Using an in vitro 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 in vitro 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. Conclusions 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 in vitro 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.

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.

Collagen VI is a ubiquitous microfibrillar collagen that forms a network in most interstitial connective tissues, including soft organs and cartilage. The extracellular and intracellular distribution of collagen VI in human liver was studied by light and electron microscopy using the indirect immunoperoxidase method. In normal adult liver, collagen VI was seen mainly in portal spaces and formed a continuous layer in the sinusoids. Fetal liver contained more of collagen VI in the sinusoid than newborn and adult livers. In alcoholic fibrotic and cirrhotic livers, collagen VI antibodies intensely stained fibrous septa that invaded the lobule. Immunoelectron microscopy on normal liver showed that collagen VI antibodies labeled microfibrillar material and occasionally the surface of cells including hepatocytes. In both perinatal and fibrotic livers, electron-dense deposits were abundant in the space of Disse, intensely staining fibrils located around bundles of banded collagen. In both normal and fibrotic adult livers, collagen VI was abundant in the rough endoplasmic reticulum of Ito cells, while hepatocytes were constantly negative. In fetal livers, hepatocytes also contained collagen VI. These results suggest that collagen VI is a major constituent of the hepatic extracellular matrix. Furthermore, the cellular sources of collagen VI appear to be different in adult and developing livers.

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.

SUMMARYMuch of the present information on muscle differentiation in fish concerns the early embryonic stages. To learn more about the maturation and the diversification of the fish myotomal fibres in later stages of ontogeny, we investigated, by means of in situ hybridisation, the developmental expression of a large repertoire of muscle-specific genes in trout larvae from hatching to yolk resorption. At hatching, transcripts for fast and slow muscle protein isoforms, namely myosins, tropomyosins, troponins and myosin binding protein C were present in the deep fast and the superficial slow areas of the myotome, respectively. During myotome expansion that follows hatching, the expression of fast isoforms became progressively confined to the borders of the fast muscle mass, whereas, in contrast, slow muscle isoform transcripts were uniformly expressed in all the slow fibres. Transcripts for several enzymes involved in oxidative metabolism such as citrate synthase, cytochrome oxidase component IV and succinate dehydrogenase, were present throughout the whole myotome of hatching embryos but in later stages became concentrated in slow fibre as well as in lateral fast fibres. Surprisingly, the slow fibres that are added externally to the single superficial layer of the embryonic(original) slow muscle fibres expressed not only slow twitch muscle isoforms but also, transiently, a subset of fast twitch muscle isoforms including MyLC1, MyLC3, MyHC and myosin binding protein C. Taken together these observations show that the growth of the myotome of the fish larvae is associated with complex patterns of muscular gene expression and demonstrate the unexpected presence of fast muscle isoform-expressing fibres in the most superficial part of the slow muscle.

Somites are repeated, epithelial structures that are derived from the unsegmented paraxial mesoderm located lateral to the notochord. In higher vertebrates, somites differentiate into a sclerotome that subsequently forms the vertebrae and the ribs and into a dermomyotome that gives rise to a myotome, from which arises the skeletal muscle, and to a dermatome, from which arises the dermis. Fish somites have been shown to produce a sclerotome and a myotome, but very little is known regarding their participation in the formation of connective tissues, especially at the junction between the epidermis and the myotome. To investigate the formation of connective tissues in fish somites, we have examined the expression pattern of the collagen I (α1) chain. As somitogenesis proceeds rostrocaudally, collagen I (α1) expression marks the sclerotomal cells and delineates the formation of the vertebrae. Surprisingly, after the completion of the segmentation, transcript for the collagen I (α1) chain appeared in a distinct epithelial-like monolayer situated at the periphery of the developing somite facing the surface epidermis. This epithelial monolayer of somitic cells that covered the superficial slow muscle cells, did not express the myogenic transcriptional regulator myogenin and was devoid of contractile filament. As the somite increased in size, these collagen-expressing epithelial cells flattened, forming a thin cellular layer underlying the epidermis and recovering the lateral surface of the myotome. In conclusion, the lateral domain of the fish somite forms a distinct epithelial cell layer sharing many characteristics with amniote dermatome. Developmental Dynamics 233:605–611, 2005. © 2005 Wiley-Liss, Inc.

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.

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.

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.

International audience; We have isolated the cDNA encoding a myogenic factor expressed in embryonic trout muscle by hybridization with a Xenopus MyoD cDNA. Nucleotide sequence analysis and amino acid comparison showed that this cDNA called TMyoD encodes a polypeptide of 276 amino acids with 70% identity to the entire Xenopus MyoD protein and 92% identity within the basic and myc-like region. Results from Northern blotting showed that the corresponding transcript is expressed both in adult and embryonic skeletal musculature and in an in vitro myogenesis system, but is undetectable in cardiac and smooth muscles and in non muscle tissues.

