Neil I. Bower

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.

Switching to fast growth: the insulin-like growth factor (IGF) system in skeletal muscle of Atlantic salmon

SUMMARY In this study we describe the complete coding sequence for insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), insulin-like growth factor binding protein (IGFBP) 1, 2, 4, 5 and 6 and IGFBP-related protein 1 (IGFBP-rP1) of Atlantic salmon (Salmo salar L.). We also report the characterisation of two gene paralogues of IGFBP-2 and IGFBP-5. Following 22 days restricted feeding (0 d) to achieve zero growth, fish were fed to satiation and sampled at 3, 5, 7, 14, 30 and 60 days. Expression profiles for genes involved in the IGF signalling pathway in fast myotomal muscle were determined using real-time quantitative RT-PCR. The transition from zero to fast growth is characterised by constitutive upregulation of IGF-I and IGFBP-4, a transient increase in IGFBP-5.2, and downregulation of IGFBP-2.1, IGF-II, IGF2R (IGF-II receptor) and IGFR1a (IGF-I receptor a). Expression of IGFBP-2.2, IGFBP-5.1, IGFBP-6, IGFBP-rP1 and IGFR1b showed little or no response to feeding. Expression of the myogenic marker genes myogenin, MHC and MLC2 were higher with feed restriction, and decreased as an early response to feeding, before increasing to a peak at 14 days, corresponding with a peak in IGF-I expression. IGFBP-4, which contains a putative connective tissue localisation signal, was the only IGFBP constitutively upregulated following feeding, and was positively correlated with IGF-I expression. Together, these data show that switching to fast growth in Atlantic salmon skeletal muscle involves the local upregulation of IGF-I, IGFBP-5.2 and IGFBP-4, with downregulation of IGFBP-2.1.

Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis

The VEGFC/VEGFR3 signaling pathway is essential for lymphangiogenesis (the formation of lymphatic vessels from pre-existing vasculature) during embryonic development, tissue regeneration and tumor progression. The recently identified secreted protein CCBE1 is indispensible for lymphangiogenesis during development. The role of CCBE1 orthologs is highly conserved in zebrafish, mice and humans with mutations in CCBE1 causing generalized lymphatic dysplasia and lymphedema (Hennekam syndrome). To date, the mechanism by which CCBE1 acts remains unknown. Here, we find that ccbe1 genetically interacts with both vegfc and vegfr3 in zebrafish. In the embryo, phenotypes driven by increased Vegfc are suppressed in the absence of Ccbe1, and Vegfc-driven sprouting is enhanced by local Ccbe1 overexpression. Moreover, Vegfc- and Vegfr3-dependent Erk signaling is impaired in the absence of Ccbe1. Finally, CCBE1 is capable of upregulating the levels of fully processed, mature VEGFC in vitro and the overexpression of mature VEGFC rescues ccbe1 loss-of-function phenotypes in zebrafish. Taken together, these data identify Ccbe1 as a crucial component of the Vegfc/Vegfr3 pathway in the embryo.

Transcriptional Regulation of the IGF Signaling Pathway by Amino Acids and Insulin-Like Growth Factors during Myogenesis in Atlantic Salmon

The insulin-like growth factor signalling pathway is an important regulator of skeletal muscle growth. We examined the mRNA expression of components of the insulin-like growth factor (IGF) signalling pathway as well as Fibroblast Growth Factor 2 (FGF2) during maturation of myotubes in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon (Salmo salar). The transcriptional regulation of IGFs and IGFBP expression by amino acids and insulin-like growth factors was also investigated. Proliferation of cells was 15% d−1 at days 2 and 3 of the culture, increasing to 66% d−1 at day 6. Three clusters of elevated gene expression were observed during the maturation of the culture associated with mono-nucleic cells (IGFBP5.1 and 5.2, IGFBP-6, IGFBP-rP1, IGFBP-2.2 and IGF-II), the initial proliferation phase (IGF-I, IGFBP-4, FGF2 and IGF-IRb) and terminal differentiation and myotube production (IGF2R, IGF-IRa). In cells starved of amino acids and serum for 72 h, IGF-I mRNA decreased 10-fold which was reversed by amino acid replacement. Addition of IGF-I and amino acids to starved cells resulted in an 18-fold increase in IGF-I mRNA indicating synergistic effects and the activation of additional pathway(s) leading to IGF-I production via a positive feedback mechanism. IGF-II, IGFBP-5.1 and IGFBP-5.2 expression was unchanged in starved cells, but increased with amino acid replacement. Synergistic increases in expression of IGFBP5.2 and IGFBP-4, but not IGFBP5.1 were observed with addition of IGF-I, IGF-II or insulin and amino acids to the medium. IGF-I and IGF-II directly stimulated IGFBP-6 expression, but not when amino acids were present. These findings indicate that amino acids alone are sufficient to stimulate myogenesis in myoblasts and that IGF-I production is controlled by both endocrine and paracrine pathways. A model depicting the transcriptional regulation of the IGF pathway in Atlantic salmon muscle following feeding is proposed.

