Mapping and Expression of Candidate Genes for Development Rate in Rainbow Trout (Oncorhynchus mykiss).

Development rate has important implications for individual fitness and physiology. In salmonid fishes, development rate correlates with many traits later in life, including life-history diversity, growth, and age and size at sexual maturation. In rainbow trout (Oncorhynchus mykiss), a quantitative trait locus for embryonic development rate has been detected on chromosome 5 across populations. However, few candidate genes have been identified within this region. In this study, we use gene mapping, gene expression, and quantitative genetic methods to further identify the genetic basis of embryonic developmental rate in O. mykiss Among the genes located in the region of the major development rate quantitative trait locus (GHR1, Clock1a, Myd118-1, and their paralogs), all were expressed early in embryonic development (fertilization through hatch), but none were differentially expressed between individuals with the fast- or slow-developing alleles for a major embryonic development rate quantitative trait locus. In a follow-up study of migratory and resident rainbow trout from natural populations in Alaska, we found significant additive variation in development rate and, moreover, found associations between development rate and allelic variation in all 3 candidate genes within the quantitative trait locus for embryonic development. The mapping of these genes to this region and associations in multiple populations provide positional candidates for further study of their roles in growth, development, and life-history diversity in this model salmonid.

Identification and expression of Smads associated with TGF-ß/activin/nodal signaling pathways in the rainbow trout (Oncorhynchus mykiss).

The Smad proteins are essential components of the TGF-ß/activin/nodal family signaling pathway. We report the identification and expression of transcripts representing three receptor Smads (Smad2a, Smad2b, and Smad3), two common Smads (Smad4a and Smad4b), and one inhibitory Smad (Smad7). Phylogenetic analysis suggests this gene family evolved through the combination of ancient and more recent salmonid genome duplication events. Tissue distribution, embryonic expression, and expression in growth hormone (GH) treated fish were assessed by reverse transcription PCR or qPCR. All six Smad transcripts were ubiquitously expressed in adult tissues. We observed the highest expression of the receptor Smads in unfertilized eggs, generally decreasing during early embryonic development and slightly increasing around 11 days post-fertilization (dpf). Smad7 expression was low for most of embryonic development, with a dramatic increase at the onset of muscle development (6 dpf), and at hatch (24 dpf). Smad4 expression was low during early embryonic development and increased after 14 dpf. The increased expression of Smad4 and Smad7 during late embryonic development may indicate modulation of gene expression by GH axis, which initiates activity during late embryonic development. These data were supported by the modulation of these Smads in the gill filament, stomach, and muscle following a GH treatment. Additionally, these changes are concurrent with the modulation of expression of TGF-ß family members. Most significantly, the increased expression of Smad7 in the muscle is simultaneous with increased expression of MSTN1A and not MSTN1B during both embryonic development and following GH treatment. These data indicate a promyogenic role for Smad7 as previously identified in other non-fish species.

Identification, characterization and genetic mapping of TLR1 loci in rainbow trout (Oncorhynchus mykiss).

Induction of innate immune pathways is critical for early anti-microbial defense but there is limited understanding of how teleosts recognize microbial molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 1 and 2 form a heterodimer involved in recognizing peptidoglycans and lipoproteins of microbial origin. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR1 gene ortholog and its mRNA expression. Two TLR1 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA sequencing and genetic linkage analyses. Full length cDNA clone and direct sequencing of four BACs revealed an intact omTLR1 open reading frame (ORF) located on chromosome 14 and a second locus on chromosome 25 that contains a TLR1 pseudogene. The duplicated trout loci exhibit conserved synteny with other fish genomes that extends beyond the TLR1 gene sequences. The omTLR1 gene includes a single large coding exon similar to all other described TLR1 genes, but unlike other teleosts it also has a 5' UTR exon and intron preceding the large coding exon. The omTLR1 ORF is predicted to encode an 808 amino-acid protein with 69% similarity to the Fugu TLR1 and a conserved pattern of predicted leucine-rich repeats (LRR). Phylogenetic analysis grouped omTLR1 with other fish TLR1 genes on a separate branch from the avian TLR1 and mammalian TLR1, 6 and 10. omTLR1 expression levels in rainbow trout anterior kidney leukocytes were not affected by the human TLR2/6 and TLR2/1 agonists diacylated lipoprotein (Pam(2)CSK(4)) and triacylated lipoprotein (Pam(3)CSK(4)). However, due to the lack of TLR6 and 10 genes in teleost genomes and up-regulation of TLR1 mRNA in response to LPS and bacterial infection in other fish species we hypothesize an important role for omTLR1 in anti-microbial immunity. Therefore, the identification of a TLR2 ortholog in rainbow trout and the development of assays to measure ligand binding and downstream signaling are critical for future elucidation of omTLR1 functions.

