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Background: Sexual maturation causes loss of fish muscle mass and deterioration of fillet quality attributes that prevent market success. We recently showed that fillet yield and flesh quality recover in female trout after spawning. To gain insight into the molecular mechanisms regulating flesh quality recovery, we used an Agilent-based microarray platform to conduct a large-scale time course analysis of gene expression in female trout white muscle from spawning to 33 weeks post-spawning., Results: In sharp contrast to the situation at spawning, muscle transcriptome of female trout at 33 weeks after spawning was highly similar to that of female trout of the same cohort that did not spawn, which is consistent with the post-spawning flesh quality recovery. Large-scale time course analysis of gene expression in trout muscle during flesh quality recovery following spawning led to the identification of approximately 3340 unique differentially expressed genes that segregated into four major clusters with distinct temporal expression profiles and functional categories. The first cluster contained approximately 1350 genes with high expression at spawning and downregulation after spawning and was enriched with genes linked to mitochondrial ATP synthesis, fatty acid catabolism and proteolysis. A second cluster of approximately 540 genes with transient upregulation 2 to 8 weeks after spawning was enriched with genes involved in transcription, RNA processing, translation, ribosome biogenesis and protein folding. A third cluster containing approximately 300 genes upregulated 4 to 13 weeks after spawning was enriched with genes encoding ribosomal subunits or regulating protein folding. Finally, a fourth cluster that contained approximately 940 genes with upregulation 8 to 24 weeks after spawning, was dominated by genes encoding myofibrillar proteins and extracellular matrix components and genes involved in glycolysis., Conclusion: Overall, our study indicates that white muscle tissue restoration and flesh quality recovery after spawning are associated with transcriptional changes promoting anaerobic ATP production, muscle fibre hypertrophic growth and extracellular matrix remodelling. The generation of the first database of genes associated with post-spawning muscle recovery may provide insights into the molecular and cellular mechanisms controlling muscle yield and fillet quality in fish and provide a useful list of potential genetic markers for these traits., (© 2021. The Author(s).)

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., (© 2019 Landemaine et al.)

Fish axial muscle consists of a series of W-shaped muscle blocks, called myomeres, that are composed primarily of multinucleated contractile muscle cells (myofibres) gathered together by an intricate network of connective tissue that transmits forces generated by myofibre contraction to the axial skeleton. This review summarises current knowledge on the successive and overlapping myogenic waves contributing to axial musculature formation and growth in fish. Additionally, this review presents recent insights into muscle connective tissue development in fish, focusing on the early formation of collagenous myosepta separating adjacent myomeres and the late formation of intramuscular connective sheaths (i.e. endomysium and perimysium) that is completed only at the fry stage when connective fibroblasts expressing collagens arise inside myomeres. Finally, this review considers the possibility that somites produce not only myogenic, chondrogenic and myoseptal progenitor cells as previously reported, but also mesenchymal cells giving rise to muscle resident fibroblasts., (Copyright © 2019 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

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 studied in vivo by knockdown in zebrafish using morpholino. We found that N-alpha-acetyltransferase 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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The 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 hyperplasic fish 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)., Results: For 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., Conclusions: The 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.

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 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., (© 2018 The Fisheries Society of the British Isles.)

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.

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.

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.

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 persists during starvation., (© 2015. Published by The Company of Biologists Ltd.)

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.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

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 3million quality sequences with an average size of 328bp, representing more than 1Gb 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/)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (© 2011 Wiley Periodicals, Inc.)

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.

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., (Copyright 2008 Wiley-Liss, Inc.)

In this article, whole mount in situ hybridization is used to examine early blood vessel and blood cell development in the embryos of the brown trout Salmo trutta lacustris. cDNAs encoding for the angiogenic markers fli1 and flk1, and for the hematopoietic markers gata1 and gata2, were identified from an expressed sequence tag library of rainbow trout. Results show that fli1, flk1 and gata2 are activated in bilateral bands of the lateral trunk mesoderm before the onset of somitogenesis, shortly followed by gata1. These bands then converge toward the ventral midline to form the intermediate cell mass (ICM) (anterior ICM). Subsequent axial vasculogenesis and initial blood cell formation involve a clear spatial separation of fli1 and gata gene expression. Fli1 staining is most intense within the axial vessel (dorsal aorta, posterior cardinal vein) forming and lateral ICM cells, whereas binding of gata1 and gata2 probes becomes confined to the central portion of ICM cells beneath the dorsal aorta. This is followed by a first wave of angiogenesis, indicated by expression of fli1 and flk1. This gives rise to the intersegmental, dorsal longitudinal anastomotic and intestinal vessels. Further angiogenesis and hematopoiesis are activated in the "posterior ICM" of the tail. Here, the absence of gata1 indicates that hematopoiesis in this tissue generates myeloid rather than erythroid cells. The results supplement and validate previous, now historical morphological work in salmonids, thus aiding the elucidation of a comprehensive general scheme of angiogenic and hematopoietic development in the teleost embryo.

The 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.

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.

NLRR-l (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-l 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-l-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.