The Wnt Receptor Ryk Plays a Role in Mammalian Planar Cell Polarity Signaling

Background: Ryk acts as a Wnt receptor in several processes, including mouse neuronal development. Results: Ryk interacts with Wnt11 in zebrafish convergent extension and with Vangl2 in mouse cochlear development and neural tube closure. Conclusion: Ryk is required for Wnt/planar cell polarity signaling during mammalian development and signals via Vangl2 and RhoA. Significance: This study extends our knowledge of signaling downstream of Ryk. Wnts are essential for a wide range of developmental processes, including cell growth, division, and differentiation. Some of these processes signal via the planar cell polarity (PCP) pathway, which is a β-catenin-independent Wnt signaling pathway. Previous studies have shown that Ryk, a member of the receptor tyrosine kinase family, can bind to Wnts. Ryk is required for normal axon guidance and neuronal differentiation during development. Here, we demonstrate that mammalian Ryk interacts with the Wnt/PCP pathway. In vitro analysis showed that the Wnt inhibitory factor domain of Ryk was necessary for Wnt binding. Detailed analysis of two vertebrate model organisms showed Ryk phenotypes consistent with PCP signaling. In zebrafish, gene knockdown using morpholinos revealed a genetic interaction between Ryk and Wnt11 during the PCP pathway-regulated process of embryo convergent extension. Ryk-deficient mouse embryos displayed disrupted polarity of stereociliary hair cells in the cochlea, a characteristic of disturbed PCP signaling. This PCP defect was also observed in mouse embryos that were double heterozygotes for Ryk and Looptail (containing a mutation in the core Wnt/PCP pathway gene Vangl2) but not in either of the single heterozygotes, suggesting a genetic interaction between Ryk and Vangl2. Co-immunoprecipitation studies demonstrated that RYK and VANGL2 proteins form a complex, whereas RYK also activated RhoA, a downstream effector of PCP signaling. Overall, our data suggest an important role for Ryk in Wnt/planar cell polarity signaling during vertebrate development via the Vangl2 signaling pathway, as demonstrated in the mouse cochlea.

Selection of reference genes for expression studies with fish myogenic cell cultures

BackgroundRelatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.). The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation.ResultsInitial results showed several of the candidate genes exhibited stable expression throughout myogenic culture while Sdha was identified as the least stable gene. Further analysis with geNorm, Normfinder and Bestkeeper identified Ef1α, Hprt1, Ppia and RNApolII as stably expressed. Comparison of data normalised with the geometric average obtained from combinations of any three of these genes showed no significant differences, indicating that any combination of these genes is valid.ConclusionThe geometric average of any three of Hprt1, Ef1α, Ppia and RNApolII is suitable for normalisation of gene expression data in primary myogenic cultures from Atlantic salmon.

Pkd1 Regulates Lymphatic Vascular Morphogenesis during Development

Lymphatic vessels arise during development through sprouting of precursor cells from veins, which is regulated by known signaling and transcriptional mechanisms. The ongoing elaboration of vessels to form a network is less well understood. This involves cell polarization, coordinated migration, adhesion, mixing, regression, and shape rearrangements. We identified a zebrafish mutant, lymphatic and cardiac defects 1 (lyc1), with reduced lymphatic vessel development. A mutation in polycystic kidney disease 1a was responsible for the phenotype. PKD1 is the most frequently mutated gene in autosomal dominant polycystic kidney disease (ADPKD). Initial lymphatic precursor sprouting is normal in lyc1 mutants, but ongoing migration fails. Loss of Pkd1 in mice has no effect on precursor sprouting but leads to failed morphogenesis of the subcutaneous lymphatic network. Individual lymphatic endothelial cells display defective polarity, elongation, and adherens junctions. This work identifies a highly selective and unexpected role for Pkd1 in lymphatic vessel morphogenesis during development.

