Mature adipocytes inhibit differentiation of myogenic cells but stimulate proliferation of fibro-adipogenic precursors derived from trout muscle in vitro.

Interactions between tissues and cell types, mediated by cytokines or direct cell-cell exchanges, regulate growth. To determine whether mature adipocytes influence the in vitro growth of trout mononucleated muscle cells, we developed an indirect coculture system, and showed that adipocytes (5 × 106 cells/well) derived from perivisceral adipose tissue increased the proliferation (BrdU-positive cells) of the mononucleated muscle cells (26% vs. 39%; p < 0.001) while inhibiting myogenic differentiation (myosin+) (25% vs. 15%; p < 0.001). Similar effects were obtained with subcutaneous adipose tissue-derived adipocytes, although requiring more adipocytes (3 × 107 cells/well vs. 5 × 106 cells/well). Conditioned media recapitulated these effects, stimulating proliferation (31% vs. 39%; p < 0.001) and inhibiting myogenic differentiation (32 vs. 23%; p < 0.001). Adipocytes began to reduce differentiation after 24 h, whereas proliferation stimulation was observed after 48 h. While adipocytes did not change pax7+ and myoD1/2+ percentages, they reduced myogenin+ cells showing inhibition from early differentiation stage. Finally, adipocytes increased BrdU+ cells in the Pdgfrα+ population but not in the myoD+ one. Collectively, our results demonstrate that trout adipocytes promote fibro-adipocyte precursor proliferation while inhibiting myogenic cells differentiation in vitro, suggesting the key role of adipose tissue in regulating fish muscle growth.

Dynamics of pax7 expression during development, muscle regeneration, and in vitro differentiation of satellite cells in rainbow trout (Oncorhynchus mykiss).

Essential for muscle fiber formation and hypertrophy, muscle stem cells, also called satellite cells, reside beneath the basal lamina of the muscle fiber. Satellite cells have been commonly identified by the expression of the Paired box 7 (Pax7) due to its specificity and the availability of antibodies in tetrapods. In fish, the identification of satellite cells remains difficult due to the lack of specific antibodies in most species. Based on the development of a highly sensitive in situ hybridization (RNAScope®) for pax7, we showed that pax7+ cells were detected in the undifferentiated myogenic epithelium corresponding to the dermomyotome at day 14 post-fertilization in rainbow trout. Then, from day 24, pax7+ cells gradually migrated into the deep myotome and were localized along the muscle fibers and reach their niche in satellite position of the fibres after hatching. Our results showed that 18 days after muscle injury, a large number of pax7+ cells accumulated at the wound site compared to the uninjured area. During the in vitro differentiation of satellite cells, the percentage of pax7+ cells decreased from 44% to 18% on day 7, and some differentiated cells still expressed pax7. Taken together, these results show the dynamic expression of pax7 genes and the follow-up of these muscle stem cells during the different situations of muscle fiber formation in trout.

Recent advances in the crosstalk between adipose, muscle and bone tissues in fish.

Control of tissue metabolism and growth involves interactions between organs, tissues, and cell types, mediated by cytokines or direct communication through cellular exchanges. Indeed, over the past decades, many peptides produced by adipose tissue, skeletal muscle and bone named adipokines, myokines and osteokines respectively, have been identified in mammals playing key roles in organ/tissue development and function. Some of them are released into the circulation acting as classical hormones, but they can also act locally showing autocrine/paracrine effects. In recent years, some of these cytokines have been identified in fish models of biomedical or agronomic interest. In this review, we will present their state of the art focusing on local actions and inter-tissue effects. Adipokines reported in fish adipocytes include adiponectin and leptin among others. We will focus on their structure characteristics, gene expression, receptors, and effects, in the adipose tissue itself, mainly regulating cell differentiation and metabolism, but in muscle and bone as target tissues too. Moreover, lipid metabolites, named lipokines, can also act as signaling molecules regulating metabolic homeostasis. Regarding myokines, the best documented in fish are myostatin and the insulin-like growth factors. This review summarizes their characteristics at a molecular level, and describes both, autocrine effects and interactions with adipose tissue and bone. Nonetheless, our understanding of the functions and mechanisms of action of many of these cytokines is still largely incomplete in fish, especially concerning osteokines (i.e., osteocalcin), whose potential cross talking roles remain to be elucidated. Furthermore, by using selective breeding or genetic tools, the formation of a specific tissue can be altered, highlighting the consequences on other tissues, and allowing the identification of communication signals. The specific effects of identified cytokines validated through in vitro models or in vivo trials will be described. Moreover, future scientific fronts (i.e., exosomes) and tools (i.e., co-cultures, organoids) for a better understanding of inter-organ crosstalk in fish will also be presented. As a final consideration, further identification of molecules involved in inter-tissue communication will open new avenues of knowledge in the control of fish homeostasis, as well as possible strategies to be applied in aquaculture or biomedicine.

