Dickkopf -3 protein ( Dkk3), a novel target gene of zebrafish myf5 intronic microRNA, is involved in the myf5 expression during myogenesis
Date Issued
2007
Date
2007
Author(s)
Zheng, Shu-Kai
DOI
zh-TW
Abstract
Myf5, one of myogenesis regulatory factors (MRFs), plays roles in the specification and differentiation of muscular cells during myogenesis. The expression of myf5 is in a somite- and stage-specific manner under a fine-tuned regulation mechanism. Hoi (2006) identified an intronic motif (miR-In, +610/+632) within intron I (+502/+835, I300) of zebrafish myf5. The miR-In can inhibit myf5 expression and regulate muscle development through repressing the expression of myf5. However, the target gene of miR-In is totally unknown. In this study, we found a putative EST sequence of target gene of miR-In by using pull-down assay. Then, the full length of miR-In-putative target gene was identified by using 5’ and 3’ rapid amplification of cDNA ends. The sequence of this clone was blasted with NCBI database and found that it was similar to a Dickkopf 3 protein (dkk3) sequence, named dkk3. Whole mount in situ hybridization showed that the dkk3 transcripts appeared at somites in the 10 hpf zebrafish embryos. The signals of dkk3 at somites were progressively increased and were up to maximum at 16 hpf. But the dkk3 transcripts were gradually decreased after 16 hpf and became quite low after 24 hpf. This expression pattern of dkk3 gene was consistent with that of myf5, suggesting that dkk3 might be involved in the myf5 expression at somites. Knocking down dkk3 with morpholino oligonucleotie (MO) resulted in a down-regulation of myf5 in somites but not in presomitic mesoderm. The phenotype of dkk3-MO injected embryos was similar to the myf5 morphant. At the same time, the embryos of myf5-GFP transgenic zebrafish were injected dkk3 morphant and were observed that fluorescence of GFP was down expression in somites. Co-injection of myf5 mRNA and dkk3-MO could rescue the expression of myogenin in somites, suggesting that dkk3 might influence myf5 expression in somites. The dual-luciferase assay revealed that miR-In enabled to inhibit dkk3 expression through binding to 3’UTR target sequences of dkk3. This is the first report by using pull-down assay to find that dkk3 is a target gene of the miR-In and demonstrate that miR-In could inhibit myf5 expression through repressing dkk3 protein expression during myogenesis in zebrafish.
Subjects
肌肉發育調控因子
intronic microRNA
miR-In
myf5
pull-down assay
myogenesis regulatory factors
intronic microRNA:miR-In
Type
other
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