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系統識別號 U0002-0806200720480800
中文論文名稱 新型神經嵴細胞標記,capsulin,參與斑馬魚顏面發育之分子機轉探討
英文論文名稱 Capsulin, a novel neural crest cell marker, is required for craniofacial development of zebrafish
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 95
學期 2
出版年 96
研究生中文姓名 張民彥
研究生英文姓名 Min-Yen Chang
學號 694290361
學位類別 碩士
語文別 中文
口試日期 2007-06-04
論文頁數 87頁
口試委員 指導教授-陳曜鴻
委員-蔡懷楨
委員-簡素芳
中文關鍵字 斑馬魚  capsulin  顏面肌肉  神經嵴細胞標記  發育 
英文關鍵字 Zebrafish  capsulin  craniofacial development  muscle  cartilage  neural crest cell 
學科別分類 學科別醫學與生命科學生物學
中文摘要 Capsulin是一種具有basic helix I-loop-helix II(bHLH)結構之轉錄因子,其在胚胎發育早期的功能,尤其是在肌肉發育過程中的角色與定位,仍然所知有限。為了深入研究capsulin如何參與頭部肌肉的發育,我們選用斑馬魚為材料,首先,透過反轉錄-聚合酶鏈鎖反應複殖出斑馬魚capsulin之cDNA序列,並透過5’端及3’端的放大技術,利用專一性引子的作用,進而取得可轉譯出176個胺基酸的capsulin序列。經比對後得知,斑馬魚之capsulin與人類、牛、雞、小鼠、大鼠與蟾蜍分別有75%、75%、73%、76%、75%與71%之相似度。 接著進行原位雜交,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。隨後capsulin mRNA會開始分成兩排,呈V字型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。我們也使用胚胎抑制劑(morpholino)去抑制內生性capsulin的蛋白質轉譯,加上F59肌肉抗體染色後,發現在1.5 ng /embryo之注射劑量下,斑馬魚頭部肌肉完全消失的比率為52.9±3.69%。因此我們認為斑馬魚頭部肌肉的發育需要capsulin,沒有capsulin,頭部肌肉無法發育。為了更進一步瞭解capsulin參與頭部肌肉形成的分子機制,我們選用注射胚胎發育抑制劑加上原位雜交法來進行實驗。結果發現,capsulin訊號表現位置與dlx2的訊號表現位置相同,再藉由胚胎發育抑制劑的作用發現,若內生性Capsulin的蛋白質轉譯受阻,不管是dlx2(所有神經嵴細胞的標記) 或 sox9a(軟骨專一性神經嵴細胞標記)的mRNA表現偵測上,訊號表現皆有減弱,尤其在第三群神經嵴細胞(即將形成斑馬魚branchial arch)的訊息表現受到抑制較為明顯,於是得知了capsulin為一新型斑馬魚神經嵴細胞標記。TUNEL分析法的證據顯示,抑制capsulin的表現會導致神經嵴細胞的死亡。因此推測,顱神經嵴細胞的特化及形成斑馬魚頭部肌肉需要capsulin。此外我們也發現抑制住myf5或myoD之蛋白質轉譯,其capsulin mRNA的表現情形並無顯著變化。反之,若先抑制住capsulin,結果發現當在受精後24小時與30小時,myf5的mRNA在craniofacial muscles、pectoral fin muscles與hypaxial muscles的precursor cell上的表現受到抑制,而myoD則是在受精後36小時之前其在將形成斑馬魚頭部肌肉細胞的訊息表現也同樣的受到影響,並且也發現抑制capsulin蛋白質轉譯後,如果再外加myf5、myoD、myf5與myoD之mRNA共同注射後,頭部肌肉消失之比例降低。因此,認為capsulin在myf5及myoD的上游控制斑馬魚頭部肌肉表現。綜合以上的實驗,我們推測capsulin與斑馬魚神經嵴細胞特化及形成斑馬魚頭部肌肉的發育有關,且在斑馬魚頭部肌肉的調控生成上與MRFs之間有著密切的關聯性。
英文摘要 Capsulin is a basic helix-loop-helix transcription factor involved in the regulation of cell differentiation. In chicken and mice, capsulin is expressed in mesenchymal cells and developing branchiomeric muscles derived from the non-segmented head mesoderm. However, the biological functions of capsulin during craniofacial myogenesis was little known. Here, we used zebrafish as a model to study the biological functions of capsulin during early development because of their well-defined developmental stages and genetic background. Using reverse transcription-polymerase chain reaction (RT-PCR), capsulin cDNA was cloned from the mRNA of zebrafish embryos. The deduced zebrafish Capsulin amino acid sequence revealed a 176-amino acid polypeptide containing a helix-loop-helix motif. After sequences comparison, we found that the zebrafish capsulin polypeptide shares sequence identities of 75, 75, 76, 73 and 71% with the reported capsulin of human, bovine, mouse, chicken and Xenopus, respectively. Whole-mount in situ hybridization experiments using capsulin anti-sense riboprobe revealed that capsulin was first detected at the central parts of the branchial arches of the 24 hours postfertilization (hpf) embryos. At later stages, capsulin expression was detected in the heart , pectoral fin buds and both terminal ends of each branchial arch. Double whole-mount in situ hybridization revealed that capsulin expression domains were mainly overlapped with those of dlx2 and hand2 (neural crest cells’ markers), indicating that capsulin should be a novel neural crest cells marker. To