BACKGROUND
the growth hormone  axis in coho salmon  and other teleosts is normally tightly coupled to energy intake and is modulated by a large number of environmental factors  <cit> .
the normal feedback control systems are essentially disabled in growth hormone-transgenics due to the extra-pituitary expression of gh in other tissues  <cit> .
high constitutive expression of gh results in increased aggressiveness, appetite and foraging activity  <cit>  leading to continuous fast growth when fish are fed to satiation  <cit> .
gh stimulates insulin-like growth factor- <dig>  secretion in the liver.
circulating igfs bind to igf receptors and activate the signalling cascades that regulate protein synthesis.
plasma igf <dig> and amino acids from the diet further stimulate igf synthesis in muscle and other tissues via paracrine action  <cit> .
gh-transgenics exhibit increased plasma insulin-like growth factor- <dig> , tissue igf <dig> mrna and gh-receptor  levels relative to wild-type fish   <cit> .
feeding coho salmon gh-transgenics below appetite  with a similar ration size as wild-type fish  results in an uncoupling of the growth hormone and nutritional signals regulating growth.
tr fish grow more slowly and show lower circulating igf <dig> than gh-transgenic fish fed to satiation  , which indicates the nutritional regulation of igf <dig> expression remains functional at the target tissue level  <cit> .
tr animals showed higher circulating gh  levels and unchanged ghr mrna relative to gh-transgenics fed to satiation  , but retained higher feeding motivation and foraging activity than wt  <cit> .
fast skeletal muscle fibres are formed in teleost fish immediately following somite formation and then in the juvenile/adult stages by processes of stratified and mosaic hyperplasia   <cit> .
myoblast-myoblast fusion results in the formation of a nascent myotube which elongates and gains additional nuclei through myoblast-myotube fusion.
activation of terminal differentiation and sarcomere formation then results in the production of an immature muscle fibre.
typically, myotube production in fast muscle continues until the fish reaches around 40- <dig> % of their maximum attainable adult body length  <cit> .
hill et al.
<cit>  provided indirect evidence that gh-transgenic coho salmon fed to satiation showed enhanced muscle growth by hyperplasia.
in a recent study we tested this hypothesis by measuring the number and size distribution of fast myotomal muscle fibres in three groups of coho salmon of similar body length: 1-year old gh-transgenics fed to satiation , 2-year old restriction-fed gh-transgenics  and wild type   <cit> .
tf coho salmon recruited fast muscle fibres at twice the rate as wt fish, but showed a similar contribution of hyperplasia and hypertrophy to reach a given body length i.e.
the hypothesis of an increased importance of hyperplasia in transgenics was not supported.
unexpectedly, tr recruited  <dig> % fewer fibres with a  <dig> % higher fibre diameter than either wt or tf fish and had larger diameter fibres across the whole range of fibre sizes, i.e.
increased hypertrophy was evident for all cohorts of fibres produced during ontogeny  <cit> .
there is direct experimental evidence from inhibitor and nuclear magnetic resonance  studies that larger diameter muscle fibres have lower costs of ionic homeostasis than smaller ones due to their lower surface to volume  ratio  <cit> .
thus the ~ <dig> % reduction in fibre s/v ratio in tr relative to wt fish would be expected to produce proportional savings in routine maintenance costs  <cit> .
previously we suggested that adjustments in fibre size might permit the reallocation of energy from maintenance to locomotion which would help explain why calorie-restricted transgenics grow at the same rate as wt fish whilst exhibiting markedly higher activity levels  <cit> .
preliminary, studies indicated that several genes associated with myotube formation were downregulated in tr relative to wt fish  <cit> .
in order to gain a broader understanding of fast skeletal muscle gene regulation in tr compare to wt fish we have now carried out rna-seq in  <dig> tr and  <dig> wt individual coho salmon for which we had associated data on the number and size of fast muscle fibres  <cit> , and validated the results by qpcr analysis.
rna was sequenced using illumina hiseq <dig> and mapped reads  were used to study global differences in digital gene expression  between groups.
following enriched gene ontology  analysis we tested specific hypotheses about the effects of uncoupling growth hormone and nutrient signals by analysing the expression of genes related to growth hormone signalling, protein translation, protein-folding and myoblast fusion.
RESULTS
transcriptome analysis
total rna from tr and wt fish  with known differences in muscle cellularity  <cit>  were sequenced in  <dig> lines of illumina hiseq <dig> .
a total of  <dig> , <dig> paired-end reads were de novo assembled into  <dig>  contigs with average length > <dig>  bp and n50 >  <dig>  bp .
using blastx against the ncbi non-redundant  database we successfully identified a total of  <dig>  contigs and, from them, a total of  <dig>  were also annotated with gene ontology  terms .
a total of  <dig>  unique genes were identified in the fully annotated transcriptome .
the teleost linage underwent a whole-genome duplication  relative to the last common ancestor with humans around 350-320mya  <cit> , increasing gene content by 15- <dig> %  <cit> .
the salmonids experienced a fourth lineage-specific wgd  around 88mya, and approximately  <dig> % of the resulting paralogues are still present in extant species  <cit> .
several examples of gene expansion due to the salmonid wgd have been described in detail  <cit> .
in order to estimate the representation of 4r paralogues in the transcriptome  <dig> well-characterized atlantic salmon  paralogues gene families were blasted  against the coho salmon de novo transcriptome.
seventeen out of  <dig> paralogues were unequivocally identified  and three more appeared with very low e-values , though not all can be expected to be expressed in skeletal muscle.
<dig>  protein-coding genes have been reported for the human genome of which  <dig>  are expressed in the muscle transcriptome  <cit> .
the  <dig> % higher gene content of the coho skeletal muscle transcriptome  than in humans likely reflects this history of the succesive wgd.
furthermore, the continuous expression of gh might also increase the number of genes expressed at detectable levels in the muscle.table  <dig> sequencing and de novo trinity assembly metrics
<dig> ,759
<dig> ,226
<dig> ,210
<dig> ,286
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
<dig>
309
tr: restriction-fed growth hormone-transgenic coho salmon; wt: wild-type coho salmon; n50: average length that contains at least half of the sum of the lengths for all contigs; bp: base pair; cds: coding sequence; kaas: keeg automatic annotation service
igfbp1a1
igfbp1a2
igfbp1b1
igfbp1b2
igfbp2a
igfbp2b1
igfbp2b2
igfbp3a1
igfbp3a2
igfbp3b1
igfbp3b2
igfbp4
igfbp5a
igfbp5b1
igfbp5b2
igfbp6a1
igfbp6a2
igfbp6b1
igfbp 6b2
akirin 1
akirin 1
akirin 1
akirin 1
akirin 2
akirin 2
akirin 2
akirin 2
hsp90aa <dig>
hsp90aa <dig>
hsp90aa <dig>
hsp90aa <dig>
hsp90b1
hsp90b2
aqp8
aqp8aa1
aqp8ab
aqp8b
myod1
myod2
myo1c
id: percentage of identity at amino acid level between s.salar and o.kitsuch orthologues
alignment: total number of amino acids successfully aligned during the tblastn between s.salar and o.kitsuch orthologues
n/a: paralogues not identified in the transcriptome
annotated contigs were blasted against the kegg database using the online web-server kaas to map them against the main metabolic and signaling pathways, yielding a total of  <dig> kegg maps where coho salmon contigs were present .
comparative analysis between annotated contigs against the annotated zebrafish proteome  allowed us to identify  <dig>  genes estimated to contain over  <dig> % of the predicted coding sequence  .
we also found that  <dig> contigs were over > <dig> % of the cds, indicating that some coho salmon genes were between 1- <dig> % longer than their zebrafish orthologues .
digital gene expression
paired-end reads from individual fish libraries were mapped against the de novo transcriptome .
mapped reads were normalized by contig length and library depth following a negative binomial using deseq and used for dge analysis.
global dge comparison between tr and wt yielded an initial list of  <dig> contigs that were differentially expressed .
contigs with < <dig> normalized reads mapped and a fold-change <2log <dig> were discarded.
redundant contigs for all genes from the initial global dge list were identified in the annotated transcriptome and their deseq-counts values were individually investigated.
those genes for which all-redundant “sister” contigs were found to show consistent, but not necessarily significant, changes in expression between groups were retained .