Much 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.

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., (Copyright 2005 Wiley-Liss, Inc)

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 (alpha1) chain. As somitogenesis proceeds rostrocaudally, collagen I (alpha1) expression marks the sclerotomal cells and delineates the formation of the vertebrae. Surprisingly, after the completion of the segmentation, transcript for the collagen I (alpha1) 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.

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.

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.

Muscle 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.

Recently, we have demonstrated in rainbow trout that environmental temperature may, independently of nutritional status, directly stimulate plasma growth hormone (GH) that is recognised as being an insulin-like growth factor (IGF) system regulator. The aim of this study was to determine whether temperature may directly regulate the IGF system or indirectly regulate it through plasma GH or nutritional status. For this purpose, rainbow trout were reared at 8, 12, or 16 degrees C and fed either ad libitum (similar nutritional status) to evidence the global effect of temperature, or with the same ration (1.2% body weight/day), to determine the temperature effect in fish with the same growth rate. Endocrine and autocrine/paracrine regulations of the IGF system were determined by measuring plasma IGF1 and IGF2, liver and muscle IGF1 and IGF2 mRNA as well as IGFRIa, IGFRIb mRNA, and the quantity of IGF type I receptor in muscle. Our results show that neither rearing temperature nor the nutritional status of fish affected the expression of both IGF receptor genes in muscle. Nevertheless, the quantity of IGF type I receptor determined by a binding study, appeared to be inversely proportional (P<0.05) to the rearing temperature without any relationship with nutritional status, suggesting a direct effect of temperature on its turnover. After 2 weeks of treatment, the levels of IGF1 mRNA in muscle at 8 degrees C were 2-fold higher (P<0.05) than at 16 degrees C in both ad libitum and restricted feed fish, whereas after 6 weeks, this difference was no longer observed. In both experiments, the levels of plasma IGF2 were 10-fold higher than the levels of plasma IGF1 (mean 105+/-3.0 versus 13.5+/-0.6 ng/ml), and plasma levels were correlated with their respective mRNA liver concentrations (r2=0.14 and 0.25, respectively; P<0.01). In the ad libitum feeding experiment, plasma and mRNA levels of IGF1 were related to the rearing temperature (P<0.05), while for IGF2 no effect was seen. In contrast, in the restricted feeding experiment, plasma and IGF2 mRNA levels were inversely proportional to the rearing temperature (P<0.0001) while plasma IGF1 was unaltered. Levels of plasma IGF1 were related to the growth rate in both experiments, while levels of plasma IGF2 appeared to be associated with the nutritional status of the fish. Our results suggest that the autocrine/paracrine expression of IGF1 and IGF2 in muscle is not a key regulator of the growth promoting effect of temperature. Conversely, temperature seems to promote growth through IGF1 secretion by the liver following GH stimulation, and impairment of nutritional status would prevent the IGF1 stimulation by temperature. In addition, the growth-promoting effect of temperature did not affect plasma IGF2, which appeared to be more related to the metabolic status of the fish.

In fish, the GH/IGF system installs very early during development suggesting that this system could promote embryonic growth and development. In contrast to mammals, the embryonic growth rate of poikilotherms depends considerably on the incubation temperature. Therefore, the aim of this study was to determine if variations of embryo growth in response to temperature could be associated with modifications in the gene expression of the GH/IGF system. In this study, using whole mount in situ hybridisation, we demonstrated that embryo incubation temperature (4, 8, and 12 degrees C) did not change the timing of GH-1 and GH-2 mRNA expression in somatotroph cells (stage 24). Similarly, at hatching (stage 30), we did not observe an obvious difference in GH protein and GH-1 and GH-2 transcript amounts in relation to the incubation temperature. Furthermore, from stage 22 to 25, the highest temperature led to a specific up-regulation of IGF-2 (2-fold between 4 and 12 degrees C), and both IGF-RIa and IGFRIb mRNA (1.5-fold between 4 and 12 degrees C), while no difference was observed for IGF-1 mRNA. Conversely, at hatching, the highest temperature specifically down-regulated IGF-2 (3-fold between 4 and 12 degrees C) and both IGF receptor mRNAs (2 fold between 4 and 12 degrees C) present in the head, while no difference was observed in the trunk. Our results demonstrated that different incubation temperatures during trout embryonic development did not change the stage of somatotroph cell appearance. Before hatching, IGF-2 and both IGF receptors, but not IGF-1 mRNA, were specifically up-regulated by high temperatures and could be related to the enhancement of embryonic growth rate., (Copyright 2003 Wiley-Liss, Inc.)

