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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/17518

Title: 斑馬魚arnt2a/b/c、hif1α及hif3α基因對嗅覺器官發育的影響
Functions of zebrafish arnt2 a/b/c、hif1α and hif3α genes in olfactory development
Authors: Jian-Shiung Wu
吳建勳
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;胚胎;發育;嗅覺
zebrafish;embryo;development;olfactory;arnt;hif
Date: 2003
Issue Date: 2011-07-04
Abstract: 最近研究發現,有一群bHLH – PAS基因調控蛋白在脊椎動物胚胎發育過程中扮演著相當重要的角色。其中又以ARNT為核心蛋白,它會和其他的bHLH – PAS蛋白形成異構偶合體,而調控多種的生理反應,例如:毒物代謝、缺氧反應、生理時鐘反應、血管形成以及神經發育等。在本實驗室先前的實驗發現,若分別將arnt2a/b/c 12ng antisense MO(morpholino)或hif1 a +hif3 a 28ng MO注射入斑馬魚胚胎,對斑馬魚胚胎進行基因knock-down的實驗,發現對於胚胎顱面的形態有相當嚴重的影響。所以本實驗首先將morphant 胚胎,於掃瞄式電子顯微鏡(SEM)下觀察,發現相較於wild-type而言,鼻窩均有不一樣的改變,且又以打arnt2a/b/c 12ng MO那組有更嚴重的影響,鼻窩幾乎已經消失,因此推測這些基因對於嗅覺器官的發育扮演著重要的角色。 所以為了更清楚去瞭解其中的基因調控機制問題,本實驗利用全覆式原位雜合的方法,觀察嗅覺上皮以及嗅球發育的相關標記基因表現,如:omp、anxV、coe2、neuroD、emx1、dlx3等,結果發現,干擾arnt2a/b/c基因表現之後,嗅覺器官的發育明顯的受到影響,而且在發育早期於嗅基板形成時期就已經干擾了嗅覺器官發育,其次再利用神經脊細胞的標記基因foxd3去檢視,也發現神經脊細胞明顯的減少,而顱部神經脊細胞已經知道對於嗅基板的誘發佔有一定重要的角色,且這些細胞將來也有部分會形成鼻子軟骨的構造,因此實驗結果顯示ARNT2A/B/C在嗅覺器官發育過程扮演著重要角色。但是在干擾hif1a + hif3a基因表現的實驗中,則未觀察到嗅覺器官發育顯著的差異,推測hif1a + hif3a對於嗅覺器官的發育只是非間接的影響。 除此之外,在一些神經系統發育標記基因的觀察下,如:ngn1、coe2、l1.1等,發現受到了arnt2a/b/c MO的影響,這些基因的表現量也是明顯的降低,顯示了ARNT2A/B/C對於神經系統發育的重要性。
Recently, a novel bHLH-PAS transcriptional factor family was revealed to play an important role in vertebrate development. SIM, HIF and AHR can form heterodimers with a central bHLH/PAS partner protein, ARNT, to modulate variety of biological functions, including neurogenesis, angiogenesis and vasculogenesis, and biological circadian rhythm. Previous studies in our laboratory have shown that repression of arnt2a/b/c or hif1a+hif3a by morpholino oligonucleotides (MO) caused serious defect in embryonic cranial phenotype. In this study, I observed the cranial defect of arnt2a/b/c