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標題: | 斑馬魚 dll4 突變魚在淋巴管和腸道造成之缺陷 The Zebrafish dll4 Mutation Causes Defects in Lymphatic Vessels and Intestine |
作者: | Hui-Sheng Young 楊惠珊 |
指導教授: | 陳秀男(Shiu-Nan Cheng) |
關鍵字: | 轉基因斑馬魚,淋巴管,腸道血管,細胞分化命運, Transgenic zebrafish,delta-like 4,lymphatic vessel,intestinal vessel,cell fate, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 摘 要
我們利用轉基因之斑馬魚 TG( fli 1:EGFP )y1研究一株在 dll4 (delta-like 4) 基因產生突變的魚,此轉基因是表達 GFP 在血管的內皮細胞上,利於觀察和研究血管的生成。先前的研究發現 dll4 為顯性的突變,其尾鰭的動脈血管是斷斷續續的,且在血管分岔的地方,其動脈血管甚至會消失。然而,在 dll4/dll4 突變魚(homozygote)當中,受精後大約 7 到 14 天之間會開始陸陸續續死亡,但卻沒有明顯的血管缺陷情況,而我們主要想去探討 dll4/dll4 突變魚到底是因為什麼樣的缺陷,而造成其早期死亡。 我們主要發現到在 dll4/dll4 突變魚有兩個 phenotypes,分別為淋巴管(lymphatic vessel)以及腸道血管(intestinal vessel)的缺陷,在 dll4/dll4 突變魚當中,我們觀察到其淋巴管大部分呈現片段不連續的情況,有的甚至會完全消失,另外有少數雖然有淋巴管但是卻沒有形成 lumen。已知 VEGF-C 和 Prox1 是淋巴管發育過程中不可或缺的基因。於是,我們進一步探討 Prox1、VEGF-C 和 dll4 三者之間如何交互作用。首先利用 morpholino (MO) knockdown VEGF-C 或 Prox1,發現淋巴管的發育皆會受到影響,而當注射含有VEGF-C cDNA 的 plasmid 去過度表現 VEGF-C 時,則造成淋巴管的過量增生。證明了 VEGF-C 及 Prox1 在斑馬魚的淋巴管發育也扮演重要角色。接著我們設計了 rescue 實驗,當注射 Prox1-MO,大約 99%的胚胎無淋巴管的發育,而同時注射 Prox1-MO 和 含有 VEGF-C cDNA 的 plasmid 時,則使得大約 62%有正常淋巴管的發育,顯示 VEGF-C 可能作用在 Prox-1 下游。利用相同的方式,我們發現 dll4 可以 rescued Prox1-MO 以及 VEGF-C-MO,相反地 含有 Prox1 和 VEGF-C cDNA 的 plasmid 皆無法 rescued dll4/dll4 突變魚或 dll4-MO 淋巴管發育的缺陷。根據以上的實驗結果,我們推測可能是由 Prox1 先作用於 VEGF-C,再作用於 dll4,進而影響到淋巴管的發育。另外,我們利用 Q-PCR 分別去看不同時期 VEGF-C 和 Prox1 的表現量,發現 wt 和 dll4/dll4 突變魚之間沒有明顯差異,間接證明了 VEGF-C 和 Prox1 可能為 dll4 的上游基因。 在腸道血管方面,我們觀察到相對於 wt 魚之下,dll4/dll4 突變魚的腸道血管較粗,為了探討 dll4/dll4 突變魚早期的腸道發育或是功能上是否有缺陷,我們利用 cdx-1b 基因來判斷腸道早期上皮細胞發育和分化過程情形,並藉由檢查一系列 delta/notch 基因在腸道的表現,來觀察腸道中、晚期發育和分化過程情形,最後利用 IFABP 基因來觀察腸道的後期發育有無問題,結果發現在腸道早期上皮細胞發育和分化過程並沒有很明顯差異,而 delta/notch 的 in situ 結果則顯示出腸道中、晚期分化過程有出現不同細胞分化命運(cell fate)的情況,最後在腸道的後期發育,IFABP 的 in situ 結果顯示出沒有明顯的差異,Q-PCR 定量結果也證明了在 wt 和 dll4/dll4 突變魚當中,IFABP 的表現量並沒有顯著差異。 我們發現到除了已知的 Prox1 以及 VEGF-C 之外,Delta-Notch signaling 也參與淋巴管的發育,且我們的實驗證明 dll4 是作用在 Prox1 以及 VEGF-C 的下游,另外 dll4 也會影響腸道上皮細胞發育和分化的命運,因此,斑馬魚 dll4/dll4 突變魚很可能是因為在淋巴管和腸道造成缺陷,而導致胚胎的死亡。 Abstract We use the transgenic zebrafish TG(fli1:EGFP)y1 that express GFP in vessel endothelial cells to study the function of dll4 (delta-like 4) gene in vascular development. Previous research suggested that dll4j16e1 is a dominant mutation causing disconnected arteries in the caudal fins of adult fish. The dll4/dll4 homozygotes, however, died between 7 to 14 days post fertilization without evident deficiencies in vessels. Hence, my project is to identify causes to the embryonic lethality of dll4/dll4 homozygotes. We discovered abnormalities in the lymphatic and intestinal vessels of dll4/dll4 mutants. In dll4/dll4 homozygote embryos, most of the lymphatic vessels appear fragmental or even absence, while less than 10% of them developed lymphatic vessels seemly without lumens. It is known that VEGF-C and Prox1 