臺灣博碩士論文加值系統

果蠅的基因tinman (tin)與脊椎動物之Nkx2-5為同源基因，扮演著果蠅的心臟發育與維持功能的樞紐角色。胚胎發育時，如果tin的功能缺失，原本將形成心臟的先驅細胞亦會跟著缺失，導致心臟無法發育成熟；可是在胚胎發育晚期，tin的功能缺失卻只導致各式心臟細胞的型態異常，而使心臟無法發揮正常功能，但胚胎仍能存活。這表示tin在胚胎發育早期是心臟先驅細胞進行特化所需，而在晚期則專司功能之分化。普遍相信，tin應非單獨直接作用在未來發育成心臟的細胞，而被認為是活化許多下游基因，或與其他轉錄因子協同合作發揮效用，以達到健全果蠅心臟形態與功能之目的。在本研究中，我們發現了一個或許在tin的下游作用，並且跟心臟生成相關的基因—Ｈim。Ｈim基因在中胚層之表現，類似但稍晚於tin，並在胚胎發育末期，tin與Him共同表現於心臟與圍心細胞之中。比較tin與Him基因表現的時空挪移，我們提出一個假設：Ｈim是受tin所活化而影響的下游基因。尤有甚者，我們發現藉由RNAi降低Him之表現量會促進心臟細胞之生成；相反的，在中胚層專一提高Him基因的表現量，卻會抑制心臟細胞的生成。既然tin與Him兩者的表現位置互相重疊，我們認為Him可能是tin下游的直接標的。有關Him與心臟相關的專一性加強子已被鑑定出來，序列分析比對發現三個tin的認知結合序列存在Him的加強子區域中。在tin的突變株遺傳背景底下，或者共轉一tin競爭型抑制基因，皆會降低Him強化子的活性。反之，異位表現tin卻能活化Him與心臟相關的強化子。另外，分生實驗證明tin能與Him強化子上具保守性的認知序列接合。因此，我們推斷tin是Him的上游調控者。

The homeobox containing gene, tinman (tin) plays a pivotal role in heart development as well as cardiac function of Drosophila. Loss of tin function during embryogenesis, leads to the abolishment of heart precursor cells and the mature heart. However, depleting the function of tin at late embryogenesis, the heart formed initially, but lack of various cardial cell types. Thus, the heart can not perform normal physiological function. This suggests that tin is required early for the specification of cardial precursors, lately for the diversification of cardial cell and function. It is generally believed that tin can not act alone in all aspects of developing heart. It has been proposed that tin exerts its function by activating down-stream genes or acting synergistically with other genes to fulfill its role for the formation of functional heart in Drosophila. In this study, we have identified a cardiogenic gene, Him, that may act downstream of tin. Like tin, Him does exhibit pan-mesodermal expression pattern but its expression is later than that of tin. Later, the expressions of tin and Him are restrict in cardiac cells and pericardial cells. Comparing the dynamic expression patterns of both tin and Him, we hypothesized that Him is a downstream target of tin. Furthermore, we have found that down-regulation of Him by RNA interference promotes cardial cell fates. By contrast, mesodermally specific expression of Him suppresses cardial cell fate. This suggests that Him functions as a cardial cell suppressor. Since the both expression patterns of tin and Him are overlapped, we believed that Him may be a directed target of tin. The cardial specific enhancer of Him has been identified. Sequence analysis revealed that three tin consensus binding sites are present in the enhancer regions. The enhancer activity were down regulated in tin mutant background or when dominant negative allele of tin are co-expressed. By contrast, ectopic tin activates Him cardial enhancer. In addition, Tin can binds to the consensus binding sites in vitro. Thus, we concluded that tin is an upstream regulator of Him.

Abstract………………………………………………………...………3
壹、中文摘要…..…………………………………………....…...…….5
貳、緒論
一、 先天性心臟病……………………………………………………7
二、 果蠅的心臟發育過程……………………………………………8
三、 研究果蠅心臟發育的重要性……………………………………9
四、 研究背景……………………………………………..…….……10
參、研究目的………………………………………………………….17
肆、材料與方法
一、果蠅胚胎收集與固定………………………………….…………18
二、果蠅胚胎原位雜合染色……………………………….…………18
三、基因轉植果蠅品系之建立………………………………….……20
四、果蠅胚胎的顯微注射…………………………………….………22
五、Electrophoresis Mobility Shift Assay(EMSA) ………..….………23
六、Him基因的enhancer在果蠅心臟表現之研究……………….…26
伍、結果
一、 果蠅胚胎組織化學染色……………………………..……….…28
二、果蠅胚胎原位雜合染色………………………………….………28
三、Him gene enhancer同源序列之分析……………........….....……29
四、EMSA…………………..............................................................…30
五、Him基因的enhancer在果蠅心臟表現之研究…………….……30
陸、討論
一、 Him enhancer序列具有高度保守性………….…………...........32
二、 Him enhancer 直接受tin的調控………………………….....…33
三、 Him在pathway 上所扮演的角色……….……………......……34
柒、參考文獻………………………………………………………..…37
捌、附圖……………………………………………………………..…41

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