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

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

Evolution of haploid–diploid life cycles when haploid and diploid fitnesses are not equal

Many organisms spend a significant portion of their life cycle as haploids and as diploids (a haploid–diploid life cycle). However, the evolutionary processes that could maintain this sort of life cycle are unclear. Most previous models of ploidy evolution have assumed that the fitness effects of new mutations are equal in haploids and homozygous diploids, however, this equivalency is not supported by empirical data. With different mutational effects, the overall (intrinsic) fitness of a haploid would not be equal to that of a diploid after a series of substitution events. Intrinsic fitness differences between haploids and diploids can also arise directly, for example because diploids tend to have larger cell sizes than haploids. Here, we incorporate intrinsic fitness differences into genetic models for the evolution of time spent in the haploid versus diploid phases, in which ploidy affects whether new mutations are masked. Life-cycle evolution can be affected by intrinsic fitness differences between phases, the masking of mutations, or a combination of both. We find parameter ranges where these two selective forces act and show that the balance between them can favor convergence on a haploid–diploid life cycle, which is not observed in the absence of intrinsic fitness differences

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

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

What determines growth potential and juvenile quality of farmed fish species?

Enhanced production of high quality and healthy fry is a key target for a successful and competitive expansion of the aquaculture industry. Although large quantities of fish larvae are produced, survival rates are often low or highly variable and growth potential is in most cases not fully exploited, indicating significant gaps in our knowledge concerning optimal nutritional and culture conditions. Understanding the mechanisms that control early development and muscle growth are critical for the identification of time windows in development that introduce growth variation, and improve the viability and quality of juveniles. This literature review of the current state of knowledge aims to provide a framework for a better understanding of fish skeletal muscle ontogeny, and its impact on larval and juvenile quality as broadly defined. It focuses on fundamental biological knowledge relevant to larval phenotype and quality and, in particular, on the factors affecting the development of skeletal muscle. It also discusses the available methodologies to assess growth and larvae/juvenile quality, identifies gaps in knowledge and suggests future research directions. The focus is primarily on the major farmed non-salmonid fish species in Europe that include gilthead sea bream, European sea bass, turbot, Atlantic cod, Senegalese sole and Atlantic halibut

Bioeconomic Modelling Applied to Fisheries with R/FLR/FLBEIA

The main objectives of the study presented in this report were to test the FLBEIA API, condition an operating model for the North Sea mixed fisheries and provide feedback on bioeconomic modelling limitations. Additionally, Fishrent and Fcube were also tested. FLR, FLBEIA, Fishrent and Fcube are software packages implemented by the scientific community studying fisheries to run bioeconomic models. A large test was carried out on FLBEIA by both running existing examples and trying to implement a bioeconomic model for the North Sea. In general the group felt FLBEIA is on the correct path to provide a bioeconomic modeling framework, although some work is still required. FLBEIA is not ready yet for production. A list of bugs and improvements was assembled. Conditioning a bioeconomic operating model for the North Sea showed the difficulties of merging economic and biological information. Inconsistencies on the effort definition seem to create additional problems when relating both sources of information. This subject must be further explored. The exercise was successful but data problems prevented the performance of a full economic analysis, although trend analysis on economic indicators for each scenario tested was possible. Nevertheless, these results must be taken carefully.JRC.G.4-Maritime affair

Scientific, Technical and Economic Committee for Fisheries (STECF)-Evaluation of Fishing Effort Regimes in European Waters Part 1 (STECF-12-09)

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Modelling the growth of popliteal artery aneurysms

Background: Popliteal artery aneurysms (PAAs) comprise up to 85 per cent of all peripheral aneurysms. Few longitudinal studies track their progression. This study aimed to track the growth of asymptomatic PAAs in a hospital‐based ultrasound service, and compare models of aneurysm growth. Methods: This retrospective single‐centre cohort study included patients who had a PAA on arterial duplex ultrasound imaging of the lower limbs between 1 January 2011 and 1 January 2016. Progression of PAA size and progression to event or intervention were the primary outcome measures. Results: Some 282 images were analysed: 47 limbs with PAA were included in a cohort of 32 patients (15 had bilateral PAAs). Twenty patients also had an abdominal aortic aneurysm (AAA). Linear multilevel modelling estimated that PAA growth was 2·4 (95 per cent c.i. 1·6 to 3·7) mm a year. Growth was estimated at 0·8 (0·1 to 1·5) mm per year in patients without an AAA and 3·5 (2·9 to 4·2) mm per year in those with a known AAA (previous open repair, previous endovascular aneurysm repair or AAA under surveillance) (P < 0·001). Conclusion: Growth rates of PAA were heterogeneous but were optimally predicted by multilevel modelling. Patients with an existing AAA may have faster PAA progression than those without

Dietary antioxidant supplementation promotes growth in senegalese sole postlarvae