are indispensable for lymphatic vessel development in the mouse. Therefore, we next investigated the interplay between Prox1, VEGF-C, and dll4. First, we tested whether VEGF-C or Prox1 are also required for lymphatic vessel development in zebrafish by using morpholino knockdown. The results show more than 90% of VEGF-C or prox1 morpholino injected zebrafish embryos develop no lymphatic vessels. Besides, over-expression of VEGF-C in zebrafish embryo by microinjecting the plasmid containing VEGF-C cDNA resulted in excess lymphatic endothelial cells and enlarged lymphatic vessels. These data demonstrated that both VEGF-C and Prox1 play important roles in zebraifsh lymphatic vessel development. We next tested whether overexpression of VEGF-C would rescue Prox1 MO. While almost 99% embryos developed no lymphatic vessels in Prox1-MO alone; 62% of VEGF-C; prox1 MO develop normal lymphatic vessels, suggesting VEGF-C might function downstream to Prox-1. Using the same methods, we further found that dll4 can rescue the lymphatic defects of Prox1-MO and VEGF-C-MO. In contrast, the overexpression of Prox1 or VEGF-C could not rescue the dll4/dll4 lymphatic vessels phenotypes. Accordingly, we proposed that Prox1 might act upstream of VEGF-C followed by dll4 during the development of lymphatic vessels. In consistent with this idea, we found the expression of VEGF-C and Prox1 by QPCR is not significantly altered in dll4/dll4 embryos. Interestingly, the Prox1 expression level is elevated in Prox1 MO suggesting a negative feedback regulation by Prox1 itself. In dll4/dll4 intestine, the intestinal vessels appear larger. In light of the recent finding that delta-notch signaling controls the intestinal cell fate specification, we went on to examine whether dll4 mutation also causes cell fate defects. Using the early marker for intestinal differentiation cdx-1b gene we showed normal pattern of cdx-1b in dll4 mutants. Interestingly, the expressions of a series of delta/notch genes suggested there were cell fate changes happened in the middle and late intestinal differentiate progression. Finally, the expression of IFABP (instestinal fatty acid binding protein) gene detected by in situ and QPCR is not significantly different between wt and dll4/dll4 embryos suggesting normal function in fatty acid absorption in dll4/dll4. We conclude that dll4 mediated Delta-Notch signaling also plays a role in lymphatic vessel development which is downstream to Prox1 and VEGF-C. dll4 might involve in the cell fate differentiation of intestinal epithelial cells. It is likely that the defects in lymphatic vessels and intestine cause the embryonic lethality of dll4/dll4 zebrafish embryos. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28820 |
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顯示於系所單位: | 漁業科學研究所 |
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