Daniel J. Macqueen

REVIEW Growth and the regulation of myotomal muscle mass in teleost fish

Summary Teleost muscle first arises in early embryonic life and its development is driven by molecules present in the egg yolk and modulated by environmental stimuli including temperature and oxygen. Several populations of myogenic precursor cells reside in the embryonic somite and external cell layer and contribute to muscle fibres in embryo, larval, juvenile and adult stages. Many signalling proteins and transcription factors essential for these events are known. In all cases, myogenesis involves myoblast proliferation, migration, fusion and terminal differentiation. Maturation of the embryonic muscle is associated with motor innervation and the development of a scaffold of connective tissue and complex myotomal architecture needed to generate swimming behaviour. Adult muscle is a heterogeneous tissue composed of several cell types that interact to affect growth patterns. The development of capillary and lymphatic circulations and extramuscular organs – notably the gastrointestinal, endocrine, neuroendocrine and immune systems – serves to increase information exchange between tissues and with the external environment, adding to the complexity of growth regulation. Teleosts often exhibit an indeterminate growth pattern, with body size and muscle mass increasing until mortality or senescence occurs. The dramatic increase in myotomal muscle mass between embryo and adult requires the continuous production of muscle fibres until 40–50% of the maximum body length is reached. Sarcomeric proteins can be mobilised as a source of amino acids for energy metabolism by other tissues and for gonad generation, requiring the dynamic regulation of muscle mass throughout the life cycle. The metabolic and contractile phenotypes of muscle fibres also show significant plasticity with respect to environmental conditions, migration and spawning. Many genes regulating muscle growth are found as multiple copies as a result of paralogue retention following whole-genome duplication events in teleost lineages. The extent to which indeterminate growth, ectothermy and paralogue preservation have resulted in modifications of the genetic pathways regulating muscle growth in teleosts compared to mammals largely remains unknown. This review describes the use of compensatory growth models, transgenesis and tissue culture to explore the mechanisms of muscle growth in teleosts and provides some perspectives on future research directions.

A well-constrained estimate for the timing of the salmonid whole genome duplication reveals major decoupling from species diversification

Whole genome duplication (WGD) is often considered to be mechanistically associated with species diversification. Such ideas have been anecdotally attached to a WGD at the stem of the salmonid fish family, but remain untested. Here, we characterized an extensive set of gene paralogues retained from the salmonid WGD, in species covering the major lineages (subfamilies Salmoninae, Thymallinae and Coregoninae). By combining the data in calibrated relaxed molecular clock analyses, we provide the first well-constrained and direct estimate for the timing of the salmonid WGD. Our results suggest that the event occurred no later in time than 88 Ma and that 40–50 Myr passed subsequently until the subfamilies diverged. We also recovered a Thymallinae–Coregoninae sister relationship with maximal support. Comparative phylogenetic tests demonstrated that salmonid diversification patterns are closely allied in time with the continuous climatic cooling that followed the Eocene–Oligocene transition, with the highest diversification rates coinciding with recent ice ages. Further tests revealed considerably higher speciation rates in lineages that evolved anadromy—the physiological capacity to migrate between fresh and seawater—than in sister groups that retained the ancestral state of freshwater residency. Anadromy, which probably evolved in response to climatic cooling, is an established catalyst of genetic isolation, particularly during environmental perturbations (for example, glaciation cycles). We thus conclude that climate-linked ecophysiological factors, rather than WGD, were the primary drivers of salmonid diversification.

Embryonic temperature affects muscle fibre recruitment in adult zebrafish: genome-wide changes in gene and microRNA expression associated with the transition from hyperplastic to hypertrophic growth phenotypes

