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    Title: 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
    Other Titles: Capsulin, a novel neural crest cell marker, is required for craniofacial development of zebrafish
    Authors: 張民彥;Chang, Min-yen
    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;capsulin;顏面肌肉;神經嵴細胞標記;發育;Zebrafish;capsulin;craniofacial development;muscle;cartilage;neural crest cell
    Date: 2007
    Issue Date: 2010-01-11 02:31:08 (UTC+8)
    Abstract: 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。
    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.
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