斑馬魚Six6基因之選殖、表現及啟動子之顱顏組織專一性調控之研究

Abstract

Six6基因屬於Six基因家族的一員。Six基因家族最早在1995年利用果蠅sine oculis (So)基因作同源篩選，找出老鼠相關的同源基因。已知So基因在果蠅視覺系統的發育扮演著相當重要的角色。雖然老鼠所有的Six基因家族成員都與So基因有同源性，可是卻只有Six3和Six6基因會表現在神經外胚層視覺原基(optic primordium)。並且有證據顯示Six3和Six6基因在功能上不是重複的。除了在視覺原基表現外，在早期腦部的組織也有表現，顯示該基因對胚胎頭部神經組織發育具有重要性。 本論文主要目的在尋找斑馬魚基因體中Six6基因的同源基因，並且分析這些基因啟動子之活性及其可能位於Six6基因上游的順式調控因子。我首先發現基因庫中包含人類、小鼠、大鼠和非洲爪蛙等四個物種Six6基因都和附近相鄰的基因包括Six1和Six4形成一個連鎖群(linkage)，然而在斑馬魚基因庫中卻沒有發現命名為Six6基因的同源基因，然而在UCSC生物資訊網頁，以人類SIX6基因作檢索，發現兩個未知名的斑馬魚同源基因(編號zgc：110344和zgc：63871)。於是利用胺基酸的序列比對分析，確定了斑馬魚這兩個未知名的基因正是Six6基因的同源基因。藉由位於同一個連鎖群的Six4基因之平行演化同源基因(paralogues)位置(locus)，分別將透過胺基酸序列比對分析的未知名的基因命名為(Six6.1基因和Six6.2)。 利用原位雜合反應偵測內生性mRNA的表現位置。結果發現斑馬魚Six6.1和Six6.2基因的內生性mRNA都會表現在間腦腹側、下視丘與將來發育為腦下垂體前葉的區域，此外，斑馬魚Six6.1基因會表現在神經視網膜組織。值得注意的是斑馬魚Six6.2基因雖然如同Six6.1基因會表現在間腦腹側、下視丘與腦下垂體前葉的區域，可是表現量都比較弱，而且在神經視網膜並沒有發現Six6.2基因的表現。 接著我以PCR的方法從斑馬魚的基因體中分別複製出包含Six6.2基因轉錄起點(-728/+165)的上游DNA片段和包含Six6.1基因轉錄起點(-1199/+204)的上游DNA片段，將其和綠色螢光蛋白報導基因(EGFP, enhanced green fluorescent protein)鍵結形成結構體，再將這兩個結構體以顯微注射的方式注入斑馬魚之受精卵中。結果在過渡性轉殖實驗中，發現Six6.1啟動子結構體能驅使綠色螢光蛋白報導基因在斑馬魚胚胎的頭部腦組織和眼睛神經視網膜有明顯的專一性表現；而Six6.2啟動子結構體則驅使綠色螢光蛋白報導基因在斑馬魚胚胎的頭部腦組織表現。 將內生性mRNA的表現位置和過渡性轉殖實驗中綠色螢光蛋白報導基因表現位置相互比較的結果位置大致上相符，顯示斑馬魚包含Six6.1基因轉錄起點(-1199/+204)的上游DNA片段和包含Six6.2基因轉錄起點(-728/+165)的上游DNA片段的區域內即包含有大部分調控該基因表現之順式調控因子。另一方面，利用Dot-Matrix方法分析，此兩基因上游皆含有兩段保守的DNA序列片段，我們推測此兩基因的組織專一性順式調控序列座落於這些保守片段中。

Six6 gene belongs to the Six gene family, of which the members were identified in mouse and other vertebrates by homologous screening with Drosophila sine oculis (so) gene as a probe. It has been shown that sine oculis plays an import role during Drosophila eye development. In contrast, only vertebrate six3 and six6 are expressed in the optic primodium and are implicated in eye development. Besides, these two genes also participate in brain development, suggesting that they are crucial for neuronal differentiation. The aim of this study is to search the zebrafish six6 orthologue and to analyze the expression and its tissue-specific gene regulation. In the first attempt, NCBI data-base search did not result in the identification of zebrafish six6 gene. However, two zebrafish six6 homologues (zgc: 110344 and zgc: 63871) were presented in the UCSC bio-informatics web site using human SIX6 gene as a reference. It has been well known that six4, six1 and six6 form a genetic linkage as six4-six1-six6 arrangement. The two putative zebrafish six6 homologues were designated as six6.1 and six6.2 according to the presence of the genetic linkage in genome and alignment of amino acid sequences. The 3’UTR cDNAs of the genes were isolated by PCR for anti-sense RNA probe preparation. In situ hybridization results revealed that zebrafish six6.1 and six6.2 transcripts are expressed in ventral diencephalon, hypothalamus and presumptive adenohypophysis. In addition, six6.1 transcripts are located in the neural retina. In regard to the gene regulation, the upstream promoter fragments of the two genes were cloned by PCR amplification and were analyzed in zebrafish embryo with GFP reporter gene by transgenic assay. The results showed that the reporter is expressed in similar patterns as those of endogenous mRNA, suggesting that the respective upstream promoter regions harbor the regulatory elements for six6.1 and six6.2. In parallel, two conserved DNA regions are located in the upstream regions of the two genes by Dot-Matrix analyses. The transgenic assays suggested that the proper cis-regulatory elements for the two genes reside in these conserved locations.