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研究生:鄭曉芸
研究生(外文):Hsiao-Yun
論文名稱:新穎同源箱基因Irxl1於斑馬魚胚胎發育之鑑定、表現及功能分析
論文名稱(外文):Identification, Expression and Functional Analysis of a Novel Homeobox Gene, Irxl1, in Zebrafish Embryonic Development
指導教授:潘惠錦
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生物醫學科學學系碩士班
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:85
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同源箱基因產物為轉錄因子,在胚胎發育過程中具備相當重要之調節功能。Iroquois homeobox-like 1 (Irxl1)為一新發現屬於TALE次家族之同源箱基因,且與Iroquois家族蛋白最相似。在目前有關小鼠之研究顯示Irxl1 mRNA會高度表現於頭部間葉組織、體節、四肢、腎臟、睪丸等組織,然而其功能目前尚未明瞭。為了探討Irxl1基因於發胎發育上所扮演之角色,我們選擇以斑馬魚作為模式動物。首先我們在NCBI資料庫中以小鼠的Irxl1同源基因進行BLAST搜尋,找到斑馬魚的Irxl1基因,接著以RT-PCR與RACE-PCR選殖出斑馬魚Irxl1基因並確認其存在兩種剪接形式。經由序列比對分析,斑馬魚Irxl1基因位於第12條染色體上,由7個exons所組成,其同源箱區之序列與哺乳類動物的同源基因100%相同。胚胎發育過程中,斑馬魚Irxl1 RNA最早於受精後18小時開始表現並一直持續至5天。兩種isoforms皆會廣泛表現於成魚組織,包括腦、眼睛、肌肉、卵巢、睪丸與鰓等。斑馬魚胚胎於不同時期之全胚體原位雜交分析顯示,Irxl1 RNA主要表現於前兩對咽喉鰓弓。利用反意morpholino oligonucleotides將Irxl1基因knock down會造成許多胚胎型態上之缺陷,如頭部變小、後腦室鼓起、體軸彎曲、卵黃柄變細以及狹長型的心臟,而畸形之幼魚只能活五至七天。這些畸形胚胎的腦部分化與分區以及組織特異性的基因皆能正常表現,但腦部型態發育上卻出現異常。最嚴重的缺陷發生於咽喉鰓弓之中胚層與軟骨組織。抑制Irxl1之轉譯功能會造成咽喉鰓弓肌肉與頭部神經嵴細胞之數目減少。此外,alcian blue染色結果顯示下顎軟骨組織於胚胎受精後五天出現嚴重缺陷,這個表型與小鼠之Twirler (Tw) mutation非常相似,而Irxl1可能為Twirler之連結基因。綜合這些觀察結果,我們推測斑馬魚Irxl1蛋白質可能會調節參與腦部與下顎型態發育之因子。

Homeobox genes are transcription factors that play important roles in the regulation of embryonic development. Iroquois homeobox-like 1 (Irxl1) is a new member of the TALE superfamily of homeobox genes that is most-closely related to the Iroquois class. Recent studies in mice showed that Irxl1 mRNA was highly expressed in craniofacial mesenchyme, somites, limb buds, kidneys and testis cords; however, its function has not been well characterized. To investigate the role of Irxl1 during embryonic development, we choose zebrafish as a vertebrate model. The zebrafish Irxl1 gene was identified by a BLAST search of the NCBI database with homology to the mouse ortholog. Using RT-PCR and rapid amplification of cDNA ends (RACE), full-length cDNA of two splicing variants were cloned. Sequence analysis showed that zebrafish Irxl1 gene was located on chromosome 12 and contained 7 exons with homeodomain exhibiting 100 % sequence identity to human and mouse orthologs. During embryonic development, the transcription of Irxl1 was detected from 18 hours postfertilization (hpf) to 5 days postfertilization (dpf). Both isoforms were broadly expressed in adult fish tissues, including brain, eyes, muscle, ovary, testis and gills. Whole-mount in situ hybridization analysis of zebrafish embryos at various developmental stages revealed that Irxl1 was expressed mainly in the first two pharyngeal arches. Knocking down of Irxl1 by antisense morpholino oligonucleotides resulted in several morphological defects in zebrafish embryos. The gross phenotype included small head, inflated ventricle of hindbrain, curved body axes, shrinkage of yolk stalk and string-like heart. Most deformed larvae only survive for 5 to 7 days. In these affected embryos, differentiation and regional specification of the nervous system were normal as judged by the expression of several tissue-specific genes, but morphogenesis was defective. The most prominent defect were observed in the mesoderm and cartilage in pharyngeal arches. Inhibition of Irxl1 translation resulted in reduction of the numbers of pharyngeal muscles and cranial neural crest cells. Moreover, alcian blue staining revealed severe cartilage defects in the arches at 5 dpf. This phenotype is reminiscent of a mouse Twirler (Tw) mutation, to which the Irxl1 gene is linked. These observations suggest that the Irxl1 protein may regulate factors that are involved in brain and arch morphogenesis in zebrafish.