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; P<0.001) and in restricted fish (14.0+/-0.3 ng/ml, 11.3+/-0.3 ng/ml, 10.0+/-0.2 ng/ml; P<0.0001), 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; P<0.001) 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; P<0.0001). 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; P<0.0001) 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 Growth hormone (GH)/insulin-like growth factor (IGF) system promotes embryonic growth in higher vertebrates. Such a system exists in salmonids, but exhibits an additional level of complexity resulting from a recent whole genome tetraploidisation. Thus, two nonallelic GH genes are present in the trout genome. Although the two GH genes are similar, the possibility remains that the two genes have evolved separately, acquiring a distinct expression pattern. In this study, using whole mounted in situ hybridisation, we observed a one stage delay between the appearance of GH-2 (Stage 22) and GH-1 (Stage 23) soon after pituitary formation (Stage 21). In addition, by double in situ hybridisation, we clearly evidenced two types of somatotroph, one expressing only GH-2 and the other type both GH-1 and GH-2 at Stage 24. Consequently, at this stage more cells expressed GH-2 than GH-1 as confirmed by quantitative RT-PCR. However at hatching, as in adult, the difference between the expression of the two GH genes was no longer observed. In addition, our immunohistochemical studies did not show any delay between the expression of the mRNA and its translation as a protein at Stage 24. A comparison of the expression pattern of the IGF system components (IGF-1, IGF-2, and the receptor type I) determined by real time RT-PCR, have shown an IGF-1 mRNA increase concomitantly to the appearance of GH expression. On the whole, our results demonstrate a differential regulation of GH-1 and GH-2 genes in rainbow trout embryo. The relationship observed between the expression of different component of the GH/IGF system seems to indicate that this system could be functional early on during embryonic development., (Copyright 2003 Wiley-Liss, Inc.)

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.

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.

The 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.

We have isolated MLC1(F) (tMLC1(F)), MLC3(F) (tMLC3(F)) and skeletal actin cDNAs from the teleost Oncorhynchus mykiss. Sequence analysis indicates that tMLC1(F) and tMLC3(F) are not produced from differentially spliced mRNAs as reported in avians and rodents but are encoded by different genes. Results from RNase protection analysis showed that the corresponding transcripts are expressed in fast skeletal muscles. Whole-mount in situ hybridisation revealed distinct expression patterns of the myosin alkali light chains and skeletal actin genes during skeletal muscle development in the embryo.

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.

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.

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, TId1 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 phenotype 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.

Previously we identified a trout myogenic factor related to MyoD. We now present a cDNA encoding a novel trout myogenic factor (TMyoD2) expressed in embryonic muscle. Nucleotide analysis and amino acid comparison showed that this cDNA encodes a MyoD-like protein of 275 amino acids that is distinct but related to TMyoD with 78% identity over the entire length. The protein sequence conservation between TMyoD2 and TMyoD was calculated to be 90% within the basic/helix-loop-helix domain that is involved in DNA binding and heterooligomerisation. At the nucleotide level, comparison of TMyoD with TMyoD2 reveals that the translated regions are flanked by highly divergent 3' and 5' ends. The substantial differences observed at translated and especially untranslated regions strongly suggest that TMyoD and TMyoD2 mRNA originate from different loci. The TMyoD and TMyoD2 mRNA are likely transcribed from two distinct genes which were duplicated during the tetraploïdization of the salmonid genome.

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 musculature 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.

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.

Basement membranes contain three major components (ie collagen IV, laminin, and the heparan sulfate proteoglycan termed perlecan). Although the distribution and origin of both collagen IV and laminin have been well documented in the liver, perlecan has been poorly investigated, so far. We have studied the distribution and cellular origin of perlecan in rat livers in various conditions as well as in hepatocyte primary culture. By immunolocalization in both adult and 18-day-old fetal liver, perlecan was found in portal spaces, around central veins, and throughout the lobule. Immunoelectron microscopy revealed its presence at the level of basement membranes surrounding bile ducts and blood vessels, and in the space of Disse discontinuously interacting with hepatocyte microvilli. Precursors of perlecan were detected in the rough endoplasmic reticulum of bile duct cells and both vascular and sinusoidal endothelial cells. Both hepatocytes and Ito cells were negative. Northern-blot analysis confirmed the lack of appreciable expression of perlecan in hepatocytes isolated from either fetal or adult livers. In 18-month-diethylnitrosamine-treated rat liver, perlecan was abundant in neoplastic nodules. Electron microscopic investigation revealed an almost continuous layer of perlecan in the space of Disse and intracellular staining in sinusoidal endothelial cells, only. Perlecan mRNAs were detectable in malignant nodules, and absent in hepatocytes from nontumorous areas. Hepatocytes expressed high levels of perlecan mRNAs only when put in culture. This expression was reduced in conditions that allow improvement of hepatocyte survival and function (ie addition of corticoids, dimethylsulfoxide or nicotinamide to the medium, or in coculture with liver epithelial cells from biliary origin). Immunolocalization by light and electron microscopy showed that deposition of the proteoglycan occurred in coculture, in basement membranelike structures located around hepatocyte cords. In vitro attachment assay of hepatocytes on purified perlecan substrate indicated that these cells may interact with the proteoglycan through integrins which belong to the beta 1 family. These data suggest that deposition of perlecan in the space of Disse requires cellular cooperation. This article on perlecan, the third major component of hepatic basement membranes, shows a unique cellular origin in the liver and, as found for both collagen IV and laminin, an expression in adult hepatocytes when place in culture.