further investigate the biological functions of capsulin during early embryogenesis, we used morpholino to knock down endogenous capsulin translation. Subtle changes of cartilages and muscle fibers were easily observed when embryos were stained with Alcian blue and monoclonal antibody F59, respectively. Our data showed that knock down of capsulin led to loss of all cranial muscles. In addition, cartilages abnormalities were observed in capsulin-morphants, including loss of ceratobrachial, and enlarged angles of ceratohyal. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were also performed, and revealed that large amounts of cell apoptosis appeared in the head region of capsulin-morphants. Furthermore, whole-mount in situ hybridization experiments were carried out using anti-sense dlx2, sox9a (cranial neural crest cells’ marker) and myf5 (myocyte-specific marker) as riboprobes. Results showed that capsulin-morphants displayed reduction of cranial neural crest cells which are required for 2nd and 3rd arches formation; myf5 transcripts were down-regulated at the precursor cells of craniofacial-, pectoral fin- and hypaxial-muscles.In addition,we found that co-injection the capsulin-MO with the mRNAs of myf5、myoD、myf5 and myoD led to recovery of no cranialmuscles phenotype.These observations clearly indicated that knock down of capsulin translation induced parts of cranial neural crest cells apoptosis and consequently affected cartilages and craniofacial muscles formation. From these result, we concluded that capsulin is required for craniofacial organization, especially for cranial myogenesis in zebrafish embryos.
論文目次 第一章 前言 ------------------------------------------- 1
第二章 材料與方法 ---------------------------------- 8
第一節 實驗用斑馬魚之養成 ---------------------------------- 8
2.1.1 實驗用斑馬魚之飼養(Danio rerio)-------------- 8
2.1.2 斑馬魚之胚胎收集(Danio rerio)----------------- 8
第二節 Capsulin 基因之構築 --------------------------------- 8
2.2.1 斑馬魚胚胎核酸萃取 --------------------------------- 8
2.2.2 pGEM-capsulin 之RLM-RACE (Ambion) ---------- 9
2.2.3 PCR 產物接合與capsulin coding region 之選殖 ----- 10
2.2.4 聚合酶鏈鎖反應 ------------------------------------------ 12
2.2.5 製作Riboprobe -------------------------------------------- 12
第三節 偵測capsulin 基因之表現 ------------------------------ 14
2.3.1 固定及脫水以保存胚胎 --------------------------------- 14
2.3.2 Whole-mount in situ Hybridization ---------------------- 14
2.3.3 Whole-mount Double in situ Hybridization ------------ 15
2.3.4 抗體染色 --------------------------------------------------- 17
2.3.5 Alcain blue staining 軟骨染色 --------------------------- 19
2.3.6 TUNEL assay ------------------------------------------------ 19
第四節 Capsulin 訊號之偵測 ------------------------------------- 20
2.4.1 顯微注射實驗 --------------------------------------------- 20
2.4.2 製備DNA 模板 -------------------------------------------- 21
2.4.3 RNA 合成 --------------------------------------------------- 22
2.4.4 SDS 聚丙烯醯氨凝膠電泳( Sodium dodecyl sulfate
polyacrylamide gel electrophoresis) ---------------------------- 23
2.4.5 西方點墨法(Western Blotting) -------------------------- 24
2.4.6 螢光顯微鏡及照相系統 ------------------------------- 25
2.4.7 序列分析軟體 ------------------------------------------- 25
第三章 結果 -------------------------------------------- 26
第一節 斑馬魚capsulin 之分子結構以及與其他脊椎動物的親
緣關係 ------------------------------------------------------ 26
第二節 藉由胚胎原位雜交法(whole- mount in situ
hybridization)觀察capsulin 之時空分佈 ------------- 27
第三節 Capsulin 為斑馬魚顱神經嵴細胞之標記 ------------ 29