Universal reference method for real-time PCR gene expression analysis of preimplantation embryos

Real-time PCR expression profiling in individual preimplantation embryos poses two main challenges. First, the amount of RNA from blastocysts (between 100 and 200 cells) is too small to quantify, and secondly, a reference gene with stable expression across preimplantation embryos produced by different reproductive technologies is required. We have developed a method using RNA and DNA spikes that allows for accurate normalization of gene expression without the use of an internal housekeeping gene in preimplantation blastocysts. Prior to the simultaneous extraction of RNA and DNA, plant-specific RNA and DNA spikes are added to the tissue. After synthesis of cDNA, target gene transcript and the exogenous RNA spike are measured using real-time PCR. To account for differences in the number of cells in each sample, the genomic gene copies of 18S-DNA are measured by quantitative PCR and normalized to the DNA spike. While the DNA spike accounts for extraction efficiency, the 18S genomic target indicates the number of cells prior to extraction. The values obtained from normalizing the target gene to the RNA spike can be adjusted for cell number, allowing the RNA spike to be used as reference gene. This universal reference approach allows the use of an exogenous spike as a pseudo-housekeeping gene for normalization of gene expression data.

Discovery and characterization of nutritionally regulated genes associated with muscle growth in Atlantic salmon

A genomics approach was used to identify nutritionally regulated genes involved in growth of fast skeletal muscle in Atlantic salmon (Salmo salar L.). Forward and reverse subtractive cDNA libraries were prepared comparing fish with zero growth rates to fish growing rapidly. We produced 7,420 ESTs and assembled them into nonredundant clusters prior to annotation. Contigs representing 40 potentially unrecognized nutritionally responsive candidate genes were identified. Twenty-three of the subtractive library candidates were also differentially regulated by nutritional state in an independent fasting-refeeding experiment and their expression placed in the context of 26 genes with established roles in muscle growth regulation. The expression of these genes was also determined during the maturation of a primary myocyte culture, identifying 13 candidates from the subtractive cDNA libraries with putative roles in the myogenic program. During early stages of refeeding DNAJA4, HSPA1B, HSP90A, and CHAC1 expression increased, indicating activation of unfolded protein response pathways. Four genes were considered inhibitory to myogenesis based on their in vivo and in vitro expression profiles (CEBPD, ASB2, HSP30, novel transcript GE623928). Other genes showed increased expression with feeding and highest in vitro expression during the proliferative phase of the culture (FOXD1, DRG1) or as cells differentiated (SMYD1, RTN1, MID1IP1, HSP90A, novel transcript GE617747). The genes identified were associated with chromatin modification (SMYD1, RTN1), microtubule stabilization (MID1IP1), cell cycle regulation (FOXD1, CEBPD, DRG1), and negative regulation of signaling (ASB2) and may play a role in the stimulation of myogenesis during the transition from a catabolic to anabolic state in skeletal muscle.

Phasing of muscle gene expression with fasting-induced recovery growth in Atlantic salmon

BackgroundMany fish species experience long periods of fasting in nature often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as myofibrillar proteins act as a major energy source. To investigate the mechanisms of metabolic reorganisation with fasting and refeeding in a saltwater stage of Atlantic salmon (Salmo salar L.) we analysed the expression of genes involved in myogenesis, growth signalling, lipid biosynthesis and myofibrillar protein degradation and synthesis pathways using qPCR.ResultsHierarchical clustering of gene expression data revealed three clusters. The first cluster comprised genes involved in lipid metabolism and triacylglycerol synthesis (ALDOB, DGAT1 and LPL) which had peak expression 3-14d after refeeding. The second cluster comprised ADIPOQ, MLC2, IGF-I and TALDO1, with peak expression 14-32d after refeeding. Cluster III contained genes strongly down regulated as an initial response to feeding and included the ubiquitin ligases MuRF1 and MAFbx, myogenic regulatory factors and some metabolic genes.ConclusionEarly responses to refeeding in fasted salmon included the synthesis of triacylglycerols and activation of the adipogenic differentiation program. Inhibition of MuRF1 and MAFbx respectively may result in decreased degradation and concomitant increased production of myofibrillar proteins. Both of these processes preceded any increase in expression of myogenic regulatory factors and IGF-I. These responses could be a necessary strategy for an animal adapted to long periods of food deprivation whereby energy reserves are replenished prior to the resumption of myogenesis.