Analysis ofmyostatingene structure, expression and function in zebrafish

Abstract

SUMMARYMyostatin is a member of the TGF-β family that functions as a negative regulator of skeletal muscle development and growth in mammals. Recently,Myostatin has also been identified in fish; however, its role in fish muscle development and growth remains unknown. We have reported here the isolation and characterization of myostatin genomic gene from zebrafish and analysis of its expression in zebrafish embryos, larvae and adult skeletal muscles. Our data showed that myostatin was weakly expressed in early stage zebrafish embryos, and strongly expressed in swimming larvae, juvenile and skeletal muscles of adult zebrafish. Transient expression analysis revealed that the 1.2 kb zebrafish myostatin 5′ flanking sequence could direct green fluorescent protein (GFP) expression predominantly in muscle cells, suggesting that the myostatin 5′ flanking sequence contained regulatory elements required for muscle expression. To determine the biological function of Myostatin in fish, we generated a transgenic line that overexpresses the Myostatin prodomain in zebrafish skeletal muscles using a muscle-specific promoter. The Myostatin prodomain could act as a dominant negative and inhibit Myostatin function in skeletal muscles. Transgenic zebrafish expressing the Myostatin prodomain exhibited no significant change in myogenic gene expression and differentiation of slow and fast muscle cells at their embryonic stage. The transgenic fish, however,exhibited an increased number of myofibers in skeletal muscles, but no significant difference in fiber size. Together, these data demonstrate that Myostatin plays an inhibitory role in hyperplastic muscle growth in zebrafish.

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

“…Although only a few developmental stages were sampled (eyed, hatched/sac present, and swim-up fry), their results also indicate that the expression of both genes rises substantially after eyeing and rtMSTN-1b mRNA levels are significantly higher than previously reported. Myostatin expression in mammals is first detected within the developing myotome (Kambadur et al 1997, although former attempts to localize myostatin message in fish somites have produced mixed results (Xu et al 2003, Amali et al 2004, Kerr et al 2005. Nevertheless, our results are the first to identify a temporal expression pattern in fish that is consistent with a functional role during the early stages of muscle development as levels of both rtMSTN-1a and rtMSTN-1b rise substantially throughout somitogenesis and begin to subside just before this developmental period ends.…”

“…The annotated gene and promoter sequences were then deposited with GenBank and assigned the accession numbers DQ136028 and DQ138300 respectively. Each gene is organized into three exons of similar size with appropriate intron/exon splice sites, a pattern that is conserved with other fish species (Maccatrozzo et al 2001b, Xu et al 2003, Kerr et al 2005 and mammals , Gonzalez-Cadavid et al 1998, Stratil & Kopecny 1999, Jeanplong et al 2001. The three rtMSTN-1a exons are 490, 368, and 1600 bp in size respectively, and are separated by 1072 and 992 bp introns (Fig.…”

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

“…Myostatin signaling deficiency through mutation or knock-out results in the increase of the skeletal muscles mass where the muscles are 100-200% larger than normal through hyperplasia and hypertrophy, as seen in mammals including humans. [29][30][31][32][33][34][35][36] In dogs with only a single functional myostatin allele, improved muscle function was observed, although not as an overt increase in muscle mass as seen with homozygotes. 34 Pharmacological inhibition of myostatin activity in rodents from either myostatin neutralizing antibodies, mutant myostatin propeptides or even decoy myostatin receptor-fusion proteins, results in increased muscle mass and improved muscle function.…”

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

“…Attempts to determine expression patterns with in situ hybridization have failed or have suggested ubiquitous expression, likely because the level of myostatin expression is too low to be detected with high specificity using this method (Amali et al, 2004;Vianello et al, 2003;Xu et al, 2003). Most other analyses have therefore used RT-PCR to evaluate expression of myostatin.…”