The effects of chronic immune stimulation on muscle growth in rainbow trout.

Successful production of aquaculture species depends on efficient growth with low susceptibility to disease. Therefore, selection programs have focused on rapid growth combined with disease resistance. However, chronic immune stimulation diminishes muscle growth (a syndrome referred to as cachexia), and decreases growth efficiency in production animals, including rainbow trout. In mammals, recent results show that increased levels of pro-inflammatory cytokines, such as those seen during an immune assault, specifically target myosin and MyoD and inhibit muscle growth. This suggests that increased disease resistance in fish, a desired trait for production, may actually decrease the growth of muscle, the main aquacultural commodity. To test this possibility, a rainbow trout model of cachexia was developed and characterized. A six-week study was conducted in which rainbow trout were chronically immune stimulated by repeated injections of LPS. Growth indices were monitored, and whole body and muscle proximate analyses, real-time PCR, and Western blotting were conducted to examine the resulting cachectic phenotype. Muscle ratio was decreased in fish chronically immunostimulated, however expression levels of MyoD2 and myosin were not decreased compared to fish that were not immunostimulated, indicating that while muscle accretion was altered, the mechanism by which it occurred was somewhat different than that characterized in mammals. Microarray analysis was used to compare gene expression in fish that had been chronically immunostimulated versus those that had not to identify possible alternative mechanisms of cachexia in fish.

Alterations in expression of genes associated with muscle metabolism and growth during nutritional restriction and refeeding in rainbow trout.

Rainbow trout, as well as many other species of fish, demonstrate the ability to survive starvation for long periods of time. During starvation, growth rate is decreased and muscle exhibits signs of wasting. However, upon resumption of feeding, accelerated growth is often observed. Alterations in muscle metabolism occur during feed restriction and refeeding, although the ways in which these alterations affect the molecular pathways that control muscle growth have not been fully determined. To analyze changes in muscle metabolism and growth during starvation and refeeding, real-time PCR was used to test the expression of six metabolic-related genes and eight muscle-specific genes in rainbow trout white muscle prior to and after 30 days of starvation, and after 4 and 14 days of refeeding. The six metabolic-related genes chosen are indicative of specific metabolic pathways: glycolysis, glycogenesis, gluconeogenesis, the pentose phosphate pathway, and fatty acid formation. The eight muscle specific genes chosen are key components in muscle growth and structural integrity, i.e., MRFs, MEFs, myostatins, and myosin. Alterations in expression of the tested metabolic-related genes and muscle-specific genes suggest that during both starvation and refeeding, changes in specific metabolic pathways initiate shifts in muscle that result mainly in the modification of myotube hypertrophy. The expression levels of many of the metabolic-related genes were altered during the refeeding period compared to those observed before the starvation period began. However, the accelerated growth often observed during refeeding is likely driven by changes in normal muscle metabolism, and the altered expression observed here may be a demonstration of those changes.

Quantitative expression analysis of genes affecting muscle growth during development of rainbow trout(Oncorhynchus mykiss).

The molecular characterization of the hyperplasia and hypertrophy that characterize postembryonic muscle development in rainbow trout is of great interest to aquaculturists because of the commercial value of the species. Determination of temporal expression levels of the genes that control muscle development is an important step in molecular analysis. Real-time quantitative reverse transcriptase polymerase chain reaction was used to characterize expression in the muscle of 3 MRF, 2 MEF, and 2 myostatin genes during 9 stages of trout development. Expression of genes that promote muscle growth (MRF and MEF) peaked in swim-up fry, and in some cases again in 25-g, 140-g, and spawning fish. Myostatin genes, which restrict muscle growth, were expressed at very low levels early in development, but their expression levels were elevated in 140-g and spawning fish. Expression levels and the known function of each tested gene were used to infer the extent of hyperplasia, hypertrophy, and restriction of muscle growth during each stage. Both hyperplasia and hypertrophy appeared to peak in swim-up fry and spawning females, and hyperplasia also appeared to peak in 25-g fish. These results should provide valuable information for developmental biologists and those interested in understanding muscle growth in fish.

Sequence, conservation, and quantitative expression of rainbow trout Myf5.

