Submitted to: Journal of Endocrinology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/21/2006
Publication Date: 6/30/2006
Citation: Garikipati, D.K., Gahr, S.A., Rodgers, B.D. 2006. Identification, characterization, and quantitative expression analysis of rainbow trout myostatin-1a and myostatin-1b genes. Journal of Endocrinology. 190:879-888. Interpretive Summary: Muscle growth results from the proliferation of myoblasts and their subsequent differentiation into muscle fibers. This process is regulated by mechanisms that include cell-to-cell interactions, cell-to-matrix interactions, and extracellular secreted factors such as myostatin (MSTN; also known as growth/differentiating factor (GDF)-8). In the Rainbow Trout, at least two homologues of the myostatin transcript have been identified, however, very little is know about the regulatory region of the gene or expression during embryonic development. In this study, we determined the sequence of the full length MSTN1a and MSTN1b gene including the promoter regions. We additionally report the quantitative assessment of the expression of each gene during embryonic/larval development and in a number of adult tissues. These studies indicate strong sequence conservation among all vertebrate myostatin genes. The expression patterns described and the subsequence analysis of the different promoters further support a role for both the MSTN-1 genes during fish myogenesis, although the ubiquitous expression pattern in different adult tissues suggests that the functional role of myostatin is far more diverse than that in mammals. The presence of multiple fish genes that are differentially expressed throughout development and adult tissues also suggests that the precise role of a particular gene may vary between tissues.
Technical 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.