Somatic growth is a balance between protein synthesis and degradation, and it is largely influenced by nutritional clues. Antioxidants levels play a key role in protein turnover by reducing the oxidative damage in the skeletal muscle, and hence promoting growth performance in the long-term. In the present study, Senegalese sole postlarvae (45 days after hatching, DAH) were fed with three experimental diets, a control (CTRL) and two supplemented with natural antioxidants: curcumin (CC) and grape seed (GS). Trial spanned for 25 days and growth performance, muscle cellularity and the expression of muscle growth related genes were assessed at the end of the experiment (70 DAH). The diets CC and GS significantly improved growth performance of fish compared to the CTRL diet. This enhanced growth was associated with larger muscle cross sectional area, with fish fed CC being significantly different from those fed the CTRL. Sole fed the CC diet had the highest number of muscle fibers, indicating that this diet promoted muscle hyperplastic growth. Although the mean fiber diameter did not differ significantly amongst treatments, the proportion of large-sized fibers (>25 μm) was also higher in fish fed the CC diet suggesting increased hypertrophic growth. Such differences in the phenotype were associated with a significant up-regulation of the myogenic differentiation 2 (myod2) and the myomaker (mymk) transcripts involved in myocyte differentiation and fusion, respectively, during larval development. The inclusion of grape seed extract (GS diet) resulted in a significant increase in the expression of myostatin1. These results demonstrate that both diets (CC and GS) can positively modulate muscle development and promote growth in sole postlarvae. This effect is more prominent in CC fed fish, where increased hyperplastic and hypertrophic growth of the muscle was associated with an upregulation of myod2 and mymk genes.FCT: IF/00482/2014/CP1217/CT0005; UIDB/04326/2020; VALORMAR (ref. 024517) through Compete 2020, Lisboa 2020, CRESC Algarve 2020, Portugal 2020 and the European Union’s ERDF, and IFAPA project RTA2017-00054-C03-01 funded from MCIU/AEI/FEDER, UE.info:eu-repo/semantics/publishedVersio

Analysis artefacts of the INS-IGF2 fusion transcript

BACKGROUND: In gene expression analysis, overlapping genes, splice variants, and fusion transcripts are potential sources of data analysis artefacts, depending on how the observed intensity is assigned to one, or more genes. We here exemplify this by an in-depth analysis of the INS-IGF2 fusion transcript, which has recently been reported to be among the highest expressed transcripts in human pancreatic beta cells and its protein indicated as a novel autoantigen in Type 1 Diabetes. RESULTS: Through RNA sequencing and variant specific qPCR analyses we demonstrate that the true abundance of INS-IGF2 is >20,000 fold lower than INS in human beta cells, and we suggest an explanation to the nature of the artefacts which have previously led to overestimation of the gene expression level in selected studies. We reinvestigated the previous reported findings of detection of INS-IGF2 using antibodies both in Western blotting and immunohistochemistry. We found that the one available commercial antibody (BO1P) raised against recombinant INS-IGF2 show strong cross-reaction to native proinsulin, and we did not detect INS-IGF2 protein in the human beta cell line EndoC-βH1. Furthermore, using highly sensitive proteomics analysis we could not demonstrate INS-IGF2 protein in samples of human islets nor in EndoC-βH1. CONCLUSIONS: Sequence features, such as fusion transcripts spanning multiple genes can lead to unexpected results in gene expression analysis, and care must be taken in generating and interpreting the results. For the specific case of INS-IGF2 we conclude that the abundance of the fusion transcript/protein is exceedingly lower than previously reported, and that current immuno-reagents available for detecting INS-IGF2 protein have a strong cross-reaction to native human proinsulin. Finally, we were unable to detect INS-IGF2 protein by proteomics analysis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12867-015-0042-8) contains supplementary material, which is available to authorized users

An Update on MyoD Evolution in Teleosts and a Proposed Consensus Nomenclature to Accommodate the Tetraploidization of Different Vertebrate Genomes

DJM was supported by a Natural Environment Research Council studentship (NERC/S/A/2004/12435).Background: MyoD is a muscle specific transcription factor that is essential for vertebrate myogenesis. In several teleost species, including representatives of the Salmonidae and Acanthopterygii, but not zebrafish, two or more MyoD paralogues are conserved that are thought to have arisen from distinct, possibly lineage-specific duplication events. Additionally, two MyoD paralogues have been characterised in the allotetraploid frog, Xenopus laevis. This has lead to a confusing nomenclature since MyoD paralogues have been named outside of an appropriate phylogenetic framework. Methods and Principal Findings: Here we initially show that directly depicting the evolutionary relationships of teleost MyoD orthologues and paralogues is hindered by the asymmetric evolutionary rate of Acanthopterygian MyoD2 relative to other MyoD proteins. Thus our aim was to confidently position the event from which teleost paralogues arose in different lineages by a comparative investigation of genes neighbouring myod across the vertebrates. To this end, we show that genes on the single myod-containing chromosome of mammals and birds are retained in both zebrafish and Acanthopterygian teleosts in a striking pattern of double conserved synteny. Further, phylogenetic reconstruction of these neighbouring genes using Bayesian and maximum likelihood methods supported a common origin for teleost paralogues following the split of the Actinopterygii and Sarcopterygii. Conclusion: Our results strongly suggest that myod was duplicated during the basal teleost whole genome duplication event, but was subsequently lost in the Ostariophysi ( zebrafish) and Protacanthopterygii lineages. We propose a sensible consensus nomenclature for vertebrate myod genes that accommodates polyploidization events in teleost and tetrapod lineages and is justified from a phylogenetic perspective.Publisher PDFPeer reviewe