Pierre-Yves Rescan

Effect of refeeding on IGFI, IGFII, IGF receptors, FGF2, FGF6, and myostatin mRNA expression in rainbow trout myotomal muscle.

Fish endure long periods of fasting and demonstrate an extensive capacity for rapid and complete recovery after refeeding. The underlying mechanisms through which nutrient intake activates an increase in somatic growth and especially in muscle growth is poorly understood. In this study we examined the expression profile of major muscle growth regulators in trout white muscle 4, 12, and 34 days after refeeding, using real-time quantitative RT-PCR. Mean insulin-like growth factor I (IGFI) mRNA level in muscle increased dramatically 8- and 15-fold, 4 and 12 days, respectively, after refeeding compared to fasted trout. This declined thereafter. Conversely, only a weak but gradual increase in mean insulin-like growth factor II (IGFII) mRNA level was observed during refeeding. Inversely to IGFI, mean IGF receptor Ia (IGFRIa) mRNA level declined after ingestion of food. In contrast, IGF receptor Ib (IGFRIb) mRNA level was not affected by refeeding. Mean fibroblast growth factor 2 (FGF2) mRNA level increased by 2.5-fold both 4 and 12 days after refeeding, whereas fibroblast growth factor 6 (FGF6) and myostatin mRNA levels were unchanged. Subsequent to IGFI and FGF2 gene activation, an increase in myogenin mRNA accumulation was observed at 12 days post-refeeding suggesting that an active differentiation of myogenic cells succeeds their proliferation. In conclusion, among the potential growth factors we examined in this study, IGFI and FGF2 were identified as candidate genes whose expression may contribute to muscle compensatory growth induced by refeeding.

Effects of environmental temperature on IGF1, IGF2, and IGF type I receptor expression in rainbow trout (Oncorhynchus mykiss).

Recently, we have demonstrated in rainbow trout that environmental temperature may, independently of nutritional status, directly stimulate plasma growth hormone (GH) that is recognised as being an insulin-like growth factor (IGF) system regulator. The aim of this study was to determine whether temperature may directly regulate the IGF system or indirectly regulate it through plasma GH or nutritional status. For this purpose, rainbow trout were reared at 8, 12, or 16 degrees C and fed either ad libitum (similar nutritional status) to evidence the global effect of temperature, or with the same ration (1.2% body weight/day), to determine the temperature effect in fish with the same growth rate. Endocrine and autocrine/paracrine regulations of the IGF system were determined by measuring plasma IGF1 and IGF2, liver and muscle IGF1 and IGF2 mRNA as well as IGFRIa, IGFRIb mRNA, and the quantity of IGF type I receptor in muscle. Our results show that neither rearing temperature nor the nutritional status of fish affected the expression of both IGF receptor genes in muscle. Nevertheless, the quantity of IGF type I receptor determined by a binding study, appeared to be inversely proportional (P<0.05) to the rearing temperature without any relationship with nutritional status, suggesting a direct effect of temperature on its turnover. After 2 weeks of treatment, the levels of IGF1 mRNA in muscle at 8 degrees C were 2-fold higher (P<0.05) than at 16 degrees C in both ad libitum and restricted feed fish, whereas after 6 weeks, this difference was no longer observed. In both experiments, the levels of plasma IGF2 were 10-fold higher than the levels of plasma IGF1 (mean 105+/-3.0 versus 13.5+/-0.6 ng/ml), and plasma levels were correlated with their respective mRNA liver concentrations (r2=0.14 and 0.25, respectively; P<0.01). In the ad libitum feeding experiment, plasma and mRNA levels of IGF1 were related to the rearing temperature (P<0.05), while for IGF2 no effect was seen. In contrast, in the restricted feeding experiment, plasma and IGF2 mRNA levels were inversely proportional to the rearing temperature (P<0.0001) while plasma IGF1 was unaltered. Levels of plasma IGF1 were related to the growth rate in both experiments, while levels of plasma IGF2 appeared to be associated with the nutritional status of the fish. Our results suggest that the autocrine/paracrine expression of IGF1 and IGF2 in muscle is not a key regulator of the growth promoting effect of temperature. Conversely, temperature seems to promote growth through IGF1 secretion by the liver following GH stimulation, and impairment of nutritional status would prevent the IGF1 stimulation by temperature. In addition, the growth-promoting effect of temperature did not affect plasma IGF2, which appeared to be more related to the metabolic status of the fish.

