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Published on: October 19, 2013
dickkopf-3-related gene regulates the expression of zebrafish myf5 gene through phosphorylated p38a-dependent Smad4
Ren-Jun Hsu1, Chiu-Chun Lin, Ying-Fang Su
1Institute of Molecular and Cellular Biology, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 106, Taiwan.
Abstract:
Myf5 is a myogenic regulatory factor that functions in myogenesis. An intronic microRNA, miR-In300, located within zebrafish myf5 intron I, has been reported to silence myf5 through the targeting of dickkopf-3-related gene (dkk3r). However, the molecular mechanism underlying the control of myf5 expression by dkk3r is unknown. By injecting dkk3r-specific morpholino-oligonucleotide (dkk3r-MO) to knock down Dkk3r, we found that the phosphorylated p38a protein was reduced. Knockdown of p38a resulted in malformed somites and reduced myf5 transcripts, which photocopied the defects induced by injection of dkk3r-MO. To block the MAPK pathway, phosphorylation of p38 was inhibited by introduction of SB203580, which caused the down-regulation of myf5 expression. The GFP signal was dramatically decreased in somites when we injected p38a-MO into embryos derived from transgenic line Tg(myf5(80K):GFP), in which the GFP was driven by the myf5 promoter. Although these p38a-MO-induced defects were rescued by co-injection with p38a mRNA, they were not rescued with p38a mRNA containing a mutation at the phosphorylation domain. Moreover, overexpression of Smad2 or Smad3a enhanced myf5 expression, but the defects induced by the dominant negative form of either Smad2 or Smad3a equaled those of embryos injected with either dkk3r-MO or p38a-MO. These results support the involvement of Smad2·Smad3a in p38a mediation. Overexpression of Smad4 enabled the rescue of myf5 defects in the dkk3r-MO-injected embryos, but knockdown of either dkk3r or p38a caused Smad4 protein to lose stability. Therefore, we concluded that Dkk3r regulates p38a phosphorylation to maintain Smad4 stability, in turn enabling the Smad2·Smad3a·Smad4 complex to form and activate the myf5 promoter.
Insights
Dickkopf-related gene (Dkk3r) regulates myogenesis by controlling p38a phosphorylation, which maintains Smad4 stability. This allows the Smad2·Smad3a·Smad4 complex to activate the myf5 promoter, crucial for muscle development.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- Myf5 is a key regulator of myogenesis.
- A microRNA, miR-In300, targets dickkopf-3-related gene (dkk3r) to regulate Myf5.
- The precise mechanism by which dkk3r controls myf5 expression remains unclear.
Purpose of the Study:
- To elucidate the molecular mechanism by which dkk3r influences myf5 expression.
- To investigate the role of p38a and Smad proteins in dkk3r-mediated myf5 regulation.
Main Methods:
- Knockdown of dkk3r and p38a using morpholino-oligonucleotides (MO).
- Inhibition of p38 phosphorylation using SB203580.
- Analysis of myf5 promoter activity using a Tg(myf5(80K):GFP) transgenic zebrafish line.
- Overexpression and dominant-negative studies of Smad proteins.
Main Results:
- Dkk3r knockdown reduced phosphorylated p38a levels.
- p38a knockdown mimicked dkk3r-MO induced defects in somite formation and myf5 expression.
- Inhibition of p38 phosphorylation down-regulated myf5 expression.
- Smad4 protein stability was dependent on dkk3r and p38a levels.
- Smad2, Smad3a, and Smad4 complex formation was essential for myf5 promoter activation.
Conclusions:
- Dkk3r regulates p38a phosphorylation, which is critical for maintaining Smad4 stability.
- The Smad2·Smad3a·Smad4 complex, regulated by Dkk3r and p38a, activates the myf5 promoter.
- This pathway is essential for proper myogenesis in zebrafish.
