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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/16551

Title: tbx5基因對斑馬魚胚胎時期心臟發育之影響及心臟缺損模式之建立
The expression patterns of tbx5gene and its impacts on embryonic development and establishment of heart defect model on zebrafish (Danio rerio) by tbx5 gene knockdown technique
Authors: Yu-Chi Li
李玉琪
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 心臟缺損模式;胚胎心臟發育
heart defect model;heart looping;tbx5
Date: 2004
Issue Date: 2011-06-30T08:39:51Z
Abstract: Tbx5屬於T-box家族的轉錄因子,在脊椎動物胚胎發育時期表現於心臟、前肢和眼睛。本研究之目的是以斑馬魚作為模式魚種探討心臟發育調控因子對心臟發育的影響。經由RT-PCR分析結果顯示tbx5 基因在1細胞時期已可偵測到,推測tbx5基因在胚胎發育早期可能為重要的調控因子。在14 hpf時 tbx5 mRNA表現量會開始增加。在22 hpf達到最高量;且在24 hpf迅速降低至基礎表現量。利用RNA全覆式原位雜交法可偵測tbx5基因在不同胚胎發育時期分別表現於心臟、胸鰭及眼睛。利用專一性反意股寡核酸(morpholino antisense oligos)抑制胚胎時期tbx5基因表現時會造成斑馬魚胚胎之軀幹(trunk)缺損包括軀幹縮短。注射tbx5-MO造成胚胎心臟缺損,包括:心臟looping缺損(heart looping defect)、心室與心房變小、心包膜積水(pericardium effusion)、腹部積水(dropsy of ventral position)、心跳頻率減緩、無明顯血液流動。注射tbx5-MO亦會造成胚胎胸鰭缺損,包括:胸鰭生長方向折曲向前、胸鰭縮短及無胸鰭之胚胎個體。注射tbx5-MO濃度越高使斑馬魚胚胎活存率越低。注射tbx5-MO濃度越高造成斑馬魚胚胎心臟looping缺損、心包膜積水與腹部積水之不正常比例越高,顯示tbx5基因對心臟發育的影響具有劑量相關(dose-dependent)之特性。注射tbx5-MO造成斑馬魚胚胎心臟缺損與造成胸鰭缺損可能無關聯性。本研究將注射tbx5-MO造成斑馬魚胚胎心臟發育缺損依嚴重程度分為5個等級。Degree 0為正常胚胎;Degree I的胚胎心臟有looping缺損的現象,且心包膜或腹部積水有輕微積水。Degree II的胚胎心臟有looping缺損的現象,且心包膜和腹部有嚴重積水。Degree III的胚胎心臟無looping進行,心臟構造變為細長導致心腔變小。Degree IV等級的胚胎其軀幹嚴重變形,心臟幾乎無法發育。tbx5基因表現量減少對所造成心臟缺損嚴重程度具有劑量相關之特性。tbx5基因表現量減少也導致心臟發育之相關調控基因dHAND、nkx2.5與肌肉生成相關基因mlc2、amhc、vmhc、IGF、myoD及myostatin等基因的表現量均降低。本研究結果顯示心臟looping缺損的徵狀可能是因tbx5表現量不足,進而造成心肌細胞命運決定因子nkx2.5,心肌纖維主要結構之amhc及vmhc基因,及肌肉生長調控因子IGF的基因表現不正常所致。本研究結果亦顯示tbx5基因表現量不足會影響心臟正常發育且會造成不同程度的心臟發育缺損,包括心臟looping缺損、心臟構造變為細長導致心腔變小、心包膜與腹部積水。本研究已利用斑馬魚心臟發育缺損之嚴重程度建立了心臟缺損等級,未來可應用在脊椎動物之遺傳突變(genetic mutations)造成心臟缺損的研究上。
Tbx5 is a transcription factor and belongs to T-box family. In the present study, we try to elucidate the molecular mechanisms of heart developmental regulators on early developmental process in zebrafish. Semi-quantitative RT-PCR analysis indicated that the expression of tbx5 gene increases gradually start from 14 hpf. The highest expression of tbx5 was detected at 22 hpf and decreased sharply at 24 hpf, and then maintained at a basal level through out to the hatching. Whole mount in situ hybridization assay indicated that tbx5 gene mRNA messages were located in the heart, eyes and pectoral fin buds. Tbx5 morpholino antisense oligos (MO) was injected into 1~8 cells stage eggs and the survival rate decreased following higher dose of tbx5-MO treated. Heart conformation changed, heart looping defect, pericardium effusion, dropsy of ventral position, pectoral fin defected and shortened trunk were found in tbx5 gene knockdown zebrafish embryos. Higher percentage of heart looping defect, pericardium effusion and dropsy of ventral position was found following higher dose of tbx5-MO treated group with dose-dependent manner. Five levels of heart defects can be distinguished following tbx5-MO treated and it can be used to identify vertebrate heart defect caused by various genetic mutations. Degree 0 is normal embryo. Degree I embryos have the mild pericardium effusion and dropsy of ventral position, and heart looping defect. Degree II embryos have the severe pericardium effusion and dropsy of ventral position, and heart looping defect. Heart without looping and conformation become lengthened and thin was found in Degree III embryos. Cardiac and somatic malformation was found in Degree IV. Most Degree IV heart defect embryos die at later development stage. Reduction of tbx5 gene expression by tbx5-MO treated resulted in different degree of heart defect with dose-dependent manner in zebrafish embryos. Lower expression level of dHAND, nkx2.5, mlc2, amhc, vmhc, IGF, myoD and myostatin gene was found in tbx5 gene knockdown zebrafish. The insufficient expressions of the cardiac myocyte cell fate determination factor nkx2.5, cardiac muscle fiber major components amhc and vmhc, and muscle growth regulator IGF might be the key factors which cause heart looping defect, atrial septal defect and ventricular septal defect following reducing tbx5 gene expression. We have demonstrated that the insufficient expression of tbx5 gene may cause different degrees of heart defect and zebrafish can be served as a model for study heart defect in vertebrates.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M92330004
http://ntour.ntou.edu.tw/ir/handle/987654321/16551
Appears in Collections:[水產養殖學系] 博碩士論文

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