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

Abstract

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.

Embryonic and tissue-specific regulation of myostatin-1 and -2 gene expression in zebrafish

“…Both transcripts were nearly undetectable during gastrulation. This indicates that neither is necessary at this stage and is consistent with previous assessments of developing embryos from different fish species including tilapia (Rodgers et al, 2001), zebrafish (Biga et al, 2005), rainbow trout (Garikipati et al, 2006), brook trout (Roberts and Goetz, 2003) and even in mammals. Indeed, in situ hybridization studies of myostatin expression in mice determined that it is first detected in the myotome compartment of somites .…”

“…Curiously, the selective knockdown of MSTN-1 expression with antisense morpholino oligonucleotides was reported to increase the expression of some muscle-specific genes at 10 hpf and to increase the developmental rate and size of somites (Amali et al, 2004)-a surprising finding given that (1) zfMSTN-1 expression is virtually undetectable at this developmental time and lower than the corresponding level of zfMSTN-2 expression (Fig. 3) and (2) that other studies of zebrafish (Kerr et al, 2005), tilapia (Rodgers et al, 2001), rainbow trout (Garikipati et al, 2006 ), brook trout (Roberts and Goetz, 2003) and even mice all suggest that myostatin is not expressed during gastrulation. Xu et al (2003) also generated null fish by overexpressing a dominant-negative zfMSTN-1 (i.e.…”

“…The exact cellular source of myostatin expression in spleens cannot be definitively determined at this time. However, we recently identified myostatin expression in peripheral blood lymphocytes as well as spleens of rainbow trout (Garikipati et al, 2006), which is indicative of immune cell origins of expression rather than non-parenchymal cell origins. These data together suggest that myostatin may influence immune cell development in both fish and mammals and that it may additionally participate in stress-mediated changes in immune function.…”

Myostatin inhibits myosatellite cell proliferation and consequently activates differentiation: evidence for endocrine-regulated transcript processing

“…Most bony fish species possess at least two myostatin genes (MSTN-1 and -2; Kerr et al 2005) as a result of early genome duplication events , Postlethwait et al 1998, while rainbow trout and other salmonids have four due to their recent tetraploidization (Postlethwait et al 1998, Kerr et al 2005, Garikipati et al 2006. Although MSTN-2b is a pseudogene, functions of the other paralogs are presumably intact and each gene is differentially expressed in a tissue-and developmental-specific manner (Rodgers & Garikipati 2008).…”

Expression profiles of myostatin and calpastatin genes and analysis of shear force and intramuscular fat content of yak longissimus muscle

“…CAST is an endogenous inhibitor of Ca 2+ -dependent protease (Barnoy et al, 1997). Garikipati et al (2006) reported the widespread expression of MSTN in over twenty tissues of rainbow trout and especially high level in spleen and eyes. In the present experiment, the expression of MSTN was also observed in all tissues examined.…”