研究Duxbl基因在出生後小鼠肌肉發育及轉殖斑馬魚的功能

Explore Functions of The Mouse Double Homeobox gene, Duxbl, in mouse posnatal myogenesis and in transgenic Zebrafish

周晉毅(Chin-Yi Chou)

指導教授 ： 王淑紅

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2014年)

https://doi.org/10.6834/CSMU.2014.00089

摘要

同源箱基因是一群具有調節基因轉錄能力的基因，其所轉譯出來的蛋白質含有的同源箱域(homeodomain proteins)具有辨識DNA與調節基因表現的能力，因此同源箱基因可以調控胚胎發育過程中細胞分化以及決定細胞的命運。先前實驗室選殖到一個可以轉譯出二個同源箱域的雙同源箱基因Duxbl (murine double homeobox gene)，是第一個在齧齒類哺乳動物中發現的雙同源箱基因。小鼠的Duxbl基因與人類雙同源箱基因DUX4的序列有高達67%的相似度。先前的研究已經證實Duxbl基因在胚胎發育過程中，表現在小鼠軀幹、四肢的延長的骨骼肌細胞以及肌小管中，這說明了Duxbl基因與胚胎發育時期肌肉發育有密切的關係。在本論文中，我們進一步分析小鼠出生後Duxbl蛋白在肌纖維的表現，發現未活化的肌纖維之衛星細胞，沒有Duxbl蛋白的表現，而培養活化之肌纖維中可以測到Duxbl與MyoD或MyoG蛋白共同染色於活化的衛星細胞，另外注射cardiotoxin造成肌纖維損傷後，再生修復過程中也發現Duxbl與MyoD蛋白共同表現於活化的衛星細胞，因此推測Duxbl參與出生後小鼠之肌纖維之生長與再生過程。在細胞實驗中，我們發現過度表現Duxbl基因會促進C2C12細胞增生及抑制細胞分化。在促進增生方面，以RT-PCR以及Western blot分析的結果顯示，Duxbl基因會促進cyclin D1和pRB表現及抑制p21的表現，因而促進C2C12細胞增生。在抑制分化方面，Duxbl蛋白會抑制MyoD調控下游基因的能力，包括M-cadherin、MyoG、p21及cyclin D3的表現，最後抑制細胞分化。另外，過度表現Duxbl於持續表現MyoD的10T1/2細胞，同樣也抑制10T1/2 MyoD細胞分化成肌肉細胞的能力，抑制MyoD下游基因M-cadherin、MyoG及p21的表現。我們也以Tol2系統建立了Duxbl以及Duxbl-s基因轉殖斑馬魚系統，分析Duxbl或Duxbl-s對於斑馬魚肌肉發育的影響，在三重複的觀察下，結果顯示Duxbl以及Duxbl-s的轉殖魚分別有9.52%及17.18%具有體節彎曲及肌肉紋路模糊的輕微異常的現象，以蘇木紫-伊紅染色觀察轉殖魚的肌纖維型態，結果發現無論是Duxbl或是Duxbl-s轉殖魚相較於控制組，肌纖維都有明顯的結構鬆散、細胞核型態模糊的異常情況發生。另外在胚胎嚴重變異的轉殖魚中，還觀察到只有一個耳石的異常的現象，正常斑馬魚有兩個耳石。從以上的結果可以知道，Duxbl參與了出生後小鼠肌肉的生長以及損傷時的修復與再生肌肉的過程，而過度表現Duxbl會促進細胞增生及抑制肌肉細胞的分化，同時Duxbl或Duxbl-s也會影響斑馬魚的肌肉發育。Duxbl影響斑馬魚肌肉發育的分子機制需要進一步探討。

關鍵字

同源箱基因 ； Duxbl基因 ； DUX4基因 ； 肌肉調控因子 ； 細胞週期調控因子 ； 衛星細胞 ； 斑馬魚

並列摘要

The homeobox gene families are characterized by a conserved DNA motif encoding the DNA-binding homeodomain. Homeodomain proteins act as transcription factors to regulate gene expressions in embryonic development and cell differentiation. Previously, we cloned and characterized a novel double homeobox gene, Duxbl (murine double homeobox gene). The homeodomain of Duxbl exhibits the highest 67% similarity to that of human DUX4, which associated with facioscapulohumeral muscular dystrophy. Previous studies have confirmed that Duxbl was expressed in skeletal muscles in trunk and limbs during mouse embryonic development. In this thesis, Duxbl expressions in postnatal mouse muscles were analyzed. The results also showed that Duxbl was expressed in activated satellite cells and colocalized with MyoD and MyoG, but no Duxbl signal was detected in non-activated satellite cells. In addition, following treatment with cardiotoxin, Duxbl and MyoD were co-localized in the satellite cells of regenerating adult skeletal muscle. In results of gain of function studies in C2C12 cells, we found that Duxbl overexpression promotes cyclin D1 and ppRB expressions, but inhibit p21 expressions by RT-PCR and Western blot analyses. Thus, Duxbl overexpression promotes C2C12 cell proliferation in growing condition. Furthermore, Duxbl overexpression inhibits C2C12 cell differentiation. The expression levels of MyoD were not influenced in Duxbl overexpressed C2C12 cells in differentiation condition. However, the expressions of MyoD-regulated downstream genes, M-cadherin, MyoG, p21, cyclin D3 were all repressed in Duxbl overexpressed C2C12 cells in differentiation condition. Moreover, Duxbl overexpression also repressed MyoD-induced M-cadherin, MyoG and p21 expressions in 10T1/2 MyoD cells under differentiation condition. On the other hand, we have established fast-fiber specific expressed Duxbl and Duxbl-s transgenic zebrafishes to study the effects of Duxbl and Duxbl-s overexpressions during zebrafish muscle development, respectively. The results show that there were 9.52% of Duxbl and 17.18% of Duxbl-s transgenic zebrafishes exhibited abnormal somites. In addition, histochemical examinations of skeletal muscle fibers showed that the organization of myofibrils in Duxbl and Duxbl-s transgenic zebrafishes appeared slightly wavy and the gap between neighboring myofibrils increased, as well as the cell nucleus number decreased when compared with control transgenic zebrafishes. We also observed only one otolith in Duxbl transgenic fish, but normal fish has two. In conclusion, Duxbl gene is involved in the embryonic myogenesis, postnatal mouse muscle growth, and repair of muscle injury. Ectopic expressions of Duxbl promote cell proliferation, inhibit differentiation of mouse muscle cells, and affect the skeletal muscle development (somite and otolith) in zebrafish. How does Duxbl expression affect muscle development in zebrafish need to be further explored.

並列關鍵字

Homeobox gene ； Duxbl gene ； DUX4 gene ； myogenic regulatory factors ； cell cycle regulators ； Satellite cell ； Zebrafish

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