MicroRNA-206 Regulates Somite Boundary Formation through Silencing the Target Gene in Zebrafish Embryos
2012
Hochschulschrift
Zugriff:
100
microRNA-206 is a muscle-specific miRNA that regulate myoblast proliferation and differentiation. According to Labeled miRNA pull-down assay (LAMP), the candidate gene rtn was screened from 16-hpf zebrafish embryo which at the transition state between proliferation and differentiation of muscle cell. Through in vitro and in vivo system, we found out that miR-206 was able to repress reporter activity and up-regulated the expression of post-transcription level through delayed the degradation of rtn mRNA. Using whole-mount in situ hybridization (WISH), the similar defect of morphology was observed by repression of endogenous miR-206 and over-expression of rtn mRNA. Although the regulation of rtn mRNA by miR-206 was confirmed, the effect of protein level and the function in zebrafish muscle development has not been study yet. First, we used western blot and found out that knockdown of endogenous miR-206 resulted in the increase of endogenous rtn protein expression. We proved that miR-206 was able to down-regulate the protein level of rtn. To verify the function of miR-206 in zebrafish muscle development through rtn, rtn mRNA and miR-206-MO were injected into one-cell stage embryos separately. According to phalloidin labeling, knockdown of miR-206 and over-expression rtn mRNA caused actin filament crossing-over between somites. The same defect was also observed by over-expression particular rtn isoform. Then, we found out that the defect of actin filament crossing-over between somite accompanied with defective somite boundary. Further quantified the ratio of defect, we discovered that the defective somite boundary were not regenerate through developmental time. Furthermore, we transplanted rtn mRNA injected cell into WT embryos. The transplanted cell developed to muscle and caused somite boundary growth abnormally. Taken together, these results suggested that rtn is one of target gene of miR-206. Through silencing rtn, miR-206 regulate somite and somite boundary formation in zebrafish embryo early development.
Titel: |
MicroRNA-206 Regulates Somite Boundary Formation through Silencing the Target Gene in Zebrafish Embryos
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Autor/in / Beteiligte Person: | Loo, Moo-Rung ; 盧慕蓉 |
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Veröffentlichung: | 2012 |
Medientyp: | Hochschulschrift |
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