The negative regulatory mechanism of myogneic regulatory factor myf5 promoter activity of Zebrafish

Although a strong, negative cis-regulatory motif located at the first intron +502/+835 (I300) of zebrafish myf5 was reported, the detailed molecular mechanism underlying the negative modulation remains unclear. To understand this repression network, we microinjected zebrafish eggs with RNA corresponding to the sense strand of I300, resulting in the dramatic reduction of luciferase reporter activity driven by the myf5 promoter. Within this I300 segment, we identified an intronic microRNA located at +610/+632 (miR-In300) and observed that it appeared predominately at the mature somites, but weakly at newly formed somites, which were opposed to the expression patterns of zebrafish myf5. By using the novel method we developed, labeled miRNA pull-down assay system (LAMP), we subsequently screened the Dickkopf-3 gene (dkk3) and identified it as the target gene of miR-In300. Inhibition of the dkk3 translation by microinjection of the dkk3-specific antisense oligonucleotide (MO) did result in down-regulation of myf5 transcripts in somites, whereas co-injection of myf5 mRNA with dkk3-MO enabled rescue of the defects induced by microinjection of dkk3-MO alone. In addition, microinjection of the miR-In300-MO enhanced myf5 transcripts in somites and Dkk3 protein level in zebrafish embryos. We concluded that miR-In300 binds to the dkk3, which, in turn, inhibits the translation of the dkk3 mRNA and results in suppressing the promoter activity of zebrafish myf5.