臺灣博碩士論文加值系統

在很多物種內 Myf5 是最早表現的肌肉特異性轉錄因子，且其轉錄機制的調控也被爭相研究。在斑馬魚的研究中，Myf5 先在前體節中胚層(PSM)被開啟，之後持續於新生體節內表現，當體節愈趨成熟後開始下降表現。但是，Myf5 在前體節中胚層是如何被開啟的卻未有被報導過。轉錄因子 Mesp 家族，包括 mespo 和 mesp-a 也是表現於前體節中胚層或體節生成小體 (somitomeres)。在這篇文章裡，我們使用核酸類似物胚胎抑制劑 (morpholino)去抑制 mespo 或 mesp-a 的蛋白質轉譯，並觀察斑馬魚 Myf5 mRNA的表現情形。首先我們注射 mespo 的胚胎抑制劑至 Myf5:BAC:gfp 這個會忠實表現內生性myf5，並以綠螢光方式呈現之轉殖魚的胚胎內，發現 Myf5-positive 綠螢光蛋白的表現被限制在前體節中胚層處，更進一步，我們使用原位雜交法 (whole mount in situ hybridization)，發現當 Mespo 蛋白質被降低後，內生性myf5 mRNA 的表現的確也被限制在尾芽(tail buds)。然而不論有無注射 mespo 的胚胎抑制劑，myoD mRNA表現都未受到影響，因此我們認為 mespo 在調控 Myf5 的轉錄表現扮演著重要的角色，當我們改注射 mesp-a 的胚胎抑制劑後，我們也觀察到同樣的結果。此外，我們也觀察到 mespo 能夠去活化 mesp-a 在體節生成小體的表現。所以當 Mespo 和 Mesp-a 的蛋白質表現都被降低時，myf5 mRNA 也會很顯著的降低。在 Mespo 蛋白質表現被降低的胚胎內，我們觀察到 her 1 和 fgf8 的 mRNA表現量也明顯的下降，所以 Mespo 蛋白質也能夠調控 her1 和 fgf8 的表現，並藉由調控 her1 和 fgf8 的表現間接的去調控體節分節過程。基於以上的觀察，我們認為 Mespo 藉由透過調控 mesp-a 及 her1 來達到其維持 myf5 在前體節中胚層的轉錄調控。

Myf5 is the first expressed muscle regulatory factor in many species and the transcription mechanism is dedicated controlled. In zebrafish, myf5 is activated in presomitic mesoderm (PSM); prolong expression at newly forming somites and down-regulation in the elder one. However, the activation mechanism of myf5 in PSM has never been reported. Mesp family, including mespo, mesp-a, are expressed in PSM or somitomere that function as transcription factors. Here, we performed morpholino (MO) knockdown experiments to examine the transcriptional effects of Mesp family on zebrafish myf5 gene. Treatment the Myf5:BAC:gfp transgenic line with mespo-MO revealed that Myf5-positive green fluorescent cells were restricted in the PSM. We furthermore using whole mount in situ hybridization to examine the expression patterns of myf5 and found that myf5 transcripts were also restricted to tail buds in mespo knockdown embryos. Compared to the wild type, myoD transcripts were unaffected, indicating that mespo played important role in regulating myf5. Similar results were observed in mesp-a knockdown embryos. Interestingly, we also found that mespo is able to activate mesp-a in somitomeres. When mespo and mesp-a were double knockdown, myf5 transcripts were down-regulated significantly. Besides, down-regulated her1 and fgf8 transcripts in mespo-morphants also showed that mespo was able to regulate her1 oscillation in anterior PSM and fgf8 expression in somites, indicating that mespo could regulate somite segmentation indirectly through regulating both her1 and fgf8. Based on these observations, we propose that transcription of myf5 on PSM is maintained by mespo through mesp-a and her1 signaling.

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Materials and Methods ------------------------------------------- 11

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