Identification, characterization and genetic mapping of TLR7, TLR8a1 and TLR8a2 genes in rainbow trout (Oncorhynchus mykiss).

Induction of the innate immune pathways is critical for early anti-viral defense but there is limited understanding of how teleost fish recognize viral molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 7 and 8 bind single-stranded RNA of viral origin and are activated by synthetic anti-viral imidazoquinoline compounds. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR7 and TLR8 gene orthologs and their mRNA expression. Two TLR7/8 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA fingerprinting and genetic linkage analyses. Direct sequencing of two representative BACs revealed intact omTLR7 and omTLR8a1 open reading frames (ORFs) located on chromosome 3 and a second locus on chromosome 22 that contains an omTLR8a2 ORF and a putative TLR7 pseudogene. We used the omTLR8a1/2 nomenclature for the two trout TLR8 genes as phylogenetic analysis revealed that they and all the other teleost TLR8 genes sequenced to date are similar to the zebrafish TLR8a, but are distinct from the zebrafish TLR8b. The duplicated trout loci exhibit conserved synteny with other fish genomes extending beyond the tandem of TLR7/8 genes. The trout TLR7 and 8a1/2 genes are composed of a single large exon similar to all other described TLR7/8 genes. The omTLR7 ORF is predicted to encode a 1049 amino acid (aa) protein with 84% similarity to the Fugu TLR7 and a conserved pattern of predicted leucine-rich repeats (LRR). The omTLR8a1 and omTLR8a2 are predicted to encode 1035- and 1034-aa proteins, respectively, and have 86% similarity to each other. omTLR8a1 is likely the ortholog of the only Atlantic salmon TLR8 gene described to date as they have 95% aa sequence similarity. The tissue expression profiles of omTLR7, omTLR8a1 and omTLR8a2 in healthy trout were highest in spleen tissue followed by anterior and then posterior kidney tissues. Rainbow trout anterior kidney leukocytes produced elevated levels of pro-inflammatory and type I interferon cytokines mRNA in response to stimulation with the human TLR7/8 agonist R848 or the TLR3 agonist poly I:C. Only poly I:C-induced IFN2 transcription was significantly suppressed in the presence of chloroquine, a compound known to block endosomal acidification and inhibit endosomal maturation. The effect of chloroquine on R848-induced cytokine expression was equivocal and so it remains questionable whether rainbow trout recognition of R848 requires endosomal maturation. TLR7 and TLR8a1 expression levels in rainbow trout anterior kidney leukocytes were not affected by poly I:C or R848 treatments, but surprisingly, TLR8a2 expression was moderately down-regulated by R848. The down-regulation of omTLR8a2 may imply that this gene has evolved to a new or altered function in rainbow trout, as often occurs when the two duplicated genes remain active.

