Project description: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 gene expression profile of hyperplastic areas of the late embryo myotome with that of adult myotomal muscle which displays only limited muscle 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 area, 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. Our microarray results were validated by in situ hybridisation of transcripts overexpressed in laser captured growth zones. 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 potentially regulate muscle regeneration in other vertebrates. To characterise the genetic basis of myotube neoformation in trout, we combined laser capture microdissection and microarray analysis to compare the gene expression profile of hyperplastic areas of the late embryo myotome (peripheral and deep domains) with that of adult myotomal muscle which displays only limited muscle hyperplasia.

Project description: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 gene expression profile of hyperplastic areas of the late embryo myotome with that of adult myotomal muscle which displays only limited muscle 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 area, 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. Our microarray results were validated by in situ hybridisation of transcripts overexpressed in laser captured growth zones. 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 potentially regulate muscle regeneration in other vertebrates.

Project description:We have constructed a rainbow trout high-density oligonucleotide microarray by using all the available tentative consensus (TC) sequences from the Rainbow Trout Gene Index database (The Computational Biology and Functional Genomics Lab., Dana Farber Cancer Institute and Harvard School of Public Health). The Rainbow Trout Gene Index integrates research data from all available international rainbow trout genomic research projects. The newly designed microarray incorporates 37,394 unique transcript-specific oligonucleotide probes, 60-mer long each. The microarray was printed according to our design by Agilent Technologies using the 4 X 44-design format and contains 1417 Agilent control spots. The performance of the new microarray platform was evaluated by analyzing gene expression associated with the rainbow trout vitellogenesis-induced muscle atrophy. These chips can be ordered from Agilent using design number 016320. This microarray is anticipated to open new avenues of research that will aid in the development of novel strategies to enhance growth efficiency and quality in salmonid species. Keywords: Development of an oligo-array for rainbow trout The performance of the new microarray platform was evaluated by analyzing transcriptome response associated with the rainbow trout vitellogenesis-induced muscle atrophy. Severe muscle deterioration accompanies the physiological responses of the energetic demands of the rainbow trout spawning/vitellogenesis. Atrophying muscle of fertile fish had 11% less extractable muscle (g/bw) and 11% less protein content compared to non-atrophying muscle of sterile fish (p<0.01). The rainbow trout was used to profile changes in gene expression of atrophying muscles. Gene expression levels were determined by comparing the amount of mRNA transcript present in the experimental sample (fertile fish) to the control (sterile fish). RNAs isolated from each experimental fish were run on separate microarrays in independent experiments, with no pooling. A total of 8 fish were used in the microarray experiments (4 replicates x 2 groups). Fluorophors (Cy3 and Cy5) were randomly assigned to RNA from each of the atrophying and nonatrophying muscles to limit the dye effect.

Project description:We investigated the effects of chronic TCDD exposure on global gene expression in developing rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout (0.18M-BM-10.01g) were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100ppb, and ten fish were sampled and pooled from each group for microarray experiments at 28 days after initiation of the exposure. Gene expression analysis was performed using the Genomics Research on All Salmonids Project (cGRASP) 16K cDNA microarrays. TCDD-responsive whole body transcripts identified in the microarray experiments have putative functions involved in various biological processes including cellular process, metabolic process, biological regulation, and response to stimulus. In addition, TCDD caused leisons in multiple organ systems in juvenile rainbow, including skin, oropharynx, liver, gas bladder, intestine, pancreas, nose and kidney. Juvenile rainbow trout (0.18M-BM-10.01g) were fed Biodiet starter (Bio-Oregon) (4% body weight per day) with TCDD added at 0, 0.1, 1, 10 and 100 ppb. Fish were sampled after 28 days. Total RNA from individual fish was isolated using TRIzol reagent (Invitrogen). Total RNA of ten control trout were pooled as a common reference which were labeled with Cy3. Total RNA of ten fish of each treatment were labeled wih Cy5. cDNA were synthesized using SuperScript II Reverse Transcriptase (Invitrogen) (1 M-NM-<g pooled RNA per synthesis). Targets were labeled using the Array 900 Expression Array Detection kit (Genisphere) following the manufacture protocol. 1M-BM-5g RNA of pooled ten control fish were labled with Cy3 and 1M-BM-5g RNA of pooled ten TCDD-treated fish were labled with Cy5, then day-swap was performed. Two arrays were run for each comparison. Microarrays were scanned at 10 M-BM-5m resolution using a ScanArray Express (PerkinElmer Life Sciences, Inc, Boston, MA). The photomultiplier tube settings (PMT) for Cy3 and Cy5 were 70 and 65-66, respectively. TIFF images of arrays were generated with ScanArray Express software (PerkinElmer). Fluorescence intensity data for Cy3 and Cy5 channels were extracted from TIFF images using ImaGene 7.5 software (BioDiscovery Inc., El Segundo, CA). Background correction, data transformation (setting background corrected values < 0.01 to 0.01), Lowess normalization, and analysis (e.g. fold-change calculations) were performed using GeneSpring GX 7.3 (Agilent Technologies, Palo Alto, CA)

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

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