SUMMARY We investigated the effects of embryonic temperature (ET) treatments (22, 26 and 31°C) on the life-time recruitment of fast myotomal muscle fibres in zebrafish Danio rerio L. reared at 26/27°C from hatching. Fast muscle fibres were produced until 25 mm total length (TL) at 22°C ET, 28 mm TL at 26°C ET and 23 mm TL at 31°C ET. The final fibre number (FFN) showed an optimum at 26°C ET (3600) and was 19% and 14% higher than for the 22°C ET (3000) and 31°C ET (3100) treatments, respectively. Further growth to the maximum TL of ∼48 mm only involved fibre hypertrophy. Microarray experiments were used to determine global changes in microRNA (miRNA) and mRNA expression associated with the transition from the hyperplasic myotube-producing phenotype (M+, 10–12 mm TL) to the hypertrophic growth phenotype (M–, 28–31 mm TL) in fish reared at 26–27°C over the whole life-cycle. The expression of miRNAs and mRNAs obtained from microarray experiments was validated by northern blotting and real-time qPCR in independent samples of fish with the M+ and M– phenotype. Fourteen down-regulated and 15 up-regulated miRNAs were identified in the M– phenotype together with 34 down-regulated and 30 up-regulated mRNAs (>2-fold; P<0.05). The two most abundant categories of down-regulated genes in the M– phenotype encoded contractile proteins (23.5%) and sarcomeric structural/cytoskeletal proteins (14.7%). In contrast, the most highly represented up-regulated transcripts in the M– phenotype were energy metabolism (26.7%) and immune-related (20.0%) genes. The latter were mostly involved in cell–cell interactions and cytokine pathways and included β-2-microglobulin precursor (b2m), an orthologue of complement component 4, invariant chain-like protein 1 (iclp), CD9 antigen-like (cd9l), and tyrosine kinase, non-receptor (tnk2). Five myosin heavy chain genes that were down-regulated in the M– phenotype formed part of a tandem repeat on chromosome 5 and were shown by in situ hybridisation to be specifically expressed in nascent myofibres. Seven up-regulated miRNAs in the M– phenotype showed reciprocal expression with seven mRNA targets identified in miRBase Targets version 5 (http://microrna.sanger.ac.uk/targets/v5/), including asporin (aspn) which was the target for four miRNAs. Eleven down-regulated miRNAs in the M– phenotype had predicted targets for seven up-regulated genes, including dre-miR-181c which had five predicted mRNA targets. These results provide evidence that miRNAs play a role in regulating the transition from the M+ to the M– phenotype and identify some of the genes and regulatory interactions involved.

Evolution of the multifaceted eukaryotic akirin gene family

BackgroundAkirins are nuclear proteins that form part of an innate immune response pathway conserved in Drosophila and mice. This studies aim was to characterise the evolution of akirin gene structure and protein function in the eukaryotes.Resultsakirin genes are present throughout the metazoa and arose before the separation of animal, plant and fungi lineages. Using comprehensive phylogenetic analysis, coupled with comparisons of conserved synteny and genomic organisation, we show that the intron-exon structure of metazoan akirin genes was established prior to the bilateria and that a single proto-orthologue duplicated in the vertebrates, before the gnathostome-agnathan separation, producing akirin1 and akirin2. Phylogenetic analyses of seven vertebrate gene families with members in chromosomal proximity to both akirin1 and akirin2 were compatible with a common duplication event affecting the genomic neighbourhood of the akirin proto-orthologue. A further duplication of akirins occurred in the teleost lineage and was followed by lineage-specific patterns of paralogue loss. Remarkably, akirins have been independently characterised by five research groups under different aliases and a comparison of the available literature revealed diverse functions, generally in regulating gene expression. For example, akirin was characterised in arthropods as subolesin, an important growth factor and in Drosophila as bhringi, which has an essential myogenic role. In vertebrates, akirin1 was named mighty in mice and was shown to regulate myogenesis, whereas akirin2 was characterised as FBI1 in rats and promoted carcinogenesis, acting as a transcriptional repressor when bound to a 14-3-3 protein. Both vertebrate Akirins have evolved under comparably strict constraints of purifying selection, although a likelihood ratio test predicted that functional divergence has occurred between paralogues. Bayesian and maximum likelihood tests identified amino-acid positions where the rate of evolution had shifted significantly between paralogues. Interestingly, the highest scoring position was within a conserved, validated binding-site for 14-3-3 proteins.ConclusionThis work offers an evolutionary framework to facilitate future studies of eukaryotic akirins and provides insight into their multifaceted and conserved biochemical functions.