The success of rainbow trout as an aquaculture species is dependent on the ability to produce fish with large amounts of high-quality lean muscle. It is therefore important to understand not only the best conditions under which to raise the fish but also the molecular control of muscle growth. Vertebrate muscle growth is initiated by the specification of myogenic precursor cells into myoblasts. The myoblasts proliferate and fuse to form multinucleated myotubes, which mature into myofibers. A family of basic helix-loop-helix (bHLH) transcription factors, the Myogenic Regulatory Factors (MRFs), controls these events. In trout, two MRF-encoding genes, TMyoD (of which there are two) and Tmyogenin, have been identified. However, the primary MRF-encoding Myf5 is not yet sequenced. Here, using degenerate PCR and 5' and 3' RACE, the cDNA sequence of trout Myf5 (TMyf5) is identified. Translation of the cDNA reveals that TMyf5 is a bHLH protein with homology to Myf5 and MRFs in other organisms. It is expressed mainly in red and white muscle, suggesting that it shares functional homology to Myf5 in other species. The molecular control of muscle growth has been well-characterized in mammals, but there are differences in the growth of fish muscle, highlighting the need for characterization of MRFs in fish species, particularly those in which understanding muscle growth will have a positive impact on the economic potential of the species.

The Drm-Bowl-Lin relief-of-repression hierarchy controls fore- and hindgut patterning and morphogenesis.

The elucidation of pathways linking patterning to morphogenesis is a problem of great interest. We show here that, in addition to their roles in patterning and morphogenesis of the hindgut, the Drosophila genes drumstick (drm) and bowl are required in the foregut for spatially localized gene expression and the morphogenetic processes that form the proventriculus. drm and bowl belong to a family of genes encoding C(2)H(2) zinc finger proteins; the other two members of this family are odd-skipped (odd) and sob. In both the fore- and hindgut, drm acts upstream of lines (lin), which encodes a putative transcriptional regulator, and relieves its repressive function. In spite of its phenotypic similarities with drm, bowl was found in both foregut and hindgut to act downstream, rather than upstream, of lin. These results support a hierarchy in which Drm relieves the repressive effect of Lin on Bowl, and Bowl then acts to promote spatially localized expression of genes (particularly the JAK/STAT pathway ligand encoded by upd) that control fore- and hindgut morphogenesis. Since the odd-family and lin are conserved in mosquito, mouse, and humans, we propose that the odd-family genes and lin may also interact to control patterning and morphogenesis in other insects and in vertebrates.

Localized JAK/STAT signaling is required for oriented cell rearrangement in a tubular epithelium.

Rearrangement of cells constrained within an epithelium is a key process that contributes to tubular morphogenesis. We show that activation in a gradient of the highly conserved JAK/STAT pathway is essential for orienting the cell rearrangement that drives elongation of a genetically tractable model. Using loss-of-function and gain-of-function experiments, we show that the components of the pathway from ligand to the activated transcriptional regulator STAT are required for cell rearrangement in the Drosophila embryonic hindgut. The difference in effect between localized expression of ligand (Unpaired) and dominant active JAK (Hopscotch) demonstrates that the ligand plays a cell non-autonomous role in hindgut cell rearrangement. Taken together with the appearance of STAT92E in a gradient in the hindgut epithelium, these results support a model in which an anteroposterior gradient of ligand results in a gradient of activated STAT. These results provide the first example in which JAK/STAT signaling plays a required role in orienting cell rearrangement that elongates an epithelium.

Drumstick is a zinc finger protein that antagonizes Lines to control patterning and morphogenesis of the Drosophila hindgut.

Elongation of the Drosophila embryonic hindgut epithelium occurs by a process of oriented cell rearrangement requiring the genes drumstick (drm) and lines (lin). The elongating hindgut becomes subdivided into domains -- small intestine, large intestine and rectum -- each characterized by a specific pattern of gene expression dependent upon normal drm and lin function. We show that drm encodes an 81 amino acid (10 kDa) zinc finger protein that is a member of the Odd-skipped family. drm expression is localized to the developing midgut-hindgut junction and is required to establish the small intestine, while lin is broadly expressed throughout the gut primordium and represses small intestine fate. lin is epistatic to drm, suggesting a model in which localized expression of drm blocks lin activity, thereby allowing small intestine fate to be established. Further supporting this model, ectopic expression of Drm throughout the hindgut produces a lin phenotype. Biochemical and genetic data indicate that the first conserved zinc finger of Drm is essential for its function. We have thus defined a pathway in which a spatially localized zinc finger protein antagonizes a globally expressed protein, thereby leading to specification of a domain (the small intestine) necessary for oriented cell rearrangement.