Capsulin與Musculin為兩個具有basic helix I-loop-helix II(bHLH)結構之轉錄因子。為了深入研究capsulin與musculin如何參與頭部肌肉的發育，我們選用斑馬魚做為模式物種，首先，利用已知之斑馬魚capsulin胺基酸序列與其它脊椎動物進行比對，得知斑馬魚之capsulin與人類、牛、小鼠、大鼠與蟾蜍分別有75%、75%、76%、75%與71%之相似度，之後再透過反轉錄-聚合酶鏈鎖反應複殖出斑馬魚musculin序列並進行比對，得知斑馬魚之musculin與人類、牛、小鼠、大鼠與蟾蜍分別有63%、63%、63%、63%與62%之相似度。接著進行胚胎原位雜交，發現在受精後24小時的胚胎中capsulin mRNA 會表現在背側頭部後方近與卵黃交接處呈現四個點狀區塊，藉由dlx2a比對後發現capsulin表現在中胚層核心的位置上。隨後capsulin mRNA會開始分成兩排往頭部延伸表現，直到受精後三天左右就只剩在心臟部位有微弱的capsulin訊號表現；musculin的表現則區分為兩部分，在發育早期會表現在中胚層的位置上，至受精後36小時藉由myoD的比對發現其表現在顎間肌(im)的位置上，至晚期則會表現在全身骨骼肌上，因此得知兩者早期皆表現在中胚層上。之後我們使用反股寡核苷酸(morpholino)去抑制內生性capsulin與musculin的蛋白質轉譯，再藉由基因轉殖魚Tg(α-actin:RFP)及F59抗體染色觀察，發現注射過capsulin MO及musculin MO後，斑馬魚頭部肌肉完全消失的比率為52.9±3.7%及26.5±6.9%，因此我們認為斑馬魚頭部肌肉的發育需要capsulin與musculin。接著利用TUNEL assay觀察得知注射過capsulin MO及musculin MO後在後腦會有細胞凋亡的狀況，分別利用capsulin mRNA及musculin mRNA進行補救後細胞凋亡會有減緩的現象，再利用腹側中胚層核心專一性標記edn-1進行觀察，發現腹側中胚層核心不受影響，之後再利用肌肉發育相關因子myf5與myoD觀察，發現皆沒有表現，而當抑制myf5或myoD時，capsulin表現情形並無顯著變化，但晚期之musculin則受到影響，也正代表著capsulin與musculin位於中胚層核心調控myf5與myoD，而myf5與myoD也控制著晚期的musculin。另外我們也透過可定量式反轉錄聚合酶鏈鎖反應(Q-PCR)發現當capsulin或musculin被抑制時，musculin或capsulin表現量會有下降的狀況，所以接著利用musculin MO2+capsulin mRNA及capsulin MO1+musculin mRNA的實驗以補救表現量下降的狀況，亦發現頭部肌肉的缺失可被補救。綜合以上的實驗，我們推測早期capsulin與musculin在斑馬魚上會位於中胚層核心調控MRFs，晚期之musculin則可承接MRFs的訊號，進而控制整個頭部肌肉的生成。

capsulin and musculin are two related transcription factors that controlled cranial muscles development in vertebrate, but their functions still unclear. Here, we used zebrafish as an animal model to study their roles during early developmental stage. After sequence comparison, we found that the zebrafish capsulin (musculin) polypeptide shares sequence identities of 75%(63%), 75%(63%), 76%(63%), 75%(63%) and 71%(62%) with the reported capsulin (musculin) of human, bovine, mice, mouse and Xenopus, respectively. Whole mount in situ hybridization experiments showed that capsulin was first detected at mesoderm of 20 hours post-fertilization (hpf) embryos. Comparing with expression pattern of dlx2a, we found that capsulin was expressed at the central parts of dlx2a of 24hpf and 30hpf embryos, indicating that capsulin was expressed at mesoderm core of cranial neural crest cells. musculin was expressed at mesoderm of early stage. At the later stage, musculin was expressed at intermandibularis (im) by comparing with the expression pattern of myoD. Using morpholino knockdown approach, Our data showed that knock capsulin and musculin down led to loss of all cranial muscles. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed that large amounts of cell apoptosis in the hindbrain region of capsulin-morphant and musculin-morphant, but ventral mesoderm core was not affected by capsulin MO and musculin MO. Molecular evidences showed that myf5 and myoD transcription were down-regulated at the precursor cells of head muscles in capsulin-morphant and musculin-morphant. musculin was down-regulated in myf5-morphant and myoD- morphant, whereas capsulin was not affected by myf5 MO and myoD MO. Taken together, we concluded that capsulin and musculin are required for zebrafish cranial muscles development.