Temperature until the ‘eyed stage’ of embryogenesis programmes the growth trajectory and muscle phenotype of adult Atlantic salmon

We investigated how adult growth in Atlantic salmon (Salmo salar L.) was affected by changing embryonic temperature from fertilization until the completion of eye pigmentation. Fertilized eggs from several hundred families were divided between four temperature treatments (2, 5, 8 or 10°C) and subsequently reared in identical conditions in replicated tanks. Fish exposed to 2 and 5°C treatments were significantly smaller at smoltification than groups at higher temperatures, but showed substantial compensatory catch-up growth. Remarkably, temperature during this short window of embryogenesis dictated adult myogenic phenotype three years later with significant treatment effects on the muscle fibre final number (FFN), maximum diameter, nuclear density and size distribution. FFN was highest for the 5°C treatment and was reduced at higher and lower treatment temperatures. Our results require direct temperature effects on embryonic tissues, such as the stem cell-containing external cell layer, in order to produce persistent effects on juvenile and adult growth.

Evolution of Ancient Functions in the Vertebrate Insulin-Like Growth Factor System Uncovered by Study of Duplicated Salmonid Fish Genomes

Whole-genome duplication (WGD) was experienced twice by the vertebrate ancestor (2 rounds; 2R), again by the teleost fish ancestor (3R) and most recently in certain teleost lineages (4R). Consequently, vertebrate gene families are often expanded in 3R and 4R genomes. Arguably, many types of “functional divergence” present across 2R gene families will exceed that between 3R/4R paralogs of genes comprising 2R families. Accordingly, 4R offers a form of replication of 2R. Examining whether this concept has implications for molecular evolutionary research, we studied insulin-like growth factor (IGF) binding proteins (IGFBPs), whose six 2R family members carry IGF hormones and regulate interactions between IGFs and IGF1-receptors (IGF1Rs). Using phylogenomic approaches, we resolved the complete IGFBP repertoire of 4R-derived salmonid fishes (19 genes; 13 more than human) and established evolutionary relationships/nomenclature with respect to WGDs. Traits central to IGFBP action were determined for all genes, including atomic interactions in IGFBP–IGF1/IGF2 complexes regulating IGF–IGF1R binding. Using statistical methods, we demonstrate that attributes of these protein interfaces are overwhelming a product of 2R IGFBP family membership, explain 49–68% of variation in IGFBP mRNA concentration in several different tissues, and strongly predict the strength and direction of IGFBP transcriptional regulation under differing nutritional states. The results support a model where vertebrate IGFBP family members evolved divergent structural attributes to provide distinct competition for IGFs with IGF1Rs, predisposing different functions in the regulation of IGF signaling. Evolution of gene expression then acted to ensure the appropriate physiological production of IGFBPs according to their structural specializations, leading to optimal IGF-signaling according to nutritional-status and the endocrine/local mode of action. This study demonstrates that relatively recent gene family expansion can facilitate inference of functional evolution within ancient genetic systems.

An update on MyoD evolution in teleosts and a proposed consensus nomenclature to accommodate the tetraploidization of different vertebrate genomes

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

Temperature influences the coordinated expression of myogenic regulatory factors during embryonic myogenesis in Atlantic salmon (Salmo salar L.)

SUMMARY Potential molecular mechanisms regulating developmental plasticity to temperature were investigated in Atlantic salmon embryos (Salmo salar L.). Six orthologues of the four myogenic regulatory factors (MRFs: individually: smyf5, smyoD1a/1b/1c, smyoG and sMRF4), the master transcription factors regulating vertebrate myogenesis, were characterised at the mRNA/genomic level. In situ hybridisation was performed with specific cRNA probes to determine the expression patterns of each gene during embryonic myogenesis. To place the MRF data in the context of known muscle fibre differentiation events, the expression of slow myosin light chain-1 and Pax7 were also investigated. Adaxial myoblasts expressed smyoD1a prior to and during somitogenesis followed by smyoD1c (20-somite stage, ss), and sMRF4 (25–30 ss), before spreading laterally across the myotome, followed closely by the adaxial cells. Smyf5 was detected prior to somitogenesis, but not in the adaxial cells in contrast to other teleosts studied. The expression domains of smyf5, smyoD1b and smyoG were not confined to the s-smlc1 expression field, indicating a role in fast muscle myogenesis. From the end of segmentation, each MRF was expressed to a greater or lesser extent in zones of new muscle fibre production, the precursor cells for which probably originated from the Pax7 expressing cell layer external to the single layer of s-smlc1+ fibres. SmyoD1a and smyoG showed similar expression patterns with respect to somite stage at three different temperatures investigated (2°C, 5°C and 8°C) in spite of different rates of somite formation (one somite added each 5 h, 8 h and 15 h at 8°C, 5°C and 2°C, respectively). In contrast, the expression of smyf5, sMRF4 and s-smlc1 was retarded with respect to somite stage at 2°C compared to 8°C, potentially resulting in heterochronies in downstream pathways influencing later muscle phenotype.