Regulation and functions of myogenic regulatory factors in lower vertebrates.

The transcription factors of the MyoD family have essential functions in myogenic lineage determination and muscle differentiation. These myogenic regulatory factors (MRFs) activate muscle-specific transcription through binding to a DNA consensus sequence known as the E-box present in the promoter of numerous muscle genes. Four members, MyoD, myogenin, myf5 and MRF4/herculin/myf6, have been identified in higher vertebrates and have been shown to exhibit distinct but overlapping functions. Homologues of these four MRFs have also been isolated in a variety of lower vertebrates, including amphibians and fish. Differences have been observed, however, in both the expression patterns of MRFs during muscle development and the function of individual MRFs between lower and higher vertebrates. These differences reflect the variety of body muscle formation patterns among vertebrates. Furthermore, as a result of an additional polyploidy that occurred during the evolution of some amphibians and fish, MyoD, myogenin, myf5 and MRF4 may exist in lower vertebrates in two distinct copies that have evolved separately, acquiring specific roles and resulting in increased complexity of the myogenic regulatory network. Evidence is now accumulating that many of the co-factors (E12, Id, MEF2 and CRP proteins) that regulate MRF activity in mammals are also present in lower vertebrates. The inductive signals controlling the initial expression of MRFs within the developing somite of lower vertebrate proteins are currently being elucidated.

Dynamic gene expression in fish muscle during recovery growth induced by a fasting-refeeding schedule.

BackgroundRecovery growth is a phase of rapid growth that is triggered by adequate refeeding of animals following a period of weight loss caused by starvation. In this study, to obtain more information on the system-wide integration of recovery growth in muscle, we undertook a time-course analysis of transcript expression in trout subjected to a food deprivation-refeeding sequence. For this purpose complex targets produced from muscle of trout fasted for one month and from muscle of trout fasted for one month and then refed for 4, 7, 11 and 36 days were hybridized to cDNA microarrays containing 9023 clones.ResultsSignificance analysis of microarrays (SAM) and temporal expression profiling led to the segregation of differentially expressed genes into four major clusters. One cluster comprising 1020 genes with high expression in muscle from fasted animals included a large set of genes involved in protein catabolism. A second cluster that included approximately 550 genes with transient induction 4 to 11 days post-refeeding was dominated by genes involved in transcription, ribosomal biogenesis, translation, chaperone activity, mitochondrial production of ATP and cell division. A third cluster that contained 480 genes that were up-regulated 7 to 36 days post-refeeding was enriched with genes involved in reticulum and Golgi dynamics and with genes indicative of myofiber and muscle remodelling such as genes encoding sarcomeric proteins and matrix compounds. Finally, a fourth cluster of 200 genes overexpressed only in 36-day refed trout muscle contained genes with function in carbohydrate metabolism and lipid biosynthesis. Remarkably, among the genes induced were several transcriptional regulators which might be important for the gene-specific transcriptional adaptations that underlie muscle recovery.ConclusionOur study is the first demonstration of a coordinated expression of functionally related genes during muscle recovery growth. Furthermore, the generation of a useful database of novel genes associated with muscle recovery growth will allow further investigations on particular genes, pathways or cellular process involved in muscle growth and regeneration.

