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    Title: LMBRD1參與斑馬魚肌肉發育之分子機轉探討
    Other Titles: LMBRD1 plays a role during zebrafish myogenesis
    Authors: 吳欣儒;Wu, Hsin-ju
    Contributors: 淡江大學生命科學研究所碩士班
    陳曜鴻;Chen, Yau-hung
    Keywords: LMBRD1;斑馬魚;肌肉;體節;myogenesis;muscle;LMBRD1;somite;Zebrafish
    Date: 2009
    Issue Date: 2010-01-11 02:31:00 (UTC+8)
    Abstract: LMBRD1蛋白質具有467個胺基酸(約53kDa),經由序列分析,其第4至13個胺基酸可能會與肌動蛋白結合,而第193至209個胺基酸可能為核位訊號(nuclear localization signal; NLS)。由小鼠的實驗研究結果得知此LMBRD1蛋白質在老鼠實驗方面最主要作用於神經系統的海馬迴(Hippocampus)。LMBRD1缺失之小鼠,其後肢微跛,推測其肌肉發育可能受影響。為了深入研究LMBRD1如何在胚胎發育早期參與肌肉的發育,我們選用斑馬魚為材料,首先,利用野生種(WT)斑馬魚分別對各時期之胚胎執行原位雜交實驗,加以探討LMBRD1與斑馬魚早期胚胎肌肉發育時期之間的相互關係,研究結果得知其signal表現皆在頭部、嗅球、體節與肌肉,但是在脊索並無任何訊息表現。為了更近一步確認LMBRD1蛋白質表現位為肌肉組織,所以藉著LMBRD1執行抗體染色,觀察結果等同於原位雜交實驗結果。緊接著為了更加暸解NESI在斑馬魚之作用機轉,我們利用Knockdown LMBRD1方式,可觀察得到斑馬魚胚胎頭部萎縮以及魚體軀幹彎曲,緊接著再利用F59、phalloidin、α-actin、α-actinin、Dystrophinu一系列之肌肉與細胞骨架之標記抗體並染色觀察之,發現斑馬魚肌纖維排列混亂、崩解,肌動蛋白排列不規則,體節間隔板排列彎曲,肌束與肌束間隔變寬,嚴重者甚至可能造成運動神經軸索無法正常延伸發育以及心肌細胞呈現較為細長,未折疊完全之情形出現。經由F59抗體標記染色後再冷凍切片觀察得知其LMBRD1-MO之慢肌較WT排列不規則,慢肌數量上也有減少之趨勢;快肌則表現量也較少。由此,我們認為這新穎的LMBRD1蛋白質對斑馬魚胚胎在早期肌肉發育的形成與心臟發育具有關鍵性的角色。
    LMBR1 domain containing 1 protein(LMBRD1),also known as NESI,is involved in a novel nuclear export pathway.Characteristic phenotypes of the heterozygous knockout mice include stumbling,lifting the hind-legs,and dragging tails during movement suggest that LMBRD1 plays significant roles in motor coordination,learning,and memory during neuronal differentiation.To further examine its biological functions during early embryogenesis,we used zebrafish as a model system because of their well-defined developmetal stages and genetic background.We stained the embryos with a peptide antibody against human LMBRD1(αhLMBRD1) to detect the spatiotemperal expression patterns of endogenous zebrafish LMBRD1 polypeptide.At 24 hpf,expression was detected throughout the somatic mesoderm in the both fast and slow muscle populations later,zebrafish LMBRD1 was restricted in the future retina,otic placode,brain,muscle and pectoral fin buds,but were down-regulated in notochord.Transverse section through the trunk of a 72 hpf embryo showing the zebrafish LMBRD1 express on the surface of each myofibril.In the addition,zebrafish LMBRD1 express at high level in retina ganglion and brain cortex but is down-regulated in the mature muscle of adult fish.By the way,also injected the LMBRD1-morpholino into zebrafish embryos to observe the difference.The LMBRD1 knockdown fish displayed reduced head and curved-body phenotypes.To further investigate the molecular mechanisms that lead to curved-body phenotypes,we stained embryos by some muscle specific markers,such as F59,phalloidin,α-actin and dystrophin,to detect the morphological changes in muscle fiber,interactions among cytoskeletal components and membrane,respectively.Our results show that the muscle fibers of LMBRD1-morphant aligned disorderly and lost their integrity,septum curved,misalignment of motor neuron innervations,the myocardium slender.Fast and slow muscle numbers are also less than those of WT.These data suggest that LMBRD1 may function to regulate muscle fiber organization and play an important role zebrafish myogenesis.
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