care was taken to identify 4r and 3r-paralogues that might be annotated with the same id by exploring the alignment of the sister contigs.
after curation for redundancy and quality control, a total of  <dig> genes from tr and  <dig> from wt were considered to be differentially expressed .
rnaseq has many advantages, but it does present challenges with respect to statistical analysis and interpretation.
firstly, transcription is not tightly coupled to translation in eukaryotes, complicating inferences about the functional significance of changes in transcript abundance.
secondly, the activity of signalling pathways is often dependent on posttranslational modifications such as phosphorylation and/or changes in compartmentalisation within the cell.
in addition, functional interpretations based on transcript abundance cannot distinguish between mrna transcription vs degradation rates.
it is also easy to fall into the trap of providing a series of post hoc stories when reporting transcriptomic data rather than testing a priori hypotheses.
standard corrections for multiple testing coupled with long gene lists also leads to a very high barrier for establishing statistical significance, leading to type- <dig> statistical errors.
we therefore used a multistep process to analyse the dge results from tr and wt fish fed the same ration.
firstly, enriched gene ontology  analysis was used to identify initial categories of those genes obtained from the global analysis for further investigation .
for the tr fish, go analysis revealed an enrichment in genes related to “macromolecule metabolic process” , “rna metabolic process” , “regulation of translation” , “translation factor activity, nucleic acid binding”  or “rna binding”  .
wt animals had a higher expression of genes related to “autophagy” , “cellular metabolic process” , “catabolic process”  and “vacuole organization”  .
based on go enrichment analysis and previous works  <cit>  we decided to specifically test the expression of genes related to myoblast fusion, protein synthesis, protein degradation by the proteasome system, protein folding and also the gh-igf receptors and ligands.
kaas annotation results and a literature survey of the results of functional analyses were used to identify the genes to include.
<dig> genes were identified in total , including 4r and 3r paralogues where identified .
deseq-counts  from individual animals for each gene were then retrieved in order to test specific a priori hypotheses.table  <dig> gene ontology analysis
biological process
molecular function
cellular component
biological process
molecular function
cellular component
gene ontology analysis from genes with significant differences in expression between restriction-fed growth hormone-transgenic coho salmon  and wild-type coho salmon ; go: gene ontology; fdr: false discovery rate
fig.
<dig> maximum likelihood phylogenetic trees for two 4r paralogues.
a
.
growth hormone receptor  b
.
caveolin  <dig> .
posterior values are indicated in the nodes of each branch.
species abbreviations are as follow; s. salar: salmo salar; o. mykiss: oncorhynchus mykiss; o. kisutch: oncorhynchus kisutch; a. mexicanus: astyanax mexicanus; d. rerio: danio rerio; t.rubripes: takifugu rubripes; g. aculeatus: gasterosteus aculeatus; o. niloticus: oreochromis niloticus; h. sapiens: homo sapiens; m. musculus: mus musculus
before analysing in detail the differences in dge between genes a principal components analysis  was carried to determine if the categories selected were informative of the differences between tr and wt individuals .
components  <dig> and  <dig> generated from pca analysis explained  <dig>  % of the variability of the data and were able to distinguish tr and wt in two separated clusters .
this is an indication that the data used was informative with respect to the differences between groups, which is highly apparent in the hierarchical clustering shown in fig.
2b.fig.
<dig> principal components analysis  and hierarchical heat map cluster of all the genes manually analysed for dge.
a
.
principal components analysis of the dge from all  <dig> genes analysed to test our specific hypothesis.
in brackets is indicated the percentage of variation explained by each of the components.
circles represent individual fish from restricted diet transgenics  and wild-type  groups b
.
hierarchical heat map cluster showing individual dge values for tr  and wt  coho salmon.
rows are standardized to have a mean of  <dig> and standard deviation of 1; red indicates high and green indicates low dge values.
each rectangle represents individual dge values
growth hormone system and protein synthesis
nineteen genes associated with the growth hormone system  were identified in the transcriptome and filtered for redundancy as previously described.
in order to capture all the biological relevant changes, the requirement that differences should be higher than 2log2-fold change was relaxed but not the requirement that average deseq-counts should be > <dig>  as expected due to the transgenic nature of the coho salmon, gh <dig> gene transcripts were  <dig> -fold higher in tr compared to wt  <cit>  .
similarly, previous studies have also reported an increase in growth hormone receptor  expression in muscle  <cit> .
however, that study didn’t distinguish between ghr paralogues whereas we identified ghra.
<dig> and ghrb.
<dig>  two of the potential  <dig> salmonid paralogues for the receptor .
we found that ghra.
<dig> was  <dig> -fold higher in tr than wt fish  whereas ghrb.
<dig> showed unaltered expression, indicating differential regulation of paralogues.
igf2/igf1r and the insulin receptor  were downregulated 2- and 3-fold respectively  in tr than wt fish, most likely as part of a negative feedback response to increased growth hormone expression .fig.
<dig> gh and igf receptors and ligands.
a
.
hierarchical heat map cluster showing individual dge values for tr  and wt  coho salmons.
rows are standardized to have a mean of  <dig> and standard deviation of 1; red indicates high and green indicates low dge values.
each rectangle represents individual dge values b
.
bar graphs showing dge values from tr and wt groups  for growth hormone , growth hormone receptor a.
<dig> , insulin-like growth factor binding protein  <dig>  and growth hormone receptor b.
<dig> .
significant differences are indicated with one  or two asterisk  according to the degree of significance
the mtorc <dig> complex integrates cues from growth factors to activate ribosome biogenesis and protein synthesis via phosphorylation of ribosomal protein s <dig> kinase  <dig>  <cit> .
mtorc1-mediated phosphorylation of the eukaryotic initiation factor 4e  binding protein  induces its dissociation from eif4e and promotes cap-dependent translation  <cit> .
mtorc <dig> is also an important part of the amino acid sensing machinery in the cell comprising rags, ragulator complex and v-atpase  <cit> .
the amino acid-rag axis activates mtorc <dig> through a mechanism involving its translocation to the lysosomal surface  <cit> .
thus in the absence of sufficient levels of amino acids, the incorrect positioning of mtorc <dig> renders it insensitive to activation by growth factors  <cit> .
the present transcriptome data provides strong evidence that elevated gh levels resulted in a widespread increase in translation initiation factor mrna with  <dig> out of  <dig>  genes significantly higher expressed in tr than wt fish, including eif4e <dig> and eif4e <dig> .
it is clear from the hierarchical cluster of all  <dig> genes that  <dig> % of the genes seem to have a higher expression in tr, even if not significant, than wt with only a handful of contigs more expressed in this condition, such as eif <dig> .
in addition,  <dig> out of  <dig> protein chaperones genes  with roles in protein folding were significantly elevated in tr relative to wt fish, including many with known roles in muscle differentiation and sarcomere assembly such as unc <dig> and hsp90aa <dig> that are also regulated by nutrition   <cit> .
the majority of e3-ubiquitin ligases analysed, enzymes involved in protein degradation through the proteasome system, were more highly expressed in wt than tr  , which indicates that tr fish were not starving.
the differences in translation initiation factors, protein chaperones and e3-ubiquitin ligases expression in tr fish were not matched by a net increased protein accretion since tr and wt fish grew at the same rate  <cit> , implying some increase in the metabolic costs of transcription relative to wt.fig.
<dig> elongation factors and eukaryotic translation factors dge.
a
.
hierarchical heat map cluster showing individual dge values for tr  and wt  coho salmon.
rows are standardized to have a mean of  <dig> and standard deviation of 1; red indicates high and green indicates low dge values.
each rectangle represents individual dge values b
.
bar graphs showing dge values from tr and wt groups  for eukaryotic translation initiation factor 4e , eukaryotic translation initiation factor  <dig> , eukaryotic translation initiation factor 5b , eukaryotic translation initiation factor 3j , eukaryotic translation initiation factor 2 s <dig>  and eukaryotic translation initiation factor 1b .
significant differences are indicated with one  or two asterisk  according to the degree of significance
fig.