目錄
致謝
中文摘要………………………………………………………………………………I
英文摘要……………………………………………………………………………..III
目錄…………………………………………………………………………………...V
圖目錄………………………………………………………………………………..VI
表目錄………………………………………………………………………………VII
序論……………………………………………………………………………………1
材料與方法……………………………………………………………………………9
結果…………………………………………………………………………………..23
討論…………………………………………………………………………………..36
參考文獻……………………………………………………………………………..45
圖目錄
圖ㄧ、斑馬魚Irxl1基因之兩種isoform結構與序列比對圖………………………..52
圖二、斑馬魚Irxl1與同源物種之胺基酸序列比對圖……………………………...53
圖三、斑馬魚Irxl1與Irx家族蛋白之序列比對圖………………………………….54
圖四、Irxl1、Irx及其他同源箱蛋白家族之分子演化樹分析圖譜………………….55
圖五、斑馬魚Irxl1基因於胚胎時期與成魚組織之表現…………………………...56
圖六、斑馬魚Irxl1於胚胎發育時期空間上之表現……………………………….57
圖七、Irxl1 morphant之分類………………………………………………………...58
圖八、His-tagged Irxl1重組蛋白之表現與純化結果……………………………….59
圖九、Irxl1 morphants腦部型態發育異常…………………………………………..60
圖十、Irxl1 knock down後,前腦的marker基因會有表現量下降之情形………….61
圖十一、Irxl1 knock down會造成中腦變小以及後腦分節不明顯……………….62
圖十二、野生型與Irxl1 morphants後腦rhombomeres之差異…………………......63
圖十三、Irxl1 knock down會造成後腦及脊髓神經元數目減少,排列散亂……….64
圖十四、野生型與Irxl1 morphants在咽喉鰓弓內胚層之差異…………………….65
圖十五、Irxl1 knock down會造成頭部肌肉部分缺失………………………….......66
圖十六、Irxl1 knock down後,dlx2會有表現量下降之情形………………………..67
圖十七、Irxl1 knock down導致軟骨細胞減少及形狀異常………………………...68
圖十八、Irxl1 knock down影響斑馬魚頭部軟骨之發育…………………………...69
圖十九、Acridine orange染色技術檢測25 hpf胚胎細胞凋亡之情況……………..70
圖二十、同時注射Irxl1- MO和Irxl1 mRNA之畸形率與表型統計圖…………….71
附圖一、蛋白純化所利用之質體pET29(b)…………………………………………77
附圖二、Rescue實驗所利用之質體pCS2+………………………………………….78
附圖三、Morpholino oligo (MO)之設計位置……………………………………….79
附圖四、RT-PCR方法檢測注射Irxl1-MOII之morphants………………………….80
附圖五、MEME分析結果…………………………………………………………...81
附圖六、三個高度保留motif之序列圖……………………………………………..82
附圖七、Irxl1 primers位置圖………………………………………………………..83
表目錄
表一、斑馬魚Irxl1與其同源蛋白之胺基酸序列比對……………………………...72
表二、斑馬魚Irxl1與Irx家族蛋白之胺基酸序列比對(Identity)…………………..73
表三、斑馬魚Irxl1與Irx家族蛋白之胺基酸序列比對(Positivity)………………74
表四、Irxl1 morphant之表型統計…………………………………………………...75
表五、同時注射Irxl1- MO和Irxl1 mRNA之表型統計…………………………..76
附表一、PCR所用之Primer列表……………………………………………………84
附表二、全胚體原位雜交所用之探針列表及其來源………………………………85

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