A second generation genetic map for rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Genetic maps characterizing the inheritance patterns of traits and markers have been developed for a wide range of species and used to study questions in biomedicine, agriculture, ecology and evolutionary biology. The status of rainbow trout genetic maps has progressed significantly over the last decade due to interest in this species in aquaculture and sport fisheries, and as a model research organism for studies related to carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. We constructed a second generation genetic map for rainbow trout using microsatellite markers to facilitate the identification of quantitative trait loci for traits affecting aquaculture production efficiency and the extraction of comparative information from the genome sequences of model fish species. RESULTS: A genetic map ordering 1124 microsatellite loci spanning a sex-averaged distance of 2927.10 cM (Kosambi) and having 2.6 cM resolution was constructed by genotyping 10 parents and 150 offspring from the National Center for Cool and Cold Water Aquaculture (NCCCWA) reference family mapping panel. Microsatellite markers, representing pairs of loci resulting from an evolutionarily recent whole genome duplication event, identified 180 duplicated regions within the rainbow trout genome. Microsatellites associated with genes through expressed sequence tags or bacterial artificial chromosomes produced comparative assignments with tetraodon, zebrafish, fugu, and medaka resulting in assignments of homology for 199 loci. CONCLUSION: The second generation NCCCWA genetic map provides an increased microsatellite marker density and quantifies differences in recombination rate between the sexes in outbred populations. It has the potential to integrate with cytogenetic and other physical maps, identifying paralogous regions of the rainbow trout genome arising from the evolutionarily recent genome duplication event, and anchoring a comparative map with the zebrafish, medaka, tetraodon, and fugu genomes. This resource will facilitate the identification of genes affecting traits of interest through fine mapping and positional cloning of candidate genes.

Effects of short-term growth hormone treatment on liver and muscle transcriptomes in rainbow trout (Oncorhynchus mykiss).

Although studies have established that exogenous growth hormone (GH) treatment stimulates growth in fish, its effects on target tissue gene expression are not well characterized. We assessed the effects of Posilac (Monsanto, St. Louis, MO), a recombinant bovine GH, on tissue transcript levels in rainbow trout selected from two high-growth rate and two low-growth rate families. Transcript abundance was measured in liver and muscle with the Genome Research in Atlantic Salmon Project (GRASP) 16K cDNA microarray. A selection of the genes identified as altered by the microarray and transcripts for insulin-like growth factors, growth hormone receptors (GHRs), and myostatins were measured by real-time PCR in the liver, muscle, brain, kidney, intestine, stomach, gill, and heart. In general, transcripts identified as differentially regulated in the muscle on the microarray showed similar directional changes of expression in the other nonhepatic tissues. A total of 114 and 66 transcripts were identified by microarray as differentially expressed with GH treatment across growth rate for muscle and liver, respectively. The largest proportion of these transcripts represented novel transcripts, followed by immune and metabolism-related genes. We have identified a number of genes related to lipid metabolism, supporting a modulation in lipid metabolism following GH treatment. Most notable among the growth-axis genes measured by real-time PCR were increases in GHR1 and -2 transcripts in liver and muscle. Our results indicate that short-term GH treatment activates the immune system, shifts the metabolic sectors, and modulates growth-regulating genes.

A survey of expressed sequence tags from the rainbow trout (Oncorhynchus mykiss) pituitary.

The pituitary plays significant roles in the regulation of physiological processes. In the current study, expressed sequence tag data was obtained for 1,920 clones from a normalized mixed-sex pituitary cDNA library. From these 3,840 sequences, a total of 524 contigs were assembled and 1,256 unique singletons identified. Assignment of functional annotation was performed through BLAST and gene ontology term assignment. Through in silico comparative mapping homologs were identified for 354 of the unigene sequences. These data provide the first functional information on many of the transcripts present in the rainbow trout pituitary.

Characterization of rainbow trout myostatin-2 genes (rtMSTN-2a and -2b): genomic organization, differential expression, and pseudogenization.

Myostatin is an extremely potent negative regulator of vertebrate skeletal muscle development. A phylogenetic analysis suggests that salmonids should possess four distinct genes, although only MSTN-1 orthologs have been characterized. Described herein are the rainbow trout (rt) MSTN-2a and -2b genes and subsequence analysis of their promoters and their quantitative expression profiles. Both genes are similarly organized, contain several putative myogenic response elements, and are legitimate MSTN-2 orthologs based on Bayesian analyses. However, rtMSTN-2b contains two in-frame stop codons within the first exon and unspliced variants of both transcripts were expressed in a tissue-specific manner. Complete splicing of rtMSTN-2a occurred only in brain, where expression is highest, whereas rtMSTN-2b transcripts were mostly present in unspliced forms. The presence of stop codons in the rtMSTN-2b open reading frame and the expression of mostly unspliced transcripts indicate that this particular homolog is a pseudogene. These results confirm our previous phylogenetic analysis and suggest that all salmonids likely possess four distinct myostatin genes. The tissue-specific expression and differential processing of both rtMSTN-2 transcripts as well the pseudogenization of rtMSTN-2b may reflect compensatory and adaptive responses to tetraploidization and may help limit rtMSTN-2a's influences primarily to neural tissue.