<dig> chaperones, co-chaperones and stress factors dge.
a
.
hierarchical heat map cluster showing individual dge values for tr  and wt  coho salmon.
rows are standardized to have a mean of  <dig> and standard deviation of 1; red indicates high and green indicates low dge values.
each rectangle represents individual dge values b
.
bar graphs showing dge values from tr and wt groups  for unc-45b homologue , cysteine and histidine-rich domain containing  <dig> , activator of heat shock 90 kda protein atpase homolog  <dig> , heat shock protein  <dig> , cytosolic heat shock protein  <dig> alpha , chaperonin containing tcp <dig> and subunit  <dig> .
significant differences are indicated with one  or two asterisk  according to the degree of significance
fig.
<dig> e3-ubiquitin ligases dge.
a
.
hierarchical heat map cluster showing individual dge values for tr  and wt  coho salmon.
rows are standardized to have a mean of  <dig> and standard deviation of 1; red indicates high and green indicates low dge values.
each rectangle represents individual dge values b
.
bar graphs showing dge values from tr and wt groups  for f-box protein  <dig> , e3-ubiquitin ligase itchya , f-box and leucine-rich repeat protein  <dig> , praja ring finger  <dig> e <dig> ubiquitin protein ligase , ubiquitin protein ligase e <dig> component n-recognin  <dig> , membrane-associated ring finger   <dig> e <dig> ubiquitin protein ligase , forkhead box k <dig>  and forkhead box n <dig> .
significant differences are indicated with one  or two asterisk  according to the degree of significance
myoblast fusion
myoblast fusion involves multiple sarcolemmal receptors, cell-surface proteins and intracellular signaling pathways leading to membrane and actin cytoskeleton remodelling  <cit> .
evidence from in vivo and in vitro studies involving mouse and zebrafish models was used to construct a list of genes required for myoblast fusion and progression.
a total of  <dig> genes including 4r-paralogues  involved in myoblast fusion and differentiation were identified in the transcriptome .
<dig> out of the  <dig> genes  from the list were differentially expressed between tr and wt groups of coho salmon  .
a total of  <dig> of the genes were significantly down regulated in ration-restricted transgenic fish concomitant with a reduction in the number of fast muscle fibres per myotomal cross-section  <cit> .
the majority  of these genes  were associated with signaling through the cell surface protein cadherin .fig.
<dig> myoblast fusion related genes dge.
a
.
hierarchical heat map cluster showing individual dge values for tr  and wt  coho salmon.
rows are standardized to have a mean of  <dig> and standard deviation of 1; red indicates high and green indicates low dge values.
each rectangle represents individual dge values b
.
bar graphs showing dge values from tr and wt groups  for dedicator of cytokinesis  <dig> , dedicator of cytokinesis  <dig> , v-crk avian sarcoma virus ct <dig> oncogene homolog , transmembrane protein 8c , pleckstrin homology domain containing, family o member  <dig> , caveolin 3a .
significant differences are indicated with one  or two asterisk  according to the degree of significance
morpholino antisense-oligonucleotide-mediated knockdown studies have shown that both dock <dig> and dock <dig> and their adaptor proteins crk, crk-like  and the pleckstrin homology domain containing family member 1a  are required for the fusion of fast-type myoblasts in zebrafish  <cit> .
members of the rho family of guanosine triphosphatases , including rac <dig>  operate downstream of dock <dig> to stimulate myoblast fusion  <cit> .
rac <dig> is also activated by m-cadherin-dependent adhesion through trio during c2c <dig> myoblast fusion  <cit> .
m-cadherin  and trio transcripts were also both significantly downregulated in tr relative to wt salmon .
on the basis of differences in the defects observed in dock1−/− and trio−/− mice it has been suggested that trio is required for myoblast-myoblast fusion, but not for myoblast-myotube fusion  <cit> .
a further three of the genes downregulated in tr fish were associated with integrins and focal adhesion kinase signaling .
also down regulated in tr relative to wt fish were sp <dig>  a component of mapk-erk <dig> signaling pathway, nfkb <dig> which is involved in the non-canonical nf-kb signaling and tmem8c or myomaker which is required for myoblast-myotube fusion   <cit> .
nine  out of the  <dig> genes on the list were significantly upregulated in tr relative to wt fish  .
integrin signaling is mediated by the non-receptor protein kinase fak promoting myoblast fusion and an associated increase in caveolin- <dig>  <cit> , transcripts of which were downregulated in tr relative to wt fish .
the differences in dge observed are consistent with a higher myoblast fusion activity in wt when compared with tr, consistent with the observed differences in fibre number  <cit> .
quantitative pcr data validation of dge
in order to validate the results obtained from dge we analysed the expression of  <dig> genes covering different ranges of expression  by quantitative pcr .
the qpcr data supports the dge estimations reported with similar differences between treatments for the individual genes .
correlation analysis was used to examine the relationship between the levels of expression for individual genes, that were found to be equivalent irrespective of whether the data were expressed as deseq-counts and average ct values  or dge and qpcr fold-change  .fig.
<dig> dge and qpcr correlations.
a. dge and qpcr correlations.
a. correlation between dge and qpcr ct values for individual genes for each treatment.
dge values are expressed as log <dig>  pearson r <dig> and p-value for the correlation are indicated in the top-left corner b. correlation between gene fold-change between tr and wt groups calculated from dge and qpcr values.
fold-change is expressed in a log <dig> scale.
pearson r <dig> and p-values for the correlation are indicated in the top-left corner.
circles with different colour than black represent the position of the 4r-paralogues plotted: cav3a/cav3b , cdc42a/cdc42b , ghra.2/ghrb.
<dig>  and vaspa/vaspb
energy budgeting in the restriction-fed transgenic model
tr fish are more aggressive and exhibit markedly higher levels routine swimming behaviour than wt fish and may have higher metabolic costs associated with the futile expression of mrnas and proteins required for growth .
in spite of these additional costs, tr fish grow at the same rate as wt fish which implies some compensating alterations to energy budgeting.
the source of these energy savings includes the cost of ionic homeostasis which has been estimate to contribute 20- <dig> % of the routine metabolic rate in teleosts  <cit> , a large part of which can be attributable to maintaining the resting membrane potential of fast skeletal muscle which comprises around  <dig> % of body mass in salmonids.
tr fish have fewer, but larger diameter fast muscle fibres than wt for a given body length  <cit> , which will result in reduced ion pumping costs  <cit> .
our working hypothesis is that the uncoupling of the gh-axis from energy status directly affects one or more of the signaling pathways regulating myotube formation and hypertrophic growth, and several candidate pathways, particularly signaling through cadherin, have been identified in the present study.
calorie restriction in wt fish may not result in changes in muscle cellularity because the metabolic signals normally influencing endogenous gh regulation remain intact, a hypothesis requiring further assessment.
methods
fish
experiments on coho salmon  were conducted in a non-commercial containment facility for transgenic fish at fisheries and oceans canada, west vancouver.
wild-type  fish were from the  <dig> brood of chehalis river strain   <cit> .
the strain of transgenic coho salmon used  was derived from chehalis river strain produced using the onmtgh <dig> construct comprised of 320 bp of sockeye salmon metallothionein-ß promoter fused to the 5′-utr region of the full-length type-i growth hormone  gene and the terminator from the same species, as previously described  <cit> .
fish were reared under standard hatchery conditions in fresh well water  with a natural photoperiod and fed commercially available salmon diets .
the  <dig> brood of gh transgenics  were fed the same ration to that of the wt group , resulting in similar growth rates in the two groups.
fish were fasted for 24 h prior to humane sacrifice and selected to produce two groups with a similar average fork length  and hence same the developmental stage.
all procedures used in the study were approved by the department of fisheries and oceans pacific region animal care committee.
wt and food-restricted growth hormone transgenic coho salmon  were obtained from the same group used by the authors in a previous study  <cit> .
no significant differences were found in weight or fork length between tr  and wt  used in the present study.
rna extraction and sequencing
sections of pure fast skeletal muscle were carefully dissected from dorsal epaxial myotomes of  <dig> animals from wt and tr groups matched for body length and with known differences in muscle fibre distribution  <cit> .
total rna was extracted by homogenization in 1 ml of trisure  using d-matrix tubes  and following the manufacturer’s recommendations.
total rna concentration, 260/ <dig> and 260/ <dig> ratios were estimated by nanodrop spectrometer n <dig> .
rna integrity was estimated by resolving 1 μg of sample in a  <dig> %  ethidium bromide agarose gel.
a total of 3 μg of rna per sample was sent to the sick-kids hospital next generation sequencing service  for sequencing.
individual barcoded libraries for each animal were paired-end sequenced using two lines of illumina hiseq <dig>  raw reads were deposited in the ebi-sra database under the accession number prjeb <dig>
cdna synthesis and qpcr reactions
1 μg of total rna from  <dig> individuals for each of the treatments  was reverse transcribed to cdna using the quantitec reverse transcription kit  including the gdna wipe-out step to remove remains of genomic dna and –rt and nt controls were run in parallel with 1 μg of rna but no rt enzyme or rt enzyme but no template.
cdna samples were diluted 1/ <dig> in nuclease-free water .