第四節 透過注射capsulin morpholino antisense oligonucleotide
(capsulin- MO) knock capsulin down 會導致內生性
capsulin 之表現量下降 ---------------------------------- 29
第五節 Capsulin knockdown 後斑馬魚顱神經嵴細胞因大量凋
亡而減少 --------------------------------------------------- 31
第六節 利用capsulin morpholino 抑制capsulin 蛋白質的轉譯
並確認capsulin 在斑馬魚頭部肌肉生成方面之功能33
第七節 不同濃度capsulin morpholino 注射後的存活率及肌肉
缺失的比率 ------------------------------------------------ 34
第八節 Capsulin 與MRFs 因子(myf5 及myoD)在調控斑馬魚頭
部肌肉生長上之先後順序關係 ------------------------ 35
第九節 Capsulin 與晚期的肌肉生成因子(myogenin)及肌肉的
結構蛋白標記(α-actin)在肌肉生成過程中的相互關係
----------------------------------------------------------------- 37
第十節 Capsulin、myf5 及myoD 的過度表現可補救capsulin
knockdown 所造成的缺陷 ------------------------------ 39
第十一節 Knock Capsulin down 會抑制斑馬魚頭部軟骨生成
及影響頭型尺寸 --------------------------------------- 41
第四章 討論 -------------------------------------------- 44
第一節 脊椎動物capsulin 之表現時期與位置 --------------- 44
第二節 抑制脊椎動物Capsulin 表現後所呈現的表現型態- 46
第三節 Capsulin 在整個頭部顏面肌肉生長機制中所扮演的角
色 ------------------------------------------------------------ 48
第四節 抑制capsulin 造成神經嵴細胞表現缺失因而導致顏面
組織生長缺陷 -------------------------------------------- 50
第五節 總結 -------------------------------------------------------- 52
參考文獻 ---------------------------------------------- 53
圖表目錄
Fig. 1 斑馬魚capsulin 的核苷酸及胺基酸序列。 ------------------------------------- 59
Fig. 2 斑馬魚與其他脊椎動物的capsulin 胺基酸序列比對及演化樹分析圖。--- 60
Fig. 3 斑馬魚capsulin 基因在胚胎發育時期之表現。-------------------------------- 61
Fig. 4 藉由胚胎雙重原位雜交法(whole mount double in situ hybridization)偵測capsulin
與dlx2 的表現。------------------------------------------------------------------------------ 62
Fig. 5 利用capsulin morpholino knock capsulin down 導致內生性capsulin 表現
量下降。--------------------------------------------------------------------------------- 63
Fig. 6 Knock capsulin down 造成對斑馬魚神經嵴細胞標記dlx2 表現的影響。-- 64
Fig. 7 利用TUNEL assay 偵測細胞凋亡情形。----------------------------------------- 65
Fig. 8 斑馬魚頭部肌肉的生成需要capsulin 的參與。--------------------------------- 66
Fig. 9 Capsulin knockdown 後會影響MRFs(myf5 與myoD)在斑馬魚頭部肌肉
的表現情形。--------------------------------------------------------------------------- 67
Fig.10 Capsulin knockdown 後會影響MRFs (myogenin)及結構基因α-actin 在
斑馬魚頭部肌肉的表現情形。----------------------------------------------------- 69
Fig. 11 軟骨專一性標記sox9a 在野生種斑馬魚及capsulin morphant 斑馬魚上
的表現差異。-------------------------------------------------------------------------- 70
Fig. 12 透過Alcian blue 軟骨染色觀察頭部軟骨生長情形。------------------------- 71
Fig. 13 斑馬魚capsulin 在心臟方面的表現情形。 ------------------------------------ 72
Fig. 14 斑馬魚capsulin 對於腎臟器官生長形成的影響。--------------------------- 73
Table 1.斑馬魚與其他脊椎動物capsulin 胺基酸序列的相似性。------------------- 74
Table 2.注射不同濃度capsulin morpholino 的存活率及表現率。------------------- 75
Table 3.注射capsulin MO 及不同濃度capsulin、myf5 與myoD mRNA 的表現型態。
--------------------------------------------------------------------------------------------- 76
Table 4. 野生種斑馬魚與capsulin morphant 斑馬魚頭部發育的變異情形。----- 77
附錄圖一 --------------------------------------------------------------------------------- 78
附錄圖二 --------------------------------------------------------------------------------- 79
附錄圖三 --------------------------------------------------------------------------------- 80
個人簡歷 --------------------------------------------------------------------------------- 85
期刊論文一 --------------------------------------------------------------------------------- 86
期刊論文二 --------------------------------------------------------------------------------- 87
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