Assignment of rainbow trout linkage groups to specific chromosomes.

The rainbow trout genetic linkage groups have been assigned to specific chromosomes in the OSU (2N=60) strain using fluorescence in situ hybridization (FISH) with BAC probes containing genes mapped to each linkage group. There was a rough correlation between chromosome size and size of the genetic linkage map in centimorgans for the genetic maps based on recombination from the female parent. Chromosome size and structure have a major impact on the female:male recombination ratio, which is much higher (up to 10:1 near the centromeres) on the larger metacentric chromosomes compared to smaller acrocentric chromosomes. Eighty percent of the BAC clones containing duplicate genes mapped to a single chromosomal location, suggesting that diploidization resulted in substantial divergence of intergenic regions. The BAC clones that hybridized to both duplicate loci were usually located in the distal portion of the chromosome. Duplicate genes were almost always found at a similar location on the chromosome arm of two different chromosome pairs, suggesting that most of the chromosome rearrangements following tetraploidization were centric fusions and did not involve homeologous chromosomes. The set of BACs compiled for this research will be especially useful in construction of genome maps and identification of QTL for important traits in other salmonid fishes.

Identification, characterization, and quantitative expression analysis of rainbow trout myostatin-1a and myostatin-1b genes.

Myostatin is a potent negative regulator of skeletal muscle growth. Although several cDNA clones have been characterized in different vertebrates, the genomic organization and bioactivity of non-mammalian homologs have not. The intron/exon organization and promoter subsequence analysis of two rainbow trout myostatin genes, rtMSTN-1a and rtMSTN-1b (formerly 1 and 2 respectively), as well as a quantitative assessment of their embryonic, larval, and adult tissue expression profiles are reported herein. Each gene was similarly organized into three exons of 490, 368, and 1600 bp for MSTN-1a and 486, 386, and 1419 bp for MSTN-1b. Comparative mapping of coding regions from several vertebrate myostatin genes revealed a common organization between species, including conserved pre-mRNA splice sites; the first among the fishes and the second across all vertebrate species. In silico subsequence analysis of the promoter regions identified E-boxes and other putative myogenic response elements. However, the number and diversity of elements were considerably less than those found in mammalian promoters or in the recently characterized zebrafish MSTN-2 gene. A quantitative analysis of the embryonic expression profile for both genes indicates that rtMSTN-1a expression is consistently greater than that of rtMSTN-1b and neither gene is significantly expressed throughout gastrulation. Expression of both steadily increases fourfold during somitogenesis and subsides as this period ends. After eyeing, however, rtMSTN-1a mRNA levels ultimately rise 20-fold by day 49 and peak before hatching and yolk sac absorption (YSA). Levels of rtMSTN-1b rise similarly, but do not peak before YSA. An analysis of adult (2-year-old fish) tissue expression indicates that both transcripts are present in most tissues although levels are highest in brain, testes, eyes, muscle, and surprisingly spleen. These studies suggest that strong selective pressures have preserved the genomic organization of myostatin genes throughout evolution. However, the different expression profiles and putative promoter elements in fishes versus mammals suggests that limitations in myostatin function may have evolved recently.