The parallel evolution of dwarfism in Arctic charr is accompanied by adaptive divergence in mTOR-pathway gene expression

Ecological factors have a major role in shaping natural variation in body size, although the underlying mechanisms are poorly understood. Icelandic Arctic charr (Salvelinus alpinus L.) populations represent an ideal model to understand body‐size evolution, because adult dwarfism has arisen independently on multiple occasions in response to parallel environmental pressures. The mechanistic target of rapamycin (mTOR) pathway transmits signals from the environment to control cellular growth and is a primary candidate to be under selection for the dwarf phenotype. To test this hypothesis, we modified ‘inputs’ to this pathway in five dwarf and two generalist populations (with ancestral life history and body‐size traits), using a standardized manipulation of food intake in a common environment. The skeletal muscle transcript levels of 21 mTOR‐pathway genes were quantified in 274 individuals (∼6000 datapoints), and statistical modelling was used to elucidate sources of variation. Constitutive expression differences between populations were the main component of variation for around three‐quarters of the studied genes, irrespective of nutritional‐state and body‐size phenotype. There was evidence for stabilizing selection acting among populations, conserving the nutritionally dependent regulation of pathway genes controlling muscle atrophy. There were three genes (mTOR, 4E‐BP‐1 and IGFBP4), where the expression variation between dwarf and generalist populations exceeded the between‐population variation. Divergence in the expression of these candidate adaptive genes was most evident during a period of rapid growth following sustained fasting and was directionally consistent with their functions regulating growth and protein synthesis. We concluded that selection has operated efficiently to shape gene expression evolution in Icelandic charr populations and that the regulation of certain mTOR‐pathway genes evolved adaptively in locations favouring dwarfism, resulting in reduced muscle protein accretion under growth‐favouring conditions.

Salmonid genomes have a remarkably expanded akirin family, coexpressed with genes from conserved pathways governing skeletal muscle growth and catabolism

Metazoan akirin genes regulate innate immunity, myogenesis, and carcinogenesis. Invertebrates typically have one family member, while most tetrapod and teleost vertebrates have one to three. We demonstrate an expanded repertoire of eight family members in genomes of four salmonid fishes, owing to paralog preservation after three tetraploidization events. Retention of paralogs secondarily lost in other teleosts may be related to functional diversification and posttranslational regulation. We hypothesized that salmonid akirins would be transcriptionally regulated in fast-twitch skeletal muscle during activation of conserved pathways governing catabolism and growth. The in vivo nutritional state of Arctic charr (Salvelinus alpinus L.) was experimentally manipulated, and transcript levels for akirin family members and 26 other genes were measured by quantitative real-time PCR (qPCR), allowing the establishment of a similarity network of expression profiles. In fasted muscle, a class of akirins was upregulated, with one family member showing high coexpression with catabolic genes coding the NF-kappaB p65 subunit, E2 ubiquitin-conjugating enzymes, E3 ubiquitin ligases, and IGF-I receptors. Another class of akirin was upregulated with subsequent feeding, coexpressed with 14-3-3 protein genes. There was no similarity between expression profiles of akirins with IGF hormones or binding protein genes. The level of phylogenetic relatedness of akirin family members was not a strong predictor of transcriptional responses to nutritional state, or differences in transcript abundance levels, indicating a complex pattern of regulatory evolution. The salmonid akirins epitomize the complexity linking the genome to physiological phenotypes of vertebrates with a history of tetraploidization.