A gene with homology to myogenin is expressed in developing myotomal musculature of the rainbow trout and in vitro during the conversion of myosatellite cells to myotubes

We report the cloning of a new trout myogenic cDNA which encodes helix‐loop‐helix protein homologous to the myogenic factor myogenin. Northern analyses indicate that trout myogenin (Tmyogenin) transcripts accumulate in large amounts in the myotomal masculature of embryos and frys. In adults, transcripts concentrate within the thin lateral layer of red (slow oxydative) muscle fibres. They are present only in low amounts in white (fast glycolytic) muscle fibres which constitute the major part of the trunk musculature. Using an in vitro myogenesis system, we observed that the trout myogenin encoding gene is not activated until myosatellite cells fuse to generate multinucleated myotubes, indicating that Tmyogenin lies downstream of muscle determination factors. All these observations show that in a major taxinomic group like teleosts, a gene with homology to myogenin exists. Its activation during myogenesis suggests that it acts as a major developmental regulator of muscle differentiation.

Differential expression of two nonallelic MyoD genes in developing and adult myotomal musculature of the trout (Oncorhynchus mykiss).

Abstract Previously we identified two nonallelic MyoD encoding genes in the rainbow trout. These two MyoD genes (TMyoD and TMyoD2) were duplicated during the tetraploidization of the salmonid genome. In this study we show that TMyoD and TMyoD2 exhibit a distinct spatiotemporal pattern of expression that defines discrete cell populations in the developing somite. TMyoD expression is first detected in the mid-gastrula on either side of the elongating embryonic shield. During the anterior-to-posterior wave of somite formation the TMyoD transcript is initially present in adaxial cells of both the presomitic mesoderm and the forming somites. A lateral extension of TMyoD expression occurs only when the myotomes acquire their characteristic chevron shape pointing rostrally. By contrast, the initial expression of TMyoD2 occurs in somites that have already formed and is limited to the posterior compartment of somites. Further, in postlarval trout we observed a differential expression of TMyoD and TMyoD2 genes in muscle fibers with differing phenotype. Collectively, these data provide evidence that the two trout MyoD encoding genes have evolved to become functionally different. A comparison of the expression patterns of the two trout MyoD genes with that of myogenin allowed us to position them in the regulatory pathway leading to muscle differentiation.

Environmental temperature increases plasma GH levels independently of nutritional status in rainbow trout (Oncorhynchus mykiss)

Like many poecilotherms, salmonids exhibit seasonal variations of growth rate in relation with seasonal temperatures and plasma GH level. However, temperature alters other parameters like food intake, which may directly modify the level of plasma GH. In order to determine whether temperature regulates plasma GH levels independently of nutritional status, fish were reared at 8, 12, or 16 degrees C and either fed ad libitum (fish with different food intake) to determine the global effect of temperature, or with the same ration (1.2%/body weight) to observe the temperature effect in fish with the same growth rate. Plasma insulin level was inversely proportional to the temperature (8, 12, and 16 degrees C) in fish fed ad libitum (12.1+/-0.3 ng/ml, 10.9+/-0.3 ng/ml, 9.5+/-0.4 ng/ml; P<0.001) and in restricted fish (14.0+/-0.3 ng/ml, 11.3+/-0.3 ng/ml, 10.0+/-0.2 ng/ml; P<0.0001), probably due to a prolonged nutrient absorption, and delayed recovery of basal insulin level at low temperature. Conversely, temperature did not affect plasma T3 level of fish fed ad libitum (2.5+/-0.2 ng/ml, 2.4+/-0.1 ng/ml, 2.5+/-0.1 ng/ml at 8, 12, and 16 degrees C) while fish fed with the same ration present less T3 at 16 degrees C than at 8 degrees C (1.83+/-0.1 ng/ml versus 1.2+/-0.1 ng/ml; P<0.001) throughout the experiment; these observations indicate that different plasma T3 levels reflect the different nutritional status of the fish. The levels of GH1 and GH2 mRNA, and GH1/GH2 ratio were not different for whatever the temperature or the nutritional status. Pituitary GH content, of fish fed ad libitum did not exhibit obvious differences at 8, 12, or 16 degrees C (254+/-9 ng/g bw, 237+/-18 ng/g bw, 236+/-18 ng/g bw), while fish fed with the same ration have higher pituitary GH contents at 16 degrees C than at 8 degrees C (401+/-30 ng/g bw versus 285+/-25 ng/g bw; P<0.0001). Interestingly, high temperature strongly increases plasma GH levels (2.5+/-0.3 ng/ml at 8 degrees C versus 4.8+/-0.6 ng/ml at 16 degrees C; P<0.0001) to the same extent in both experiments, since at a given temperature average plasma GH was similar between fish fed ad libitum or a restricted diet. Our results, demonstrate that temperature regulates plasma GH levels specifically but not pituitary GH content, nor the levels of GH1 and GH2 mRNA. In addition no differential regulation of both GH genes was evidenced whatever the temperature.

