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| Title: | 斑馬魚兩型肌肉調控因子MRF4a與MRF4b的分子結構,基因表現及生物功能 |
| Other Titles: | Molecular structure, spatiotemporal expression and biological functions of zebrafish muscle regulatory factors MRF4a and MRF4b |
| Authors: | 李駿凱;Li, Chun-kai |
| Contributors: | 淡江大學生命科學研究所碩士班
陳曜鴻;Chen, Yau-hung |
| Keywords: | 斑馬魚;肌肉調控因子;肌肉發育;Zebrafish;MRFs;MRF4;muscle development |
| Date: | 2007 |
| Issue Date: | 2010-01-11 02:28:58 (UTC+8) |
| Abstract: | 本實驗室先前的研究發現在斑馬魚體內有兩型MRF4 (MRF4 a和MRF4 b)存在，兩者蛋白質序列相似度達97%，接著利用mrf4 a和mrf4 b特異性核酸探針進行原位雜交實驗偵測兩型mrf4表現位置發現mrf4 a表現位置在olfactory placode、三叉神經 (trigeminal nerve) 和RB神經元 ;mrf4 b表現位置在體節上。接著我利用核酸類似物胚胎抑制劑 (morpholino)去分別抑制兩型或同時抑制兩型mrf4，再利用各種標定物以了解兩型mrf4在斑馬魚體內分工情形。首先利用肌肉標定物F59、α-actin、tnnt1、tnnt3b，進行免疫螢光染色和原位雜交實驗，結果發現mrf4 a morphant慢肌纖維排列不受影響，α-actin表現正常，mrf4 b被抑制會造成慢肌纖維排列混亂，α-actin表現下降，tnnt1及tnnt3b則有表現下降及表現情形混亂。接著我使用神經標定物aat (anti-acetylated tubulin)、Znp1、α-bungarotoxin、Zn5和Zn12一級抗體對兩型mrf4 morphant進行染色，發現mrf4 a morphant 上RB 神經元樹突生長數目變少且長度變短，二級運動神經發育受阻礙，而mrf4 b morphant中乙醯膽鹼接受體變少，一級運動神經生長受阻礙。综合以上結果我認為兩型mrf4功能有所不同，其中mrf4 a對於神經生長有較大相關性，而mrf4 b則和肌肉生長有較大相關性。
Our previous study shows that zebrafish has two MRF4 isoforms, MRF4 a and MRF4 b. The two MRF4 proteins exhibited a 97 % identity rate. To detect the spatiotemporal expression pattern of mrf4 a and mrf4 b at mRNA level, we used in situ hybridization. Our data showed mrf4 a expressed in trigeminal nerve, olfactory placode and RB (Rohon-beard) neuron. mrf4 b expressed in somite. To test the biological function of each mrf4, we first used anti-sense morpholino to knockdown mrf4 a and mrf4 b individually.Curved body were observed after knockdown. Then we used muscle markers F59, α-actin、tnnt1、tnnt3b，and neuron markers aat (anti-acetylated tubulin), Znp1, α-bungarotoxin, Zn5, Zn12 to detect morphological change of mrf4 morphant in skeletal muscle, slow muscle, fast muscle, motor neuron, innervations, acetylcholine receptor cluster and RB neuron. Our data shows mrf4 a knockdown led to RB neuron dendrite loss, motor neuron innervation, mrf4 b knockdown led to misalignment of slow muscle, α-actin, tnnt1, tnnt3b down reglation, secondary motorneuron defect. On the basis of our observations, we suggest that mrf4 a expression is involved in neuron development, mrf4 b expression is involved in muscle development. |
| Appears in Collections: | [生命科學研究所] 學位論文
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