Genomic structure and expression of uncoupling protein 2 genes in rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Uncoupling protein 2 (UCP2) belongs to the superfamily of mitochondrial anion carriers that dissociate the respiratory chain from ATP synthesis. It has been determined that UCP2 plays a role in several physiological processes such as energy expenditure, body weight control and fatty acid metabolism in several vertebrate species. We report the first characterization of UCP2s in rainbow trout (Oncorhynchus mykiss). RESULTS: Two UCP2 genes were identified in the rainbow trout genome, UCP2A and UCP2B. These genes are 93% similar in their predicted amino acid sequences and display the same genomic structure as other vertebrates (8 exons and 7 introns) spanning 4.2 kb and 3.2 kb, respectively. UCP2A and UCP2B were widely expressed in all tissues of the study with a predominant level in macrophage-rich tissues and reproductive organs. In fry muscle we observed an increase in UCP2B expression in response to fasting and a decrease after refeeding in agreement with previous studies in human, mouse, rat, and marsupials. The converse expression pattern was observed for UCP2A mRNA which decreased during fasting, suggesting different metabolic roles for UCP2A and UCP2B in rainbow trout muscle. Phylogenetic analysis including other genes from the UCP core family located rainbow trout UCP2A and UCP2B with their orthologs and suggested an early divergence of vertebrate UCPs from a common ancestor gene. CONCLUSION: We characterized two UCP2 genes in rainbow trout with similar genomic structures, amino acid sequences and distribution profiles. These genes appeared to be differentially regulated in response to fasting and refeeding in fry muscle. The genomic organization and phylogeny analysis support the hypothesis of a common ancestry between the vertebrate UCPs.

Fasting and refeeding affect the expression of the Inhibitor of DNA Binding (ID) genes in rainbow trout (Oncorhynchus mykiss) muscle.

The Inhibitor of DNA Binding/Differentiation (ID) proteins are a family of dominant negative regulators of the basic helix-loop-helix (bHLH) transcription factors, shown in mammals to delay cell differentiation and prolong proliferation. In the current study we used real-time PCR to investigate the effects of fasting and refeeding on the expression of ID genes in rainbow trout muscle. Fry shortly following yolk-sac absorption (approximately 250 mg) were used in a pair of experiments. In the first experiment, the treatment groups included fish fed or fasted throughout the duration of the experiment, and fish fasted for 14 days followed by feeding for the remainder of the experiment. The second experiment consisted of the same treatment groups; however the fish were only fasted for 7 days prior to refeeding. In both experiments, ID gene expression in the muscle of fasted fish was significantly lower than the fed samples after 7 days. Refeeding for 3 or 7 days returned the ID expression to levels similar to the fed fish. The reduction of ID expression during a fast and the subsequent return to fed levels with refeeding suggests the ID proteins participate in the regulation of muscle growth in the rainbow trout.

Identification and expression profile of the ID gene family in the rainbow trout (Oncorhynchus mykiss).

ID proteins are negative regulators of basic helix-loop-helix transcription factors governing growth and development in mammals. However, little is known about the ID gene function and expression in fish. We report the identification and characterization of two new rainbow trout ID genes (ID1D and ID2B) and extend our expression analyses of two previously identified ID genes (ID1A and ID2A). Phylogenetic analyses indicate an evolutionary relationship between ID1A and ID1D and between ID1B and ID1C, suggesting a mechanism of divergence throughout salmonid evolution. To access the expression of these genes in adult and developing fish, we measured the relative transcript abundance of four ID1 and two ID2 genes by real-time PCR. ID1 transcripts were expressed in a variety of tissues and the ID1 paralogues showed similar patterns of expression, whereas the ID2 paralogues were differentially expressed. To access the role of the ID genes during embryonic development, gene expression was measured at early (day 0 and day 2), mid (day 9 and day 18) and late (day 30 and day 50) embryonic development. ID1A and ID1D expression remained unchanged throughout embryonic development, while ID1B and ID1C were lowest during early, highest at mid, and decreased during late embryonic development. The ID2 transcripts revealed the highest expression in unfertilized eggs and day 2 embryos, and remained low throughout the remainder of embryonic development. The sequence analyses and gene expression patterns implicate gene and genome duplication in rainbow trout ID gene evolution and suggest an extensive role for the IDs in rainbow trout growth and development.

The effects of in ovo rhIGF-I administration on expression of the growth hormone secretagogue receptor (GHSR) during chicken embryonic development.