Identification of a muscle factor related to MyoD in a fish species

We have isolated the cDNA encoding a myogenic factor expressed in embryonic trout muscle by hybridization with a Xenopus MyoD cDNA. Nucleotide sequence analysis and amino acid comparison showed that this cDNA called TMyoD encodes a polypeptide of 276 amino acids with 70% identity to the entire Xenopus MyoD protein and 92% identity within the basic and myc-like region. Results from Northern blotting showed that the corresponding transcript is expressed both in adult and embryonic skeletal musculature and in an in vitro myogenesis system, but is undetectable in cardiac and smooth muscles and in non muscle tissues.

New insights into skeletal muscle development and growth in teleost fishes

Recent research has significantly broadened our understanding of how the teleost somite is patterned to achieve embryonic and postembryonic myogenesis. Medial (adaxial) cells and posterior cells of the early epithelial somite generate embryonic superficial slow and deep fast muscle fibers, respectively, whereas anterior somitic cells move laterally to form an external cell layer of undifferentiated Pax7-positive myogenic precursors surrounding the embryonic myotome. In late embryo and in larvae, some of the cells contained in the external cell layer incorporate into the myotome and differentiate into new muscle fibers, thus contributing to medio-lateral expansion of the myotome. This supports the suggestion that the teleost external cell layer is homologous to the amniote dermomyotome. Some of the signalling molecules that promote lateral movement or regulate the myogenic differentiation of external cell precursors have been identified and include stromal cell-derived factor 1 (Sdf1), hedgehog proteins, and fibroblast growth factor 8 (Fgf8). Recent studies have shed light on gene activations that underlie the differentiation and maturation of slow and fast muscle fibers, pointing out that both adaxially derived embryonic slow fibers and slow fibers formed during the myotome expansion of larvae initially and transiently bear features of the fast fiber phenotype.

Muscle fiber differentiation in fish embryos as shown by in situ hybridization of a large repertoire of muscle-specific transcripts.

Skeletal muscles are composed of different fiber types, largely defined by differential expression of protein isoforms involved in myofibrillogenesis or metabolism. To learn more about the gene activations that underlie the differentiation and the diversification of embryonic fish myotomal fibers, we investigated the developmental expression of 25 muscle genes in trout embryos by in situ hybridization of muscle‐specific transcripts. The earliest event of muscle differentiation, at approximately the 25‐somite stage, was the expression of a variety of muscle‐specific genes, including slow‐twitch and fast‐twitch muscle isoforms. The activation of these muscle genes started in the deep somitic domain, where the slow muscle precursors (the adaxial cells) were initially located, and progressively spread laterally throughout the width of the myotome. This mediolateral progression of gene expression was coordinated with the lateral migration of slow adaxial cells, which specifically expressed the slow myosin light chain 1 and the SLIM1/FHL1 genes. Subsequently, the fast and slow skeletal muscle isoforms precociously expressed in the course of the mediolateral wave of muscle gene activation became down‐regulated in the superficial slow fibers and the deep fast fibers, respectively. Finally, several muscle‐specific genes, including troponins, a slow myosin‐binding protein C, tropomodulins, and parvalbumin started their transcription only in late embryos. Taken together, these findings show in fish embryos that a common myogenic program is triggered in a mediolateral progression in all muscle cells. The acquisition of the slow phenotype involves the additional activation of several slow‐specific genes in migrating adaxial muscle cells. These events are followed by sequential gene activations and repressions in fast and slow muscle cells. Developmental Dynamics 233:659–666, 2005.