6 μl per sample were mixed with  <dig>  μl of sensifast sybr lo-rox 2x master mix  containing 400nm of sense/antisense primers.
reactions were performed in duplicated in a mx3005p thermocycler , with 1 cycle of 2 min at 95 °c and x <dig> cycles of 5 s  <dig> °c and 20s at 65 °c, followed by a dissociation curve analysis, where a single peak was detected in all cases.
primers were designed using primer  <dig>  to amplify products between 100-200 bp from gene sequences retrieved from a de novo coho salmon skeletal muscle transcriptome .
netprimer   was used to detect primer hairpins and cross-dimmers.
when two salmonid paralogues were identified primers were design to bind to the most divergent regions of sequence.
genorm software  <cit>  was used to evaluate the stability of the four reference genes analysed rpl <dig>  rpl <dig>  ef1a and ßactin.
the rpl <dig> and ef1a were found to be the most stable reference genes .
normalization of gene expression was performed using the geometric average of rpl <dig> and ef1a.
all expression values are expressed as arbitrary units.
de novo transcriptome assembly and annotation
quality filtered raw reads were de novo assembled by sick-kids bioinformatic services  using trinity software  <cit>  .
resulting contigs were identified by blast  against the ncbi non-redundant database  and gene ontology  annotated using blast2go software default settings  <cit> .
in order to identify the number of unique genes blastx results were manually investigated to remove duplicates and those contigs annotated as hypothetical proteins or predicted proteins.
fully annotated contigs  were blast against the kegg collection of metabolic and signaling pathways with the kegg automatic annotation service  using the single-directional best hit  method against all vertebrates pathways deposited in the database  <cit> .
for identification of coding sequences in the contigs generated during the de novo assembly the complete zebrafish proteome  <cit>  was blasted  against all annotated contigs using bioedit software .
alignment data from positive hits results between coho salmon contigs and zebrafish gene amino acid sequences gave us an estimation of the percentage of coding sequences contained in the de novo contigs.
digital gene expression
read mapping, read normalization and global digital expression were carried out by the bioinformatics department of sick-kids hospital, next generation sequencing service .
quality filtered raw reads from individual libraries were mapped to the complete de novo coho salmon transcriptome and their abundance estimated using the rsem  <cit> .
deseq program from the r-bioconductor package was used to estimate global differences in digital gene expression  between tr and wt groups  <cit> .
the deseq program normalizes mapped reads for individual samples by contig length and library depth using a negative binomial distribution previous to test differences in reads mapped between conditions.
to test specific hypothesis for particular physiological processes deseq normalized counts  per individual animal were retrieved.
gene ontology  enrichment analysis
go enrichment analysis from those genes differently expressed between tr and wt after the global dge analysis was performed using the string webserver  <cit> .
in order to maximize go enrichment analysis blast results were manually annotated with their human orthologue abbreviated gene name .
paralogue identification and phylogenetic analysis
a phylogenetic analysis was carried to confirm the salmonid-specific wgd origin of potential 4r paralogues in the transcriptome.
potential 4r-paralogues were conceptually translated to their amino acid sequence.
blastp against the non-redundant ncbi database was used to confirm the identities of the translated paralogues.
teleost orthologues for the genes of interest were retrieved from ensembl including human and mouse orthologues to be used as outgroups.
potential coho salmon 4r paralogues were used as a query against the rainbow trout  protein collection  and the atlantic salmon  transcriptome shotgun assembly  data deposited in the ncbi .
positive blast hits were included in the analysis.
amino acids sequences were aligned using guidance webserver  <cit>  with mafft as msa algorithm.
only aligned sections with a score over  <dig>  were used to generate the phylogenetic tree.
evolutionary models were estimated for all alignments using mega <dig>  <cit> .
in all cases the best evolutionary model was estimated to be jtt + g .
finally maximum likelihood trees for each alignment were constructed using phyml webserver  <cit>  and displayed using figtree .
statistical analysis
for global dge between tr and wt individuals the deseq program was used following programmer recommendations and a false discovery rate  cut-off of fdr <  <dig>  was applied for significant differences.
the original list of genes differently expressed generated by deseq was manually curated and only those contigs with > <dig> deseq-counts and over 2log <dig> fold-change were used.
as expected from previous studies  <cit> , coho salmon de novo transcriptome presented a significant degree of redundancy; therefore, more than one contig shared the same blast results .
the original deseq list of genes was further curated, maintaining those genes that showed consistent expression between “sister contigs”, special care was taken to detect 3r and 4r-paralogues annotated with the same id by investigating the alignment of sister contigs.
differences in expression for specific groups of genes were estimated using deseq-counts values per individual animal for each gene .
significant differences were detected by a two-tailed test between wt and tr individuals followed by a benjamin-hochberg  correction for multiple comparisons.
significant differences were accepted when fdr <  <dig> .
hierarchical clustering analysis of gene expression data was performed using permutmatrix with gene expression normalized by row using mcquitty’s method  <cit> .
principal component analysis for gene expression of the  <dig> coho salmon analysed was performed using pasw statistic software v <dig> .
differences in gene expression analysed by qpcr were analysed by t-test and a significant threshold of p <  <dig> .
pearson correlation was used to study qpcr and dge expression data obtained for the same genes.
in order to facilitate visualization of results data was logarithmically transformed  prior to correlation analysis.
pearson correlation was estimated using pasw statistic software v <dig>
CONCLUSIONS
gh-transgenic coho salmon show increased appetite and growth relative to wild type fish.
we restricted the food intake of transgenic fish  to that of wild-type  fish fed to satiation, resulting in higher levels of muscle gh expression, but a similar final body size.
the two groups had markedly different gene expression signatures, with tr fish showing increased transcript abundance for pathways associated with protein translation and protein folding and reduced expression of genes involved with myotube fusion.
the down-regulation of genes with known function in myoblast fusion, particularly cadherin signaling, was correlated with a reduction in average muscle fibre diameter in tr relative to wt fish which is expected to reduce the costs of maintaining ionic homeostasis.
this may explain why tr fish are more active than wt fish yet grow at a similar rate.
availability of supporting data
all supporting data for the present manuscript is included as additional files.
additional files
additional file 1:
annotated transcriptome sequences , sequences were annotated using blas2go  against the ncbi non-redundant database  unigene ids present in the transcriptome .
kaas file containing mapping results  and the transcriptome was annotated against all vertebrate pathways present in the kaas online server.
additional file 2:
gene sequences containing >90% of the coding sequence .
coding sequence coverage distribution from annotated unigenes present in the coho salmon transcriptome .
additional file 3:
paired-end reads generated for each individual library and the percentage of assembly.
additional file 4:
genes with significant changes in their expression between restriction-fed growth hormone-transgenic coho salmon  and wild-type salmon  after deseq analysis of the transcriptome as a whole .
additional file 5:
maximum-likelihood phylogenetic trees, genes sequences and alignments used to resolve 4r-salmonid paralogues found during the study.
additional file 6:
genes related to myoblast fusion, the gh-igf network, chaperones/co-chaperones, initiation factors, e3-ubiquitin ligases, initiation factors and autophagy, retrieved from the transcriptome to test specific hypothesis.
wt 1– <dig> and tr 1– <dig> values represent deseq-counts for individual animals.
additional file 7:
dge results of genes involved with initiation of protein translation.
additional file 8:
dge results of genes involved with protein folding.
additional file 9:
dge results of genes involved with myoblast fusion.
additional file 10:
bar graphs of qpcr results for individual genes.
gene expression is expressed in arbitrary units .
significant differences between groups are indicated with an asterisk .
additional file 11:
quantitative pcr primer sequences, pcr efficiencies and melting temperatures for genes analysed.
additional file 12:
de novo
coho salmon transcriptome .
additional file 13:
de novo
coho salmon transcriptome .
additional file 14:
de novo
coho salmon transcriptome .
additional file 15:
de novo
coho salmon transcriptome .
additional file 16:
de novo
coho salmon transcriptome .
competing interests
the authors declare that they have no competing interests.
authors’ contributions
dgs carried out the bioinformatic and digital gene expression analysis with support of iaj.
dgs performed the statistical analysis.
rhd was responsible of the fish husbandry, generation of the experimental groups and provided the samples.
dgs and iaj wrote the manuscript with rhd inputs.
iaj and rhd conceived the study.
all authors read and approved the final manuscript.