目錄
授權書
口試委員審議通過簽名單
謝誌----------------------------------------------I
中文摘要-----------------------------------------II
英文摘要-----------------------------------------IV
目錄---------------------------------------------VI
圖表目錄------------------------------------------X

第一章 前言---------------------------------------1
1.1 脊椎動物肌肉的起源及分化。-----------------------------1
1.2 肌肉發育相關因子在肌肉發育時所扮演的角色。-------------2
1.3 神經嵴細胞在胚胎發育上所扮演的角色。-------------------3
1.4 中胚層核心的起源及在胚胎發育上所扮演的角色。-----------3
1.5 Capsulin與Musculin在脊椎動物上所表現之位置。------------4
1.6 Capsulin與Musculin在細胞實驗上所扮演的角色。------------5
1.7 Capsulin與Musculin在脊椎動物上所扮演的角色。------------6
1.8 斑馬魚的優勢。-----------------------------------------7
1.9 本論文的研究目的。-------------------------------------8
第二章 材料與方法---------------------------------9
2.1 實驗用斑馬魚(Danio rerio)之飼養。-------------------------9
2.2 斑馬魚胚胎收集。---------------------------------------9
2.3 斑馬魚RNA萃取及cDNA合成。--------------------------9
2.4 聚合酶鏈鎖反應(polymerase chain reaction, PCR) 。----------10
2.5 定量聚合酶鏈鎖反應(real-time quantitative PCR, Q-PCR) 。----11
2.6 DNA接合反應。----------------------------------------11
2.7 勝任細胞製備與大腸桿菌(Escherichia coli)轉型。------------12
2.8 小量質體萃取。----------------------------------------12
2.9 musculin coding region複殖(cloning) 。---------------------13
2.10 胚胎固定及脫水保存(embryo fixation) 。------------------13
2.11 探針(riboprobe)合成。----------------------------------14
2.12 原位雜交法(whole-mount in situ hybridization) 。------------14
2.13 抗體染色(immunostaining) 。----------------------------16
2.14 TUNEL assay。-----------------------------------------16
2.15 顯微注射實驗。---------------------------------------17
 2.15.1 Morpholino。----------------------------------------17
 2.15.2 mRNA。-------------------------------------------18
2.16 mRNA合成。------------------------------------------18
2.16.1萃取大量plasmid。-----------------------------------18
 2.16.2製備DNA模板。------------------------------------19
 2.16.3 RNA合成。----------------------------------------19
2.17 切片及樣品製備。-------------------------------------20
2.18 序列比對。-------------------------------------------21
第三章 結果--------------------------------------22
3.1 斑馬魚(zebrafish, Danio rerio) Capsulin與Musculin之分子結構與其他脊椎動物的演化關係。---------------------------------22
3.2 capsulin與musculin時間與空間上的分佈。------------------23
3.3 注射capsulin與musculin morpholino antisense oligonucleotide可專一性的knockdown內生性capsulin及musculin。-----------------25
3.4 注射不同濃度之capsulin與musculin morpholino的存活率及肌肉缺失的比例。----------------------------------------------27
3.5 Knockdown capsulin及musculin後造成後腦區域大量細胞凋亡。---------------------------------------------------------31
3.6 Knockdown capsulin及musculin後，中胚層核心依然存在。-----32
3.7 早期之capsulin及musculin位於肌肉發育相關因子(myf5及myoD)上游調控斑馬魚胚胎頭部肌肉發育。--------------------------33
3.8 晚期之musculin位於肌肉發育相關因子(myf5及myoD)下游調控斑馬魚胚胎頭部肌肉發育，capsulin則否。----------------------34
3.9 早期capsulin與musculin在斑馬魚頭部肌肉發育上的交互關係。---------------------------------------------------------35
第四章 討論--------------------------------------37
4.1 脊椎動物capsulin及musculin之表現時期及位置具有高度保守性。------------------------------------------------------37
4.2 抑制脊椎動物之capsulin及musculin造成頭部肌肉的缺失，及capsulin對器官形成的影響。---------------------------------38
4.3 capsulin及musculin與肌肉發育相關因子(MRFs)在斑馬魚頭部肌肉發育上之關聯性。----------------------------------------40
4.4 capsulin及musculin在頭部肌肉發育上具有平行的關係。------42
4.5 調控capsulin的上游基因。-------------------------------44
參考文獻-----------------------------------------46
圖表---------------------------------------------51
附錄---------------------------------------------70




圖表目錄
Fig.1 斑馬魚與其他脊椎動物的capsulin與musculin胺基酸序列比對及演化樹分析圖。------------------------------------------52
Fig.2 內生性capsulin在早期斑馬魚胚胎上的表現。-------------54
Fig.3 內生性musculin在早期斑馬魚胚胎上的表現。-------------55
Fig.4 利用反轉錄聚合酶鏈鎖反應證明MO造成內生性capsulin與musculin mRNA剪接錯誤。----------------------------------56
Fig.5 capsulin MO及musculin MO可造成斑馬魚頭部肌肉缺失。--57
Fig.6 利用TUNEL assay觀察得知capsulin及musculin MO會造成斑馬魚胚胎有細胞凋亡的現象。--------------------------------59
Fig.7 capsulin與musculin在頭部肌肉發育上所扮演的角色。------60
Fig.8 利用各模式物種及細胞實驗證明capsulin與musculin在肌肉發育上所扮演的角色。----------------------------------------61
Fig.9 capsulin在三天大的斑馬魚胚胎會表現在心臟及腎臟。------62
Fig.10 capsulin MO注射後造成腎臟萎縮的現象。---------------63
Fig.11 藉由CPA及SU5402分別抑制Hh家族及FGF家族成員，造成capsulin的mRNA表現量下降。----------------------------64
Table.1 反轉錄聚合酶反應及探針合成所使用之引子。-----------65
Table.2 用於原位雜交法所使用之探針。-----------------------66
Table.3 用於knockdown特定基因之morpholino序列。-----------67
Table.4 斑馬魚之capsulin及musculin與其他脊椎動物之胺基酸序列相似度。--------------------------------------------------68
Table.5 capsulin MO或musculin MO造成內生性musculin或capsulin表現量下降。----------------------------------------------69

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