藉由綠色螢光蛋白報導基因研究斑馬魚Six1.1基因啟動子之顱顏組織專一性表現

Functional Analysis of Six1.1 Gene Enhancer Elements During Craniofacial Development by Transgenic Zebrafish with Green Fluorescent Protein (GFP) Reporter Gene

呂智楷(Chih-Kai Lu)

指導教授 ： 張百恩

國立臺灣大學/醫學院/口腔生物科學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.01906

摘要

Six基因家族的第一個成員sine oculis (so)最早於果蠅(Drosophila)中被鑑定出來，且根據核苷酸序列同源性質(nucleotide sequence homology)，Six基因家族已於斑馬魚、人類以及小鼠等許多物種中被鑑定出來。在脊椎動物中，Six基因家族的成員會廣泛地在許多組織中表現，故可得知其對於調控胚胎正常的型態發育、器官發育及細胞的分化上扮演著重要的角色。
本篇實驗之主要目的在分析斑馬魚six1基因(其後稱為six1.1)基因啟動子之活性及其可能位於six1.1基因上下游於物種間保守序列中的順式調控因子(cis-regulatory element)。首先將斑馬魚six1.1基因上游約2.6 kb (+31/-2631)之DNA片段，和綠色螢光蛋白報導基因(EGFP, enhanced green fluorescent protein)鍵結形成結構體，並以顯微注射的方式送入斑馬魚之受精卵中。在過渡性轉殖實驗結果中，發現此結構體能驅使綠色螢光蛋白在受精後斑馬魚胚胎的體節(somite)、耳囊(otic vesicle)以及神經丘(neuromast)等位置有著組織專一性的表現。
接著將帶有斑馬魚six1.1上游2.6 kb啟動子片段之F0親代成魚，與野生型之成魚交配後便可得到F1子代，觀察其綠色螢光蛋白表現後可從中篩選到1個轉殖恆定品系(transgenic stable line)，且其胚胎在發育至72小時時期後，可明顯觀察到在耳囊腹部(ventral otic vesicle)及神經丘(neuromast)皆有綠色螢光蛋白之表現，此結果與先前報告之原位雜合反應偵測斑馬魚內生性six1.1 mRNA表現的組織位置相符。以上這些結果顯示在斑馬魚six1.1上游2.6 kb (+31/-2631)的區域內，應包含有能調控此基因在體節(somite)、耳囊(otic vesicle)及神經丘(neuromast)等組織專一性表現的順式調控因子。
之後，利用Dot Matrix軟體及UCSC網頁比對包含斑馬魚、人類、小鼠、大鼠、負鼠、雞、非洲爪蛙、青鱂魚、四齒魨及河豚等十個物種Six1基因上下游相關序列，可發現在各物種Six1基因的上下游包含有五段高度保守的片段(conserved sequence)，分別命名為UCR1 (upstream conserved region 1)、UCR2、UCR3、UCR4以及DCR (downstream conserved region)，其中UCR1、UCR2、UCR3以及UCR4位於six1.1上游及six4.1下游，意即位於six1.1及six4.1兩基因之間，而且其中UCR1及UCR2位於斑馬魚six1.1近端2.6 kb的範圍內。將各片段與斑馬魚six1.1基因近端280 bp啟動子(promoter)或是具有HSV-thymidine kinase (TK)基礎啟動子之載體相互鍵結成結構體，同樣以顯微注射的方式送入斑馬魚之受精卵，在過渡性基因轉殖實驗中，可觀察到含有各片段之結構體在體節(somite)、耳囊(otic vesicle)、神經丘(neuromast)以及咽弓區域(branchial arch)等不同的組織器官中有綠色螢光蛋白的表現，顯示這些片段所含之促進子(enhancer)，可能具有重覆性(enhancer redundancy)。綜合上述，本篇實驗於斑馬魚six1.1基因的上下游相關序列，發現有五個保守片段含有各物種間的高度保留序列，且其彼此之間具有調控功能性重覆的現象(enhancer redundancy)，由於這些片段位於six1.1及six4.1兩基因間或其鄰近區域，我們推測這些片段具有能同時調控斑馬魚six1.1及six4.1於組織專一性表現的功能。

關鍵字

Six1 ； Six4 ； 斑馬魚 ； 保守序列 ； 神經丘 ； 體節 ； 耳囊

並列摘要

The homeobox-containing Six genes, homologues to Drosophila sine oculis (so) gene, are widely expressed in many tissues during vertebrate embryogenesis. Identified in many species, Six family genes are hypothesized to play important roles in morphogenesis, organgenesis and cell differentiation.
To identify the cis-regulatory elements of zebrafish six1.1, we ligated the genomic DNA promoter region from +31 to -2631 to the egfp gene and injected this construct into zebrafish embryos. The resulting reporter-EGFP expression showed spatial restrictions similar to those of six1.1 mRNA detected by in situ hybridization (i.e., mainly in somites, ventral otic vesicle and neuromasts). This transient transgenic assay suggested that the proximal 2.6 kb promoter contains some tissue-specific cis-regulatory elements.
One stable transgenic line (F1 generation) injected with six1.1 proximal 2.6 kb promoter was obtained and the EGFP reporter expression can be observed mainly in ventral otic vesicle and neuromasts.
Dot Matrix and UCSC-bioinformatics analyses of the Six1 genes in many species revealed five conserved sequences, designated as UCR1 (upstream conserved region 1), UCR2, UCR3, UCR4 and DCR (downstream conserved region), in the vicinity of zebrafish six1.1 and six4.1 genes, that are candidate regulatory elements. These fragments inserted upstream of the zebrafish six1 proximal promoter or the thymidine kinase (TK) basal promoter with egfp reporter gene were analyzed in zebrafish embryos. The reporter EGFP expression was observed in somites, otic vesicle, neuromasts and branchial arch.
Taken together, these results suggested that 2.6 kb upstream region of zebrafish six1.1 has regulatory function. And five conserved sequences harboring enhancer elements that may have enhancer redundancy and participate in the regulatory mechanism of zebrafish six1.1 and six4.1 expression patterns.

並列關鍵字

Six1 ； Six4 ； zebrafish ； conserved sequence ； neuromast ； somite ； otic vesicle

參考文獻

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