Myomaker and Myomixer Characterization in Gilthead Sea Bream under Different Myogenesis Conditions.

Skeletal muscle is formed by multinucleated myofibers originated by waves of hyperplasia and hypertrophy during myogenesis. Tissue damage triggers a regeneration process including new myogenesis and muscular remodeling. During myogenesis, the fusion of myoblasts is a key step that requires different genes' expression, including the fusogens myomaker and myomixer. The present work aimed to characterize these proteins in gilthead sea bream and their possible role in in vitro myogenesis, at different fish ages and during muscle regeneration after induced tissue injury. Myomaker is a transmembrane protein highly conserved among vertebrates, whereas Myomixer is a micropeptide that is moderately conserved. myomaker expression is restricted to skeletal muscle, while the expression of myomixer is more ubiquitous. In primary myocytes culture, myomaker and myomixer expression peaked at day 6 and day 8, respectively. During regeneration, the expression of both fusogens and all the myogenic regulatory factors showed a peak after 16 days post-injury. Moreover, myomaker and myomixer were present at different ages, but in fingerlings there were significantly higher transcript levels than in juveniles or adult fish. Overall, Myomaker and Myomixer are valuable markers of muscle growth that together with other regulatory molecules can provide a deeper understanding of myogenesis regulation in fish.

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

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

Autophagy in farm animals: current knowledge and future challenges.

Autophagy (a process of cellular self-eating) is a conserved cellular degradative process that plays important roles in maintaining homeostasis and preventing nutritional, metabolic, and infection-mediated stresses. Surprisingly, little attention has been paid to the role of this cellular function in species of agronomical interest, and the details of how autophagy functions in the development of phenotypes of agricultural interest remain largely unexplored. Here, we first provide a brief description of the main mechanisms involved in autophagy, then review our current knowledge regarding autophagy in species of agronomical interest, with particular attention to physiological functions supporting livestock animal production, and finally assess the potential of translating the acquired knowledge to improve animal development, growth and health in the context of growing social, economic and environmental challenges for agriculture.Abbreviations: AKT: AKT serine/threonine kinase; AMPK: AMP-activated protein kinase; ASC: adipose-derived stem cells; ATG: autophagy-related; BECN1: beclin 1; BNIP3: BCL2 interacting protein 3; BVDV: bovine viral diarrhea virus; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CMA: chaperone-mediated autophagy; CTSB: cathepsin B; CTSD: cathepsin D; DAP: Death-Associated Protein; ER: endoplasmic reticulum; GFP: green fluorescent protein; Gln: Glutamine; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; IF: immunofluorescence; IVP: in vitro produced; LAMP2A: lysosomal associated membrane protein 2A; LMS: lysosomal membrane stability; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MDBK: Madin-Darby bovine kidney; MSC: mesenchymal stem cells; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NBR1: NBR1 autophagy cargo receptor; NDV: Newcastle disease virus; NECTIN4: nectin cell adhesion molecule 4; NOD1: nucleotide-binding oligomerization domain 1; OCD: osteochondritis dissecans; OEC: oviduct epithelial cells; OPTN: optineurin; PI3K: phosphoinositide-3-kinase; PPRV: peste des petits ruminants virus; RHDV: rabbit hemorrhagic disease virus; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy.

Trout myomaker contains 14 minisatellites and two sequence extensions but retains fusogenic function.

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.

Naa15 knockdown enhances c2c12 myoblast fusion and induces defects in zebrafish myotome morphogenesis.

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.

Histological, transcriptomic and in vitro analysis reveal an intrinsic activated state of myogenic precursors in hyperplasic muscle of trout.

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.

Distribution of H3K27me3, H3K9me3, and H3K4me3 along autophagy-related genes highly expressed in starved zebrafish myotubes.

