Up‐regulation of muscle‐specific transcription factors during embryonic somitogenesis of zebrafish (Danio rerio) by knock‐down of myostatin‐1

Abstract

Myostatin, a secreted growth and differentiation factor (GDF-8) belongs to transforming growth factor (TGF-␤)superfamily that plays as a negative regulator of skeletal muscle development and growth. Recently, myostatin has been isolated from fish; however, its role in muscle development and growth remains unknown. Here, we present the expression of myostatin during development and the effects of its knock-down on various genes such as muscle regulatory transcription factors (MRFs), muscle-specific proteins (MSP), and insulin-like growth factors (IGFs). The myostatin expression was found to be maternal as it starts in one-cell stage onward. The reverse transcriptionpolymerase chain reaction (RT-PCR), in situ hybridization, and Southern and Northern blots demonstrated that the myostatin expression is not only restricted to skeletal muscle, but it expressed all the tested tissues. Expression of myostatin was effected by using antisense morpholinos resulted in significant phenotypic difference in stages 18 and 20 hours postfertilization (hpf). To confirm the specificity of myostatin morpholino, furthermore, a rescue experiment was conducted. The length as well as width of somites was increased with almost no gap in between the somites. In addition, it deserves to mention that this is a first animal model that shows changes in the size of the somites. Moreover, analyses of MRFs, MSP, and IGFs in the knock-down embryos by RT-PCR revealed the up-regulation of MyoD, Myogenin, and Mck transcription, whereas IGF-2 transcription showed mild response with no effect on IGF-1, Desmin, and Myf5. In situ hybridization showed that there was an increase in the number of somites from 3 to 4 at 13 and 22 hpf. Taken together, these data suggest that myostatin plays a major role during myogenesis, apart from inhibition of proliferation as well as differentiation. Developmental Dynamics 229:847-856, 2004.

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

“…It was first detected in catfish embyros beginning at day 1 postfertilization, but this required 35 cycles of amplification and was relatively weak (Kocabas et al, 2002). Similar qualitative analyses detected zebrafish myostatin mRNA in fertilized embryos, which is consistent with maternally deposited transcripts, and throughout the early and late stages of development (Amali et al, 2004;Vianello et al, 2003;Xu et al, 2003). While these studies were able to detect myostatin in fish embryos, only Amali et al specifically addressed expression during somitogenesis, when myogenesis begins, and none attempted to quantify expression during this significant stage of muscle development.…”

“…Nevertheless, the temporal expression patterns of both genes in zebrafish suggest that they are both likely involved in the early stages of muscle development. 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.…”

“…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.…”

Expression of Growth-Related Factors in Skeletal Muscle of Pirarucu(Arapaima Gigas) during Growth

“…As pirarucu achieves large size in the adult stage, the creation of many fibers during the first developmental stages is the best strategy to improve muscle growth. Myogenin regulates the late stages of muscle fiber formation and growth, which culminates in myoblast fusion, myotube formation, and adult muscle fiber differentiation [24,[75][76][77]. In the present study, neither the myogenin gene nor protein had different expression among the groups.…”

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.…”