一個新發現的斑馬魚myf5 intronic microRNA目標基因dkk3和其參與myf5在肌肉發育時期的表現

Abstract

Myf5 是屬於肌肉發育調控因子 ( myogenesis regulatory factors) 之一，在肌肉細胞的特化與分化上都扮演著相當重要的角色。而myf5 的活化與抑制則受到嚴謹的調控，因此使得myf5在體節的表現具有時間與位置的特異性。在最近的研究發現斑馬魚 myf5 的intron I (+502/+835, I300) 含有一段intronic microRNA (miR-In, +610/+632) 的序列，可以抑制myf5表現的能力，並透過抑制myf5的表現達到調控肌肉的發育。然而，對於miR-In是透過抑制哪些基因轉譯成蛋白質，而調控myf5在體節的表現，目前仍然不是很清楚。在本篇研究我們利用pull down assay的技術找到一段可能是miR-In目標基因的3端EST序列，並利用5端及3端RACE ( rapid amplification of cDNA ends ) 定義出全長，再將這段序列和NCBI上的資料庫比對，發現和 Dickkopf3 (Dkk3) 類似因而命名為dkk3。從whole-mount in situ hybridization實驗結果發現，在10 hpf (hours post fertilization) dkk3就會在斑馬魚胚胎的體節表現，一直到16 hpf表現量最強，之後表現量開始下降，24 hpf以後dkk-3在體節的表現量就變的很淡。由此結果顯示dkk3在體節的表現和myf5有相似的情形，都是在16 hpf有很高量的表現，但到了24 hpf之後表現量就變的很低。這也暗示說dkk3 和myf5在體節的表現有關。利用胚胎發育抑制劑抑制 (morpholino, MO) knock down dkk3 的表 現會造成myf5在體節的表現下降但在presomitic mesoderm的表現則不受影響。而且注射dkk3-MO胚胎的表型和注射myf5 morphant的結果也很類似。同時，注射dkk-3morphant到myf5綠螢光轉殖魚胚胎可以明顯觀察到在體節部分的綠螢光表現下降。共同注射myf5 mRNA和dkk3-MO可以回復myf5 下游基因myogenin在體節的表現，也顯示說dkk3會影響myf5在體節的表現。我們進一步從dual-luciferase assay的結果，證實miR-In能夠藉由結合到dkk3 3端未轉譯區域的目標序列而抑制dkk3的蛋白質表現。這是在斑馬魚中第一篇藉由pull-down assay發現intronic microRNA, miR-In的目標基因dkk3。並且證實在體節發育過程， miR-In能透過抑制dkk3蛋白質的表現而抑制myf5在體節的表現。

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.