臺灣博碩士論文加值系統

本實驗室利用雙擇面光顯微術（dual illumination light sheet microscopy）拍攝果蠅胚胎細胞隨時間推移的定時影像（time-lapsed imaging），已觀察、紀錄並分析帶有螢光蛋白標定之engrailed（en）基因與eye gone（eyg）基因CD增強子片段（CD enhancer，簡稱為CD）於視覺系統發育過程中隨時間與空間（spatial-temporal）的表現。發現於第13個胚胎發育階段（growth stage）具有en與CD螢光共定位（co-localization）的一群細胞（約4至14個），是構成眼睛觸角碟原基（Eye-Antenna Disc Primordium，簡稱為EADP）結構的細胞當中，唯一一群具有en基因表現，且最早開始表現CD的細胞，我們定義該群細胞為EADP-engaged en-expressing cells，簡稱為EECs。
過去的研究認為果蠅胚胎眼睛觸角碟原基與其他組成視覺系統的結構，例如：視葉（optic lobe），皆透過內凹（invagination）的方式由胚胎表面往內部移動，目前並沒有影像方面的結果可以證實這項推論。我們與中研院孫以瀚博士及陳壁彰博士實驗室合作，使用Bessel掃描式擇面光照明顯微術（Bessel beam plane illumination microscopy）拍攝同時標定細胞邊界（cell boundary）與en基因表現之果蠅胚胎，觀察於第13至15個胚胎發育階段期間胚胎頭盤表面（procephalic lobe）之細胞動態（cell dynamics）。
由果蠅胚胎頭盤上方視角，可觀察到靠近頭盤邊緣的EECs與周圍的上皮細胞（epithelial cells）自第15個發育階段開始，由接近圓形變得狹長，逐漸形成一道拱型邊界，同時，EECs會沿著這道邊界往胚胎後端移動，但直到EECs被dorsal ridge完全覆蓋之前，EECs的形狀並未出現代表內凹發生的細胞頂部收縮（apical constriction）現象。此外利用免疫螢光染色觀察EECs與wingless（wg）基因在頭盤邊緣表現情形的初步結果發現，兩者的相對位置與en和wg基因於體節（segment）上的排列非常相似，推測EADP是由一個體節發育而來。EECs在眼睛觸角碟原基形成過程中可能具有重要功能，我們進一步利用溫度敏感型（temperature-sensitive）Gal80ts蛋白質，分別抑制（1）自第10個發育階段至胚胎孵化為止，en基因表現細胞的eyg基因功能；（2）自第15個發育階段至胚胎孵化為止，CD表現細胞的en基因表現；前者並未對成蟲複眼型態發育造成顯著的影響，後者則造成個體於胚胎發育階段死亡。

In our previous studies of the formation of the Eye-Antenna Disc Primordium (EADP) of Drosophila embryos, via dual illumination light sheet microscopy, we recorded and analyzed the spatial and temporal distributions of fluorescence-labeled engrailed（en）expressed cells and CD enhancer expressed cells. Our results revealed that among all the cells which took part in the formation of the EADP, there were 4 to 14 cells, which exhibited the co-localization of en and CD expression during the 13th growth stage; besides, these cells were the only group of cells with en-expression which also expressed CD in the earliest stage. We name these cells the EADP-engaged en-expressing cells (or EECs). To track and analyze the migration of EECs during the process of EADP invagination, we applied the Bessel beam plane illumination microscopy to take time-lapsed live images of the embryos for approximately 2 hours [from 10 to 12 hrs after egg deposition (AED)]. We found that there was a dome-like boundary formed in front of the dorsal ridge and EECs moved along this boundary to move posteriorly during the head involution. Thus, EECs might serve as a boundary marker during the process of EADP invagination. Since en is one of the segment polarity genes, it is very likely that the EADP also inherited this property (of segment polarity). Our immunostaining result of en and wg expression pattern suggests that the EADP is developed from one segment. To understand the role of EECs during the process of EADP formation, we knock-downed en and inhibited the function of eyg at stages 10 and 15, respectively, using the temperature sensitive Gal80ts protein. We observed that losing the function of eyg in en+ cells had no significant effect on the adult eye phenotype, while the inhibition of en expression in CD+ cells might lead to embryonic death.

誌謝·················································································i
摘要················································································iv
Abstract············································································vi
目錄···············································································viii
圖目錄·············································································ix
第一章背景簡介·······························································01
1.1 果蠅視覺系統發育簡介········································01
1.2 內凹機制··························································06
1.3 果蠅體節結構····················································07
1.4 本實驗室過去研究··············································08
1.5 晶格層光顯微術簡介···········································11
1.6 研究動機··························································14
第二章研究方法與架設······················································15
2.1 實驗架設與影像處理···········································15
2.2 果蠅樣品準備步驟··············································16
2.3 利用Gal80ts蛋白質調控基因表現實驗流程··············18
第三章結果與討論····························································21
3.1 ubi-GFP-CAAX於胚胎頭部之表現··························21
3.2 wg-lacZ於胚胎頭部之表現····································27
3.3抑制EECs上之en與eyg基因表現的結果················30
第四章未來工作·······························································34
參考文獻··········································································35

圖目錄
圖1-1 果蠅視覺系統發育示意圖。··········································02
圖1-2 果蠅胚胎視覺系統發育示意圖。····································04
圖1-3果蠅三齡幼蟲時期形成之成蟲盤結構示意圖。···················05
圖1-4 內凹過程之細胞型態變化示意圖。································06
圖1-5 果蠅胚胎體節結構形成示意圖。···································07
圖1-6 利用雙擇面光顯微術拍攝果蠅胚胎stage 13至16頭部發育細胞隨時間推移之定時影像。··················································09
圖1-7 高斯光束與貝索光束之比較。······································12
圖1-8 貝索光束具有自我復原的特性。···································12
圖2-1 晶格層光顯微鏡架設光路圖。·······································15
圖2-2 利用Gal80ts抑制胚胎時期eyg基因功能之實驗流程。··········19
圖2-3 利用Gal80ts抑制胚胎時期en基因功能之實驗流程。···········20
圖3-1 使用Bessel掃描式擇面光照明顯微術拍攝之stage 13胚胎三維立體呈現圖。··································································23
圖3-2以Bessel掃描式擇面光照明顯微術拍攝stage 13至15胚胎頭部細胞隨時間推移之定時攝影。·············································25
圖3-3以Bessel掃描式擇面光照明顯微術拍攝stage 15至16胚胎頭部細胞隨時間推移之定時攝影。·············································26
圖3-4使用ZEISS LSM780共軛焦顯微鏡（PLan-Apochromat, 40X油鏡，N.A.為1.4）拍攝stage 12果蠅胚胎頭部wg與en基因表現。·······28
圖3-5使用ZEISS LSM780共軛焦顯微鏡（規格同圖3-4）拍攝不同濃度抗體免疫染色結果。······················································29
圖3-6 抑制eyg基因在EECs的表現對成蟲複眼發育的影響。······31
圖3-7 抑制CD+細胞的en基因表現對果蠅成蟲數量統計。············33

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