Over the past decade, numerous nonviral cationic vectors have been synthesized. They share a high density of positive charges and efficiency for gene transfer in vitro. However, their positively charged surface causes instability in body fluids and cytotoxicity, thereby limiting their efficacy in vivo. Therefore, there is a need for developing alternative molecular structures. We have examined tetrabranched amphiphilic block copolymers consisting of four polyethyleneoxide/polypropyleneoxide blocks centered on an ethylenediamine moiety. Cryo-electron microscopy, ethidium bromide fluorescence and light and X-ray scattering experiments performed on vector–DNA complexes showed that the dense core of the nanosphere consisted of condensed DNA interacting with poloxamine molecules through electrostatic, hydrogen bonding and hydrophobic interactions, with DNA molecules also being exposed at the surface. The supramolecular organization of block copolymer/DNA nanospheres induced the formation of negatively charged particles. These particles were stable in a solution that had a physiological ionic composition and were resistant to decomplexation by heparin. The new nanostructured material, the structure of which clearly contrasted with that of lipoplexes and polyplexes, efficiently transferred reporter and therapeutic genes in skeletal and heart muscle in vivo. Negatively charged supramolecular assemblies hold promise as therapeutic gene carriers for skeletal and heart muscle-related diseases and expression of therapeutic proteins for local or systemic uses.

The lipocyte is an important source of laminin in the normal liver. We have investigated the expression of the 3 chains of laminin in isolated rat lipocytes. Both B1 and B2 chains, but not A, were found in medium from 5-day-old lipocyte primary cultures by immunoblotting and immunoprecipitation of 35S-labeled proteins after reducing SDS-polyacrylamide gel electrophoresis. An additional polypeptide of Mr=380 000 was identified by immunoprecipitation. Under non-reducing conditions only one Mr=900 000 band was revealed. High levels of B1 and B2 mRNAs were also demonstrated in 5-day-old cultured lipocytes while at the time of seeding, only B2 chain mRNAs were clearly detectable. A chain mRNA was constantly absent. These results suggest that lipocytes produce a variant form of laminin in primary culture and that the Mr=380 000 polypeptide could be unrelated to the A chain of laminin.

La production de truite en France s'oriente de plus en plus vers des poissons de grande taille destinés notamment à la fumaison. La production d’oeufs de truite conduit à l’obtention de poissons de grande taille, mais leur chair, très altérée par la maturation sexuelle, présente des défauts de qualité. La présente étude visait à déterminer si, chez la truite arc-en-ciel, la période qui suit la ponte, permet une restauration des propriétés technologiques et organoleptiques des filets compatibles avec leur transformation et leur consommation. Les caractéristiques morphologiques des poissons, la couleur et la résistance mécanique des filets frais et fumés ont été mesurées chez des truites femelles âgées de deux ans, de la ponte jusqu'à 32 semaines après la ponte. Au moment de la ponte, les femelles matures avaient un poids vif, une valeur Fatmeter® et un rendement en filets frais nettement inférieurs à ceux mesurés chez des truites témoins immatures (truites de la même cohorte qui n'avaient pas produit d’oeufs). Les mesures instrumentales de la couleur et de la résistance mécanique ont montré que, juste après la ponte, les filets frais étaient moins colorés (faible indice de rouge a*) avec une valeur élevée de la luminosité L*, et présentaient une moindre résistance mécanique que les témoins. A partir de 12 semaines post-ponte, le poids vif et la valeur Fatmeter® mesurés étaient significativement supérieurs à ceux évalués précédemment, et ont continué à augmenter jusqu’à la fin de l’expérimentation. De plus, le rendement en filets frais a augmenté après la 16ème semaine. La couleur des filets a fortement évolué au cours de la période post-ponte. La luminosité a régulièrement diminué après la 4ème semaine et s’est stabilisée à partir de la 24ème semaine tandis que l’indice de rouge a augmenté après la 8ème semaine jusqu’à la 24ème semaine post-ponte. Quant à la résistance mécanique, elle a augmenté juste après la ponte, est restée inchangée jusqu’à la 16ème semaine, a chuté fortement entre la 16ème semaine et la 24ème semaine et a conservé une valeur stable jusqu’à la fin de l’expérimentation. Pour ce qui est du fumage, le rendement de salage-fumage des filets (poids filet salé et fumé/poids filet frais avant salage et fumage) des femelles matures était nettement inférieur à celui des témoins au moment de la ponte et n’a significativement augmenté qu’à partir de la 24ème semaine. L’évolution de la qualité des filets fumés était similaire à celle des filets frais pour la couleur et la résistance mécanique mais leur luminosité a moins varié. A la fin de l’expérimentation, les truites qui ont pondu présentaient globalement les mêmes caractéristiques que les témoins immatures, attestant d’une restauration des qualités technologique et sensorielle de la chair.