Growth hormone secretion is under the control of a pair of hypothalamic factors, growth hormone releasing hormone and somatostatin. The growth hormone secretagogue receptor (GHSR) and its endogenous ligand represent a novel third method regulating the release of growth hormone. Early chicken embryonic development has been proposed to be independent of GH. However, recent evidence shows that peripheral GH secretion has paracrine/autocrine functions during embryonic development. In the current study, we used the reverse-transcriptase polymerase chain reaction to determine the expression pattern of the GHSR during embryonic development and the effects of in ovo recombinant human (rh) IGF-I administration on its expression pattern. Eggs were injected once with 100 ng rhIGF-I in 10 mM acetic acid, and 0.1% BSA per embryo on embryonic day 3. Total RNA was isolated from whole embryos on embryonic day (E) 0-6 (n=6 per day), thoracic/abdominal halves of the embryos on E7- E8 (n= 6 per day) and Pectoralis muscle on E9-E20 (n= 4 per day). We found that GHSR expression was low during E0-E4, followed by an increase on E5 and remained constant through E17. GHSR expression then increased on E18 before reducing on E20. A similar pattern was found in the rhIGF-I treated embryos with the exception of a significant increase in GHSR expression on E8. These data indicate that the GHSR may be active in regulating GH secretion during early embryonic development, and upregulation of the GHSR gene following IGF-I administration may have an important role in the determination of postnatal muscle growth.

Myostatin and TGF-beta2 gene expression patterns in response to in ovo administration of rhIGF-I during chicken embryonic development.

The objective of the study was to evaluate the impact of in ovo administration of recombinant human insulin-like growth factor-I (rhIGF-I) on myostatin and transforming growth factor-beta2 (TGF-beta2) gene expression during chicken embryogenesis with emphasis on skeletal muscle development. Eggs were injected once with 100 ng rh IGF-I in 10 mM acetic acid, 0.1% BSA per embryo on day 3 of embryonic development. Total RNA was isolated from whole embryos on each of embryonic days (E) 0 to 6 (n = 6 per day/per treatment), from thoracic/abdominal halves of the embryo at E 7 to 8 (n = 6 per day/per treatment), and from pectoralis muscle tissues at E 9 to 20 (n = 4 per day/per treatment). Reverse-transcription polymerase chain reaction (RT-PCR) was used to synthesize cDNAs. Myostatin mRNA isolated from pectoralis muscles of the rhIGF-I treated group increased on E 10 (approximately 2.5 fold) and remained high through E 13, whereas myostatin mRNA from control pectoralis muscles increased at E 9 and remained high until E 12. TGF-beta2 gene expression from in ovo rhIGF-I treated pectoralis muscles dramatically increased at E 13 (approximately 2.5 fold), in contrast to E 14 from control pectoralis muscle, and gradually declined through E 16. Our results demonstrate that in ovo administration of rhIGF-I on E 3 may alter developmental expression patterns of myostatin and TGF-beta2 genes.

IGF-I, IGF-II, and IGF-receptor-1 transcript and IGF-II protein expression in myostatin knockout mice tissues.

Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were performed to demonstrate whether a correlation exists between insulin-like growth factors (IGFs)-positive regulators of growth-and myostatin, a negative regulator of muscle growth. IGF-I, -II, and IGF-receptor-1 (IGF-R1) mRNA and IGF-II protein expressions were determined in control and myostatin knockout mice tissues. IGF-I gene expressions were similar between control and knockout mice tissues, whereas IGF-II mRNA levels were significantly higher in myostatin knockout mice kidney and soleus muscles than those of control mice (P <.01). IGF-R1 mRNA levels from control mice heart (P <.05) and kidney (P <.01) were significantly higher than in myostatin knockout mice, whereas levels were lower in pectoralis muscle of control mice than knockout mice (P <.01). The strongly IGF-II-positive cells in soleus muscle were more common in myostatin knockout mice and were seen in a few foci in control mice. IGF-II immunoreactivity in both control and myostatin knockout mice kidneys was localized to the epithelium of renal tubules and collecting ducts. Reciprocal changes in the expression of myostatin and IGF-II and IGF-R1 may underlie normal growth of skeletal muscle and other organs in mammals, and the changes in these tissues associated with disease.