The zebrafish (Danio rerio) remains the teleost fish of choice for biological investigations due to the vast array of molecular tools and resources available. To better understand the epigenetic regulation of autophagy, we utilized a primary myotube culture system generated from isolated myogenic precursor cells (MPCs) from zebrafish grown under starvation conditions using a media devoid of serum and amino acids. Here, we report starvation-induced regulation of several autophagy-related genes (atg) expression and profile the distribution of H3K27me3, H3K9me3, and H3K4me3 marks along lc3b, atg4b and p62/sqstm1 loci. These data support epigenetic regulation of autophagy in response to starvation that suggests a level of regulation that can be sustained for chronic conditions via chromatin modification.

miR-210 expression is associated with methionine-induced differentiation of trout satellite cells.

In fish, data on microRNAs (miRNAs) involved in myogenesis are scarce. In order to identify miRNAs involved in satellite cell differentiation, we used a methionine depletion/replenishment protocol to synchronize myogenic cell differentiation. Our results validated that methionine removal (72 h) from the medium strongly decreased myoD1 and myogenin expression, indicating differentiation arrest. In contrast, methionine replenishment rescued expression of myoD1 and myogenin, showing a resumption of differentiation. We performed a miRNA array analysis of myogenic cells under three conditions: presence of methionine for 72 h (control), absence of methionine for 72 h (Meth-) and absence of methionine for 48 h followed by 24 h of methionine replenishment (Meth-/+). A clustering analysis identified three clusters: cluster I corresponds to miRNA upregulated only in Meth-/+ conditions; cluster II corresponds to miRNA downregulated only in Meth-/+ conditions; cluster III corresponds to miRNAs with high expression in control, low expression in Meth- conditions and intermediate expression after methionine replenishment (Meth-/+). Cluster III was very interesting because it fitted with the data obtained for myoD1 and myogenin (supporting an involvement in differentiation) and contained seven miRNAs with muscle-related function (e.g. miR-133a) and one (miR-210) with unknown function. Based on our previously published miRNA repertoire ( Juanchich et al., 2016), we confirmed miR-133a was expressed only in white muscle and showed that miR-210 had strong expression in white muscle. We also showed that miR-210 expression was upregulated during differentiation of satellite cells, suggesting that miR-210 was potentially involved in the differentiation of satellite cells.

Gene expression profile during proliferation and differentiation of rainbow trout adipocyte precursor cells.

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.

Gene expression profiling of trout regenerating muscle reveals common transcriptional signatures with hyperplastic growth zones of the post-embryonic myotome.

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.

Characterization of an extensive rainbow trout miRNA transcriptome by next generation sequencing.

BACKGROUND: MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of gene expression in a wide variety of physiological processes. They can control both temporal and spatial gene expression and are believed to regulate 30 to 70% of the genes. Data are however limited for fish species, with only 9 out of the 30,000 fish species present in miRBase. The aim of the current study was to discover and characterize rainbow trout (Oncorhynchus mykiss) miRNAs in a large number of tissues using next-generation sequencing in order to provide an extensive repertoire of rainbow trout miRNAs. RESULTS: A total of 38 different samples corresponding to 16 different tissues or organs were individually sequenced and analyzed independently in order to identify a large number of miRNAs with high confidence. This led to the identification of 2946 miRNA loci in the rainbow trout genome, including 445 already known miRNAs. Differential expression analysis was performed in order to identify miRNAs exhibiting specific or preferential expression among the 16 analyzed tissues. In most cases, miRNAs exhibit a specific pattern of expression in only a few tissues. The expression data from sRNA sequencing were confirmed by RT-qPCR. In addition, novel miRNAs are described in rainbow trout that had not been previously reported in other species. CONCLUSION: This study represents the first characterization of rainbow trout miRNA transcriptome from a wide variety of tissue and sets an extensive repertoire of rainbow trout miRNAs. It provides a starting point for future studies aimed at understanding the roles of miRNAs in major physiological process such as growth, reproduction or adaptation to stress. These rainbow trout miRNAs repertoire provide a novel resource to advance genomic research in salmonid species.

Dynamic expression of tgf-ß2, tgf-ß3 and inhibin ßA during muscle growth resumption and satellite cell differentiation in rainbow trout (Oncorhynchus mykiss).

