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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/17492

Title: arnt2 基因在斑馬魚胚胎發育上的角色
Functional Analysis of ARNT2 in Zebrafish Development
Authors: Wen-Der Wang
王文德
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: 斑馬魚;咽弧;頭骨
zebrafish;arnt;cyp1a1;craniofacial skeleton;neural crest;PTU;branchial arch;cartilate
Date: 2003
Issue Date: 2011-07-04
Abstract: 3 中文摘要 在胚胎發育過程中,細胞的分化與器官的發育、以及對環境刺激 的反應與適應,是受到細胞內一系列嚴謹的訊息傳遞與基因調控系統 所控制。研究發現,一群bHLH-PAS 蛋白在胚胎的發育與對環境刺激 的反應與適應上扮演著相當重要的角色。其中以ARNT 為其核心蛋白,會分別與同為bHLH-PAS 蛋白的AHR、HIF 及SIM 形成異偶合蛋 白,再進一步調控標的基因表現。其中,AHR::ARNT 與毒物誘導cytochrome P450 表現有關,HIF::ARNT 則與細胞處於低氧環境下的反應有關,而SIM::ARNT 則與胚胎神經系統的發育有關。 ARNT 蛋白結構除了具有bHLH-PAS domain 外,在其C-端還具有一段transactivation domian。先前本實驗室從斑馬魚胚胎選殖出兩個C- 端缺少transactivation domian 的arnt 基因:arnt2a 與arnt2x。在斑馬魚肝臟表皮細胞珠(ZLE)內分別表現arnt2a 與arnt2x。結果顯示兩者均會抑制TCDD 活化cyp1a1 基因的程度,顯示ARNT2A 與ARNT2X 在AHR::ARNT 訊息傳遞途徑中扮演著抑制者的角色。在胚胎發育時期arnt2a 與arnt2x 會廣泛的表現於成熟未受經卵,胚胎時期的腦區、咽弧以及成魚的各器官組織中。以arnt2a/b/c 專一性的morpholino 阻斷胚胎中的ARNT2A/B/C 蛋白轉譯生成,結果發現胚胎頭部骨骼的發育發生嚴重缺陷。脊椎動物頭部骨骼的發育是由胚胎中不同胚層的細胞族群共同發育而來:包括外胚層、中胚層與內胚層。實驗結果發現arnt2a/b/c-morpholino 會影響外胚層衍生的神經脊細胞的數目,對於中胚層與內胚層的形成與分化為肌肉細胞與咽囊則無影響。另外,arnt2a/b/c-morpholino 也會阻斷神經脊細胞分化為軟骨細胞。顯示ARNT2A/B/C 對於胚胎頭部骨骼的發育扮演著相當重要的角色。另外,和ARNT 同為bHLH-PAS 蛋白的AHR 為ligand-dependent的轉錄因子,AHR 會被鹵化芳香多環化合物(HAHs)和非鹵化芳香多環化合物所活化(PAHs),被活化的AHR 會進一步開啟標的基因的表現,例如cyp1a1。實驗發現常被用來抑制胚胎色素生成的PTU 會誘導早期胚胎的表皮和後期胚胎的血管表現cyp1a1。同時以PTU 和TCDD 處理胚胎後發現,PTU 會抑制TCDDD 誘導cyp1a1 的表現。進一步研究發現PTU 和TCDD 一樣都是透過AHR::ARNT 訊息傳遞途徑誘導cyp1a1 表現。
Abstract During embryogenesis, all of the processes of cell differentiation,organogenesis and response to environmental stimulation are controlled byserious gene regulation systems. The bHLH-PAS (Per-AhR/Arnt-Sim)protein family plays important roles in vertebrate development and xenobiotic metabolism. In the bHLH-PAS mediated pathway, Arnt proteinplays as a central role, which can dimerizes with other bHLH-PAS(such as AHR, HIF, and SIM)to regulate a variety genes. The heterodimer complex of AHR::ARNT is an ubiquitous transcription factor which mediates the expression of xenobiotic-response genes, such as cytochrome p450 1a1 (cyp1a1) and cyp1a2. Previously, we obtained cDNA of two truncated ARNT factor, arnt2a and arnt2x, both of their encoded proteins contain the highly conserved bHLH and PAS A/B domains, but they all lack a conventional transactivation domain at carboxyl end. In cultured ZLE cells, transiently expressed ARNT2A and ARNT2X inhibited 2,3,7,8-TCDD-activated cyp1a1 transcription with different efficiencies. Four arnt2 cDNA were cloned in zebrafish, named arnt2a, arnt2b, arnt2c, and arnt2x. arnt2a/b/c mRNAs were exited in the mature oocytes as materal mRNA and expressed during embryogenesis. To investigate the function of ARNT2A/B/C, I have employed gene-specific antisense morpholino oligonucleotide to block ARNT2A/B/C protein translation. Injecting of arnt2a/b/c antisense morpholino into fertilized embryos at 1 cell stage led to malformation of craniofacial skeleton. Craniofacial skeleton is formed by cells that are derived from ectoderm, endoderm, mesoderm. Injection of arnt2a/b/c antisense morpholino into embryo led to reduce the amount of cranial neural crest cell, but didn’t disrupt the muscle and pharyngeal pouches formation. The aryl hydrocarbon receptor (AHR ) is a ligand-dependent transcription factor that belongs to the basic-helix-loop-helix PAS (bHLH-PAS) family. The AHR can be activated by a diverse synthetic and naturally-occurring chemicals, such as halogenated aromatic hydrocarbons (HAHs) and the non- halogenated polycyclic aromatic hydrocarbons (PAHs). The liganded AHR modulates the genetic activity of a variety of xenobiotic-responsive gene, including cyp1a1. Here, I showed evidence that 0.2 mM PTU induced a basal level of cyp1a1 transcription in blood vessels at 36 hpf. During larval stage, the liver and all pharyngeal arch vessels of PTU-treated embryos exhibited cyp1a1 transcription as well. Coincubating the embryos with PTU and TCDD led to epressing TCDD-induced cyp1a1 transcription. Mechanistic studies indicated that both of PTU- and TCDD- mediated cyp1a1 transcriptions are modulated by the same AHR-ARNT signaling pathway.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0D86360014
http://ntour.ntou.edu.tw/ir/handle/987654321/17492
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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