 the growth hormone axis in coho salmon and other teleosts is normally tightly coupled to energy intake and is modulated by a large number of environmental factors. the normal feedback control systems are essentially disabled in growth hormone-transgenics due to the extra-pituitary expression of gh in other tissues. high constitutive expression of gh results in increased aggressiveness, appetite and foraging activity leading to continuous fast growth when fish are fed to satiation. gh stimulates insulin-like growth factor- secretion in the liver. circulating igfs bind to igf receptors and activate the signalling cascades that regulate protein synthesis. plasma igf and amino acids from the diet further stimulate igf synthesis in muscle and other tissues via paracrine action. gh-transgenics exhibit increased plasma insulin-like growth factor-, tissue igf mrna and gh-receptor levels relative to wild-type fish. feeding coho salmon gh-transgenics below appetite with a similar ration size as wild-type fish results in an uncoupling of the growth hormone and nutritional signals regulating growth. tr fish grow more slowly and show lower circulating igf than gh-transgenic fish fed to satiation, which indicates the nutritional regulation of igf expression remains functional at the target tissue level. tr animals showed higher circulating gh levels and unchanged ghr mrna relative to gh-transgenics fed to satiation, but retained higher feeding motivation and foraging activity than wt. fast skeletal muscle fibres are formed in teleost fish immediately following somite formation and then in the juvenile/adult stages by processes of stratified and mosaic hyperplasia. myoblast-myoblast fusion results in the formation of a nascent myotube which elongates and gains additional nuclei through myoblast-myotube fusion. activation of terminal differentiation and sarcomere formation then results in the production of an immature muscle fibre. typically, myotube production in fast muscle continues until the fish reaches around 40- % of their maximum attainable adult body length. hill et al. provided indirect evidence that gh-transgenic coho salmon fed to satiation showed enhanced muscle growth by hyperplasia. in a recent study we tested this hypothesis by measuring the number and size distribution of fast myotomal muscle fibres in three groups of coho salmon of similar body length: 1-year old gh-transgenics fed to satiation, 2-year old restriction-fed gh-transgenics and wild type. tf coho salmon recruited fast muscle fibres at twice the rate as wt fish, but showed a similar contribution of hyperplasia and hypertrophy to reach a given body length i.e. the hypothesis of an increased importance of hyperplasia in transgenics was not supported. unexpectedly, tr recruited % fewer fibres with a % higher fibre diameter than either wt or tf fish and had larger diameter fibres across the whole range of fibre sizes, i.e. increased hypertrophy was evident for all cohorts of fibres produced during ontogeny. there is direct experimental evidence from inhibitor and nuclear magnetic resonance studies that larger diameter muscle fibres have lower costs of ionic homeostasis than smaller ones due to their lower surface to volume ratio. thus the ~ % reduction in fibre s/v ratio in tr relative to wt fish would be expected to produce proportional savings in routine maintenance costs. previously we suggested that adjustments in fibre size might permit the reallocation of energy from maintenance to locomotion which would help explain why calorie-restricted transgenics grow at the same rate as wt fish whilst exhibiting markedly higher activity levels. preliminary, studies indicated that several genes associated with myotube formation were downregulated in tr relative to wt fish. in order to gain a broader understanding of fast skeletal muscle gene regulation in tr compare to wt fish we have now carried out rna-seq in tr and wt individual coho salmon for which we had associated data on the number and size of fast muscle fibres, and validated the results by qpcr analysis. rna was sequenced using illumina hiseq and mapped reads were used to study global differences in digital gene expression between groups. following enriched gene ontology analysis we tested specific hypotheses about the effects of uncoupling growth hormone and nutrient signals by analysing the expression of genes related to growth hormone signalling, protein translation, protein-folding and myoblast fusion.  transcriptome analysis total rna from tr and wt fish with known differences in muscle cellularity were sequenced in lines of illumina hiseq. a total of, paired-end reads were de novo assembled into contigs with average length >  bp and n50 >   bp. using blastx against the ncbi non-redundant database we successfully identified a total of contigs and, from them, a total of were also annotated with gene ontology terms. a total of unique genes were identified in the fully annotated transcriptome. the teleost linage underwent a whole-genome duplication relative to the last common ancestor with humans around 350-320mya, increasing gene content by 15- %. the salmonids experienced a fourth lineage-specific wgd around 88mya, and approximately % of the resulting paralogues are still present in extant species. several examples of gene expansion due to the salmonid wgd have been described in detail. in order to estimate the representation of 4r paralogues in the transcriptome well-characterized atlantic salmon paralogues gene families were blasted against the coho salmon de novo transcriptome. seventeen out of paralogues were unequivocally identified and three more appeared with very low e-values, though not all can be expected to be expressed in skeletal muscle. protein-coding genes have been reported for the human genome of which are expressed in the muscle transcriptome. the % higher gene content of the coho skeletal muscle transcriptome than in humans likely reflects this history of the succesive wgd. furthermore, the continuous expression of gh might also increase the number of genes expressed at detectable levels in the muscle.table sequencing and de novo trinity assembly metrics,759,226,210,286 309 tr: restriction-fed growth hormone-transgenic coho salmon; wt: wild-type coho salmon; n50: average length that contains at least half of the sum of the lengths for all contigs; bp: base pair; cds: coding sequence; kaas: keeg automatic annotation service igfbp1a1 igfbp1a2 igfbp1b1 igfbp1b2 igfbp2a igfbp2b1 igfbp2b2 igfbp3a1 igfbp3a2 igfbp3b1 igfbp3b2 igfbp4 igfbp5a igfbp5b1 igfbp5b2 igfbp6a1 igfbp6a2 igfbp6b1 igfbp 6b2 akirin 1 akirin 1 akirin 1 akirin 1 akirin 2 akirin 2 akirin 2 akirin 2 hsp90aa hsp90aa hsp90aa hsp90aa hsp90b1 hsp90b2 aqp8 aqp8aa1 aqp8ab aqp8b myod1 myod2 myo1c id: percentage of identity at amino acid level between s.salar and o.kitsuch orthologues alignment: total number of amino acids successfully aligned during the tblastn between s.salar and o.kitsuch orthologues n/a: paralogues not identified in the transcriptome annotated contigs were blasted against the kegg database using the online web-server kaas to map them against the main metabolic and signaling pathways, yielding a total of kegg maps where coho salmon contigs were present. comparative analysis between annotated contigs against the annotated zebrafish proteome allowed us to identify genes estimated to contain over % of the predicted coding sequence. we also found that contigs were over > % of the cds, indicating that some coho salmon genes were between 1- % longer than their zebrafish orthologues. digital gene expression paired-end reads from individual fish libraries were mapped against the de novo transcriptome. mapped reads were normalized by contig length and library depth following a negative binomial using deseq and used for dge analysis. global dge comparison between tr and wt yielded an initial list of contigs that were differentially expressed. contigs with < normalized reads mapped and a fold-change <2log were discarded. redundant contigs for all genes from the initial global dge list were identified in the annotated transcriptome and their deseq-counts values were individually investigated. those genes for which all-redundant “sister” contigs were found to show consistent, but not necessarily significant, changes in expression between groups were retained. care was taken to identify 4r and 3r-paralogues that might be annotated with the same id by exploring the alignment of the sister contigs. after curation for redundancy and quality control, a total of genes from tr and from wt were considered to be differentially expressed. rnaseq has many advantages, but it does present challenges with respect to statistical analysis and interpretation. firstly, transcription is not tightly coupled to translation in eukaryotes, complicating inferences about the functional significance of changes in transcript abundance. secondly, the activity of signalling pathways is often dependent on posttranslational modifications such as phosphorylation and/or changes in compartmentalisation within the cell. in addition, functional interpretations based on transcript abundance cannot distinguish between mrna transcription vs degradation rates. it is also easy to fall into the trap of providing a series of post hoc stories when reporting transcriptomic data rather than testing a priori hypotheses. standard corrections for multiple testing coupled with long gene lists also leads to a very high barrier for establishing statistical significance, leading to type- statistical errors. we therefore used a multistep process to analyse the dge results from tr and wt fish fed the same ration. firstly, enriched gene ontology analysis was used to identify initial categories of those genes obtained from the global analysis for further investigation. for the tr fish, go analysis revealed an enrichment in genes related to “macromolecule metabolic process”, “rna metabolic process”, “regulation of translation”, “translation factor activity, nucleic acid binding” or “rna binding”. wt animals had a higher expression of genes related to “autophagy”, “cellular metabolic process”, “catabolic process” and “vacuole organization”. based on go enrichment analysis and previous works we decided to specifically test the expression of genes related to myoblast fusion, protein synthesis, protein degradation by the proteasome system, protein folding and also the gh-igf receptors and ligands. kaas annotation results and a literature survey of the results of functional analyses were used to identify the genes to include. genes were identified in total, including 4r and 3r paralogues where identified. deseq-counts from individual animals for each gene were then retrieved in order to test specific a priori hypotheses.table gene ontology analysis biological process molecular function cellular component biological process molecular function cellular component gene ontology analysis from genes with significant differences in expression between restriction-fed growth hormone-transgenic coho salmon and wild-type coho salmon; go: gene ontology; fdr: false discovery rate fig. maximum likelihood phylogenetic trees for two 4r paralogues. a. growth hormone receptor b. caveolin. posterior values are indicated in the nodes of each branch. species abbreviations are as follow; s. salar: salmo salar; o. mykiss: oncorhynchus mykiss; o. kisutch: oncorhynchus kisutch; a. mexicanus: astyanax mexicanus; d. rerio: danio rerio; t.rubripes: takifugu rubripes; g. aculeatus: gasterosteus aculeatus; o. niloticus: oreochromis niloticus; h. sapiens: homo sapiens; m. musculus: mus musculus before analysing in detail the differences in dge between genes a principal components analysis was carried to determine if the categories selected were informative of the differences between tr and wt individuals. components and generated from pca analysis explained % of the variability of the data and were able to distinguish tr and wt in two separated clusters. this is an indication that the data used was informative with respect to the differences between groups, which is highly apparent in the hierarchical clustering shown in fig. 2b.fig. principal components analysis and hierarchical heat map cluster of all the genes manually analysed for dge. a. principal components analysis of the dge from all genes analysed to test our specific hypothesis. in brackets is indicated the percentage of variation explained by each of the components. circles represent individual fish from restricted diet transgenics and wild-type groups b. hierarchical heat map cluster showing individual dge values for tr and wt coho salmon. rows are standardized to have a mean of and standard deviation of 1; red indicates high and green indicates low dge values. each rectangle represents individual dge values growth hormone system and protein synthesis nineteen genes associated with the growth hormone system were identified in the transcriptome and filtered for redundancy as previously described. in order to capture all the biological relevant changes, the requirement that differences should be higher than 2log2-fold change was relaxed but not the requirement that average deseq-counts should be > as expected due to the transgenic nature of the coho salmon, gh gene transcripts were -fold higher in tr compared to wt. similarly, previous studies have also reported an increase in growth hormone receptor expression in muscle. however, that study didn’t distinguish between ghr paralogues whereas we identified ghra. and ghrb. two of the potential salmonid paralogues for the receptor. we found that ghra. was -fold higher in tr than wt fish whereas ghrb. showed unaltered expression, indicating differential regulation of paralogues. igf2/igf1r and the insulin receptor were downregulated 2- and 3-fold respectively in tr than wt fish, most likely as part of a negative feedback response to increased growth hormone expression.fig. gh and igf receptors and ligands. a. hierarchical heat map cluster showing individual dge values for tr and wt coho salmons. rows are standardized to have a mean of and standard deviation of 1; red indicates high and green indicates low dge values. each rectangle represents individual dge values b. bar graphs showing dge values from tr and wt groups for growth hormone, growth hormone receptor a., insulin-like growth factor binding protein and growth hormone receptor b.. significant differences are indicated with one or two asterisk according to the degree of significance the mtorc complex integrates cues from growth factors to activate ribosome biogenesis and protein synthesis via phosphorylation of ribosomal protein s kinase. mtorc1-mediated phosphorylation of the eukaryotic initiation factor 4e binding protein induces its dissociation from eif4e and promotes cap-dependent translation. mtorc is also an important part of the amino acid sensing machinery in the cell comprising rags, ragulator complex and v-atpase. the amino acid-rag axis activates mtorc through a mechanism involving its translocation to the lysosomal surface. thus in the absence of sufficient levels of amino acids, the incorrect positioning of mtorc renders it insensitive to activation by growth factors. the present transcriptome data provides strong evidence that elevated gh levels resulted in a widespread increase in translation initiation factor mrna with out of genes significantly higher expressed in tr than wt fish, including eif4e and eif4e. it is clear from the hierarchical cluster of all genes that % of the genes seem to have a higher expression in tr, even if not significant, than wt with only a handful of contigs more expressed in this condition, such as eif. in addition, out of protein chaperones genes with roles in protein folding were significantly elevated in tr relative to wt fish, including many with known roles in muscle differentiation and sarcomere assembly such as unc and hsp90aa that are also regulated by nutrition. the majority of e3-ubiquitin ligases analysed, enzymes involved in protein degradation through the proteasome system, were more highly expressed in wt than tr, which indicates that tr fish were not starving. the differences in translation initiation factors, protein chaperones and e3-ubiquitin ligases expression in tr fish were not matched by a net increased protein accretion since tr and wt fish grew at the same rate, implying some increase in the metabolic costs of transcription relative to wt.fig. elongation factors and eukaryotic translation factors dge. a. hierarchical heat map cluster showing individual dge values for tr and wt coho salmon. rows are standardized to have a mean of and standard deviation of 1; red indicates high and green indicates low dge values. each rectangle represents individual dge values b. bar graphs showing dge values from tr and wt groups for eukaryotic translation initiation factor 4e, eukaryotic translation initiation factor, eukaryotic translation initiation factor 5b, eukaryotic translation initiation factor 3j, eukaryotic translation initiation factor 2 s and eukaryotic translation initiation factor 1b. significant differences are indicated with one or two asterisk according to the degree of significance fig. chaperones, co-chaperones and stress factors dge. a. hierarchical heat map cluster showing individual dge values for tr and wt coho salmon. rows are standardized to have a mean of and standard deviation of 1; red indicates high and green indicates low dge values. each rectangle represents individual dge values b. bar graphs showing dge values from tr and wt groups for unc-45b homologue, cysteine and histidine-rich domain containing, activator of heat shock 90 kda protein atpase homolog, heat shock protein, cytosolic heat shock protein alpha, chaperonin containing tcp and subunit. significant differences are indicated with one or two asterisk according to the degree of significance fig. e3-ubiquitin ligases dge. a. hierarchical heat map cluster showing individual dge values for tr and wt coho salmon. rows are standardized to have a mean of and standard deviation of 1; red indicates high and green indicates low dge values. each rectangle represents individual dge values b. bar graphs showing dge values from tr and wt groups for f-box protein, e3-ubiquitin ligase itchya, f-box and leucine-rich repeat protein, praja ring finger e ubiquitin protein ligase, ubiquitin protein ligase e component n-recognin, membrane-associated ring finger e ubiquitin protein ligase, forkhead box k and forkhead box n. significant differences are indicated with one or two asterisk according to the degree of significance myoblast fusion myoblast fusion involves multiple sarcolemmal receptors, cell-surface proteins and intracellular signaling pathways leading to membrane and actin cytoskeleton remodelling. evidence from in vivo and in vitro studies involving mouse and zebrafish models was used to construct a list of genes required for myoblast fusion and progression. a total of genes including 4r-paralogues involved in myoblast fusion and differentiation were identified in the transcriptome. out of the genes from the list were differentially expressed between tr and wt groups of coho salmon. a total of of the genes were significantly down regulated in ration-restricted transgenic fish concomitant with a reduction in the number of fast muscle fibres per myotomal cross-section. the majority of these genes were associated with signaling through the cell surface protein cadherin.fig. myoblast fusion related genes dge. a. hierarchical heat map cluster showing individual dge values for tr and wt coho salmon. rows are standardized to have a mean of and standard deviation of 1; red indicates high and green indicates low dge values. each rectangle represents individual dge values b. bar graphs showing dge values from tr and wt groups for dedicator of cytokinesis, dedicator of cytokinesis, v-crk avian sarcoma virus ct oncogene homolog, transmembrane protein 8c, pleckstrin homology domain containing, family o member, caveolin 3a. significant differences are indicated with one or two asterisk according to the degree of significance morpholino antisense-oligonucleotide-mediated knockdown studies have shown that both dock and dock and their adaptor proteins crk, crk-like and the pleckstrin homology domain containing family member 1a are required for the fusion of fast-type myoblasts in zebrafish. members of the rho family of guanosine triphosphatases, including rac operate downstream of dock to stimulate myoblast fusion. rac is also activated by m-cadherin-dependent adhesion through trio during c2c myoblast fusion. m-cadherin and trio transcripts were also both significantly downregulated in tr relative to wt salmon. on the basis of differences in the defects observed in dock1−/− and trio−/− mice it has been suggested that trio is required for myoblast-myoblast fusion, but not for myoblast-myotube fusion. a further three of the genes downregulated in tr fish were associated with integrins and focal adhesion kinase signaling. also down regulated in tr relative to wt fish were sp a component of mapk-erk signaling pathway, nfkb which is involved in the non-canonical nf-kb signaling and tmem8c or myomaker which is required for myoblast-myotube fusion. nine out of the genes on the list were significantly upregulated in tr relative to wt fish. integrin signaling is mediated by the non-receptor protein kinase fak promoting myoblast fusion and an associated increase in caveolin-, transcripts of which were downregulated in tr relative to wt fish. the differences in dge observed are consistent with a higher myoblast fusion activity in wt when compared with tr, consistent with the observed differences in fibre number. quantitative pcr data validation of dge in order to validate the results obtained from dge we analysed the expression of genes covering different ranges of expression by quantitative pcr. the qpcr data supports the dge estimations reported with similar differences between treatments for the individual genes. correlation analysis was used to examine the relationship between the levels of expression for individual genes, that were found to be equivalent irrespective of whether the data were expressed as deseq-counts and average ct values or dge and qpcr fold-change.fig. dge and qpcr correlations. a. dge and qpcr correlations. a. correlation between dge and qpcr ct values for individual genes for each treatment. dge values are expressed as log pearson r and p-value for the correlation are indicated in the top-left corner b. correlation between gene fold-change between tr and wt groups calculated from dge and qpcr values. fold-change is expressed in a log scale. pearson r and p-values for the correlation are indicated in the top-left corner. circles with different colour than black represent the position of the 4r-paralogues plotted: cav3a/cav3b, cdc42a/cdc42b, ghra.2/ghrb. and vaspa/vaspb energy budgeting in the restriction-fed transgenic model tr fish are more aggressive and exhibit markedly higher levels routine swimming behaviour than wt fish and may have higher metabolic costs associated with the futile expression of mrnas and proteins required for growth. in spite of these additional costs, tr fish grow at the same rate as wt fish which implies some compensating alterations to energy budgeting. the source of these energy savings includes the cost of ionic homeostasis which has been estimate to contribute 20- % of the routine metabolic rate in teleosts, a large part of which can be attributable to maintaining the resting membrane potential of fast skeletal muscle which comprises around % of body mass in salmonids. tr fish have fewer, but larger diameter fast muscle fibres than wt for a given body length, which will result in reduced ion pumping costs. our working hypothesis is that the uncoupling of the gh-axis from energy status directly affects one or more of the signaling pathways regulating myotube formation and hypertrophic growth, and several candidate pathways, particularly signaling through cadherin, have been identified in the present study. calorie restriction in wt fish may not result in changes in muscle cellularity because the metabolic signals normally influencing endogenous gh regulation remain intact, a hypothesis requiring further assessment. methods fish experiments on coho salmon were conducted in a non-commercial containment facility for transgenic fish at fisheries and oceans canada, west vancouver. wild-type fish were from the brood of chehalis river strain. the strain of transgenic coho salmon used was derived from chehalis river strain produced using the onmtgh construct comprised of 320 bp of sockeye salmon metallothionein-ß promoter fused to the 5′-utr region of the full-length type-i growth hormone gene and the terminator from the same species, as previously described. fish were reared under standard hatchery conditions in fresh well water with a natural photoperiod and fed commercially available salmon diets. the brood of gh transgenics were fed the same ration to that of the wt group, resulting in similar growth rates in the two groups. fish were fasted for 24 h prior to humane sacrifice and selected to produce two groups with a similar average fork length and hence same the developmental stage. all procedures used in the study were approved by the department of fisheries and oceans pacific region animal care committee. wt and food-restricted growth hormone transgenic coho salmon were obtained from the same group used by the authors in a previous study. no significant differences were found in weight or fork length between tr and wt used in the present study. rna extraction and sequencing sections of pure fast skeletal muscle were carefully dissected from dorsal epaxial myotomes of animals from wt and tr groups matched for body length and with known differences in muscle fibre distribution. total rna was extracted by homogenization in 1 ml of trisure using d-matrix tubes and following the manufacturer’s recommendations. total rna concentration, 260/ and 260/ ratios were estimated by nanodrop spectrometer n. rna integrity was estimated by resolving 1 μg of sample in a % ethidium bromide agarose gel. a total of 3 μg of rna per sample was sent to the sick-kids hospital next generation sequencing service for sequencing. individual barcoded libraries for each animal were paired-end sequenced using two lines of illumina hiseq raw reads were deposited in the ebi-sra database under the accession number prjeb cdna synthesis and qpcr reactions 1 μg of total rna from individuals for each of the treatments was reverse transcribed to cdna using the quantitec reverse transcription kit including the gdna wipe-out step to remove remains of genomic dna and –rt and nt controls were run in parallel with 1 μg of rna but no rt enzyme or rt enzyme but no template. cdna samples were diluted 1/ in nuclease-free water. 6 μl per sample were mixed with  μl of sensifast sybr lo-rox 2x master mix containing 400nm of sense/antisense primers. reactions were performed in duplicated in a mx3005p thermocycler, with 1 cycle of 2 min at 95 °c and x cycles of 5 s °c and 20s at 65 °c, followed by a dissociation curve analysis, where a single peak was detected in all cases. primers were designed using primer to amplify products between 100-200 bp from gene sequences retrieved from a de novo coho salmon skeletal muscle transcriptome. netprimer was used to detect primer hairpins and cross-dimmers. when two salmonid paralogues were identified primers were design to bind to the most divergent regions of sequence. genorm software was used to evaluate the stability of the four reference genes analysed rpl rpl ef1a and ßactin. the rpl and ef1a were found to be the most stable reference genes. normalization of gene expression was performed using the geometric average of rpl and ef1a. all expression values are expressed as arbitrary units. de novo transcriptome assembly and annotation quality filtered raw reads were de novo assembled by sick-kids bioinformatic services using trinity software. resulting contigs were identified by blast against the ncbi non-redundant database and gene ontology annotated using blast2go software default settings. in order to identify the number of unique genes blastx results were manually investigated to remove duplicates and those contigs annotated as hypothetical proteins or predicted proteins. fully annotated contigs were blast against the kegg collection of metabolic and signaling pathways with the kegg automatic annotation service using the single-directional best hit method against all vertebrates pathways deposited in the database. for identification of coding sequences in the contigs generated during the de novo assembly the complete zebrafish proteome was blasted against all annotated contigs using bioedit software. alignment data from positive hits results between coho salmon contigs and zebrafish gene amino acid sequences gave us an estimation of the percentage of coding sequences contained in the de novo contigs. digital gene expression read mapping, read normalization and global digital expression were carried out by the bioinformatics department of sick-kids hospital, next generation sequencing service. quality filtered raw reads from individual libraries were mapped to the complete de novo coho salmon transcriptome and their abundance estimated using the rsem. deseq program from the r-bioconductor package was used to estimate global differences in digital gene expression between tr and wt groups. the deseq program normalizes mapped reads for individual samples by contig length and library depth using a negative binomial distribution previous to test differences in reads mapped between conditions. to test specific hypothesis for particular physiological processes deseq normalized counts per individual animal were retrieved. gene ontology enrichment analysis go enrichment analysis from those genes differently expressed between tr and wt after the global dge analysis was performed using the string webserver. in order to maximize go enrichment analysis blast results were manually annotated with their human orthologue abbreviated gene name. paralogue identification and phylogenetic analysis a phylogenetic analysis was carried to confirm the salmonid-specific wgd origin of potential 4r paralogues in the transcriptome. potential 4r-paralogues were conceptually translated to their amino acid sequence. blastp against the non-redundant ncbi database was used to confirm the identities of the translated paralogues. teleost orthologues for the genes of interest were retrieved from ensembl including human and mouse orthologues to be used as outgroups. potential coho salmon 4r paralogues were used as a query against the rainbow trout protein collection and the atlantic salmon transcriptome shotgun assembly data deposited in the ncbi. positive blast hits were included in the analysis. amino acids sequences were aligned using guidance webserver with mafft as msa algorithm. only aligned sections with a score over were used to generate the phylogenetic tree. evolutionary models were estimated for all alignments using mega. in all cases the best evolutionary model was estimated to be jtt + g. finally maximum likelihood trees for each alignment were constructed using phyml webserver and displayed using figtree. statistical analysis for global dge between tr and wt individuals the deseq program was used following programmer recommendations and a false discovery rate cut-off of fdr <  was applied for significant differences. the original list of genes differently expressed generated by deseq was manually curated and only those contigs with > deseq-counts and over 2log fold-change were used. as expected from previous studies, coho salmon de novo transcriptome presented a significant degree of redundancy; therefore, more than one contig shared the same blast results. the original deseq list of genes was further curated, maintaining those genes that showed consistent expression between “sister contigs”, special care was taken to detect 3r and 4r-paralogues annotated with the same id by investigating the alignment of sister contigs. differences in expression for specific groups of genes were estimated using deseq-counts values per individual animal for each gene. significant differences were detected by a two-tailed test between wt and tr individuals followed by a benjamin-hochberg correction for multiple comparisons. significant differences were accepted when fdr < . hierarchical clustering analysis of gene expression data was performed using permutmatrix with gene expression normalized by row using mcquitty’s method. principal component analysis for gene expression of the coho salmon analysed was performed using pasw statistic software v. differences in gene expression analysed by qpcr were analysed by t-test and a significant threshold of p < . pearson correlation was used to study qpcr and dge expression data obtained for the same genes. in order to facilitate visualization of results data was logarithmically transformed prior to correlation analysis. pearson correlation was estimated using pasw statistic software v  gh-transgenic coho salmon show increased appetite and growth relative to wild type fish. we restricted the food intake of transgenic fish to that of wild-type fish fed to satiation, resulting in higher levels of muscle gh expression, but a similar final body size. the two groups had markedly different gene expression signatures, with tr fish showing increased transcript abundance for pathways associated with protein translation and protein folding and reduced expression of genes involved with myotube fusion. the down-regulation of genes with known function in myoblast fusion, particularly cadherin signaling, was correlated with a reduction in average muscle fibre diameter in tr relative to wt fish which is expected to reduce the costs of maintaining ionic homeostasis. this may explain why tr fish are more active than wt fish yet grow at a similar rate. availability of supporting data all supporting data for the present manuscript is included as additional files. additional files additional file 1: annotated transcriptome sequences, sequences were annotated using blas2go against the ncbi non-redundant database unigene ids present in the transcriptome. kaas file containing mapping results and the transcriptome was annotated against all vertebrate pathways present in the kaas online server. additional file 2: gene sequences containing >90% of the coding sequence. coding sequence coverage distribution from annotated unigenes present in the coho salmon transcriptome. additional file 3: paired-end reads generated for each individual library and the percentage of assembly. additional file 4: genes with significant changes in their expression between restriction-fed growth hormone-transgenic coho salmon and wild-type salmon after deseq analysis of the transcriptome as a whole. additional file 5: maximum-likelihood phylogenetic trees, genes sequences and alignments used to resolve 4r-salmonid paralogues found during the study. additional file 6: genes related to myoblast fusion, the gh-igf network, chaperones/co-chaperones, initiation factors, e3-ubiquitin ligases, initiation factors and autophagy, retrieved from the transcriptome to test specific hypothesis. wt 1– and tr 1– values represent deseq-counts for individual animals. additional file 7: dge results of genes involved with initiation of protein translation. additional file 8: dge results of genes involved with protein folding. additional file 9: dge results of genes involved with myoblast fusion. additional file 10: bar graphs of qpcr results for individual genes. gene expression is expressed in arbitrary units. significant differences between groups are indicated with an asterisk. additional file 11: quantitative pcr primer sequences, pcr efficiencies and melting temperatures for genes analysed. additional file 12: de novo coho salmon transcriptome. additional file 13: de novo coho salmon transcriptome. additional file 14: de novo coho salmon transcriptome. additional file 15: de novo coho salmon transcriptome. additional file 16: de novo coho salmon transcriptome. competing interests the authors declare that they have no competing interests. authors’ contributions dgs carried out the bioinformatic and digital gene expression analysis with support of iaj. dgs performed the statistical analysis. rhd was responsible of the fish husbandry, generation of the experimental groups and provided the samples. dgs and iaj wrote the manuscript with rhd inputs. iaj and rhd conceived the study. all authors read and approved the final manuscript. 
coho salmon transgenic for growth hormone express gh in multiple tissues which results in increased appetite and continuous high growth with satiation feeding. restricting gh-transgenics to the same lower ration as wild-type fish results in similar growth, but with the recruitment of fewer, larger diameter, muscle skeletal fibres to reach a given body size. the uncoupling of gh and amino acid signals likely results in additional costs of transcription associated with protein turnover in tr fish. transcripts coding for genes required for myoblast fusion were identified with significantly downregulated in tr relative to wt fish, including associated with signaling through the cell surface protein cadherin. the predicted reduction in the ionic costs of homeostasis in tr fish associated with increased fibre size were shown to involve multiple pathways regulating myotube fusion, particularly cadherin signaling. nineteen out of translation initiation factors and of protein chaperones were upregulated in tr relative to wt fish. tr coho salmon showed increased growth hormone transcripts and gene expression associated with protein synthesis and folding than wt fish even though net rates of protein accretion were similar. illumina sequencing of individually barcoded libraries from wt and tr coho salmon yielded, paired end reads which were used to construct contigs containing unique genes of which > contained > % of the coding sequence. muscle growthteleost fishgrowth hormone transgenicsskeletal muscle transcriptomefish nutritionissue-copyright-statement© the author 2015 in order to better understand the genetic mechanisms behind these different patterns of muscle growth and to investigate how the decoupling of gh and nutritional signals affects gene regulation we used rna-seq to compare the fast skeletal muscle transcriptome in tr and wt coho salmon.
 coho salmon transgenic for growth hormone express gh in multiple tissues which results in increased appetite and continuous high growth with satiation feeding. restricting gh-transgenics to the same lower ration as wild-type fish results in similar growth, but with the recruitment of fewer, larger diameter, muscle skeletal fibres to reach a given body size. in order to better understand the genetic mechanisms behind these different patterns of muscle growth and to investigate how the decoupling of gh and nutritional signals affects gene regulation we used rna-seq to compare the fast skeletal muscle transcriptome in tr and wt coho salmon.  illumina sequencing of individually barcoded libraries from wt and tr coho salmon yielded, paired end reads which were used to construct contigs containing unique genes of which > contained > % of the coding sequence. transcripts coding for genes required for myoblast fusion were identified with significantly downregulated in tr relative to wt fish, including associated with signaling through the cell surface protein cadherin. nineteen out of translation initiation factors and of protein chaperones were upregulated in tr relative to wt fish.  tr coho salmon showed increased growth hormone transcripts and gene expression associated with protein synthesis and folding than wt fish even though net rates of protein accretion were similar. the uncoupling of gh and amino acid signals likely results in additional costs of transcription associated with protein turnover in tr fish. the predicted reduction in the ionic costs of homeostasis in tr fish associated with increased fibre size were shown to involve multiple pathways regulating myotube fusion, particularly cadherin signaling. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords muscle growthteleost fishgrowth hormone transgenicsskeletal muscle transcriptomefish nutritionissue-copyright-statement© the author 2015 