Members of the TGF-ß superfamily are involved in numerous cell functions; however, except for myostatin, their roles in the regulation of muscle growth in fish are completely unknown. We measured tgf-ß1, tgf-ß2, tgf-ß3, inhibin ßA (inh) and follistatin (fst) gene expression during muscle growth recovery following a fasting period. We observed that tgf-ß1a and tgf-ß2 expression were quickly down-regulated after refeeding and that tgf-ß3 reached its highest level of expression 7days post-refeeding, mirroring myogenin expression. Inh ßA1 mRNA levels decreased sharply after refeeding, in contrast to fst b2 expression, which peaked at day 2. No significant modification of expression was observed for tgf-ß1a, tgf-ß1b, tgf-ß1c and tgf-ß6 during refeeding. In vitro, tgf-ß2 and inh ßA1 expression decreased during the differentiation of satellite cells, whereas tgf-ß3 expression increased following the same pattern as myogenin. Surprisingly, fst b1 and fst b2 expression decreased during differentiation, whereas no variation was observed in fst a1 and fst a2 expression levels. In vitro analyses also indicated that IGF1 treatment up-regulated tgf-ß3, inh ßA1 and myogenin expression, and that MSTN treatment increased fst b1 and fst b2 expression. In conclusion, we showed that the expression of tgf-ß2, tgf-ß3 and inh ßA1 is dynamically regulated during muscle growth resumption and satellite cell differentiation, strongly suggesting that these genes have a role in the regulation of muscle growth.

Evolutionary history and epigenetic regulation of the three paralogous pax7 genes in rainbow trout.

The extraordinary muscle growth potential of teleost fish, particular those of the Salmoninae clade, elicits questions about the regulation of the relatively highly conserved transcription factors of the myogenic program. The pseudotetraploid nature of the salmonid genome adds another layer of regulatory complexity that must be reconciled with epigenetic data to improve our understanding of the achievement of lifelong muscle growth in these fish. We identify three paralogous pax7 genes (pax7a1, pax7a2 and pax7b) in the rainbow trout genome. During in vitro myogenesis, pax7a1 transcripts remain stable, whereas pax7a2 and pax7b mRNAs increase in abundance, similarly to myogenin mRNAs but in contrast to the expression pattern of the mammalian ortholog. We also profile the distribution of repressive H3K27me3 and H3K9me3 and permissive H3K4me3 marks during in vitro myogenesis across these loci and find that pax7a2 expression is associated with decreased H3K27 trimethylation, whereas pax7b expression is correlated with decreased H3K9me3 and H3K27me3. These data link the unique differential expression of pax7 paralogs with epigenetic histone modifications in a vertebrate species displaying growth divergent from that of mammals and highlight an important divergence in the regulatory mechanisms of pax7 expression among vertebrates. The system described here provides a more comprehensive picture of the combinatorial control mechanisms orchestrating skeletal muscle growth in a salmonid, leading to a better understanding of myogenesis in this species and across Vertebrata more generally.

Identification of TGF-ß, inhibin ßA and follistatin paralogs in the rainbow trout genome.

Since their initial discovery, TGF-ß superfamily members have been considered multifunctional growth and differentiation factors in many cell types. Various studies have clearly demonstrated the key roles of specific TGF-ß members in muscle growth, including myostatin and inhibin as well as genes, such as follistatin. By binding to TGF-ß members, follistatin prevents TGF-ß from binding to its receptors and thus neutralizes its activity. Here, we report the identification of the gene sequences of four TGF-ß isoforms and three paralogs of TGF-ß1, which we called TGF-ß1a, TGF-ß1b and TGF-ß1c, four sequences of inhibin ßA paralogs; and two sequences of follistatin paralogs from rainbow trout. A phylogenetic analysis clearly indicated the existence of four monophyletic clades, corresponding to TGF-ß1, -ß2, -ß3 and -ß6. Based on their sequence identity TGF-ß1a and -ß1c are grouped together, whereas TGF-ß1b appears more divergent even though it is grouped within the TGF-ß1 clade. Alignments and phylogenetic analyses showed that the protein sequences of TGF-ß, inhibin ßA and follistatin are extremely well conserved (>90%) relative to each other; however, their regulation and expression patterns are different. TGF-ß2 and -ß3 showed the most abundant expression in muscle and were the main TGF-ß members expressed in this tissue. Follistatin and inhibin ßA paralogs were expressed in all tissues examined but with different patterns. Our identification of multiple copies of TGF-ß, inhibin ßA and follistatin with different expression patterns suggests non-redundant functions for these paralogs in rainbow trout.

Myomaker mediates fusion of fast myocytes in zebrafish embryos.

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.

Preparation of primary myogenic precursor cell/myoblast cultures from basal vertebrate lineages.

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e. teleost fish) and full regeneration following appendage loss (i.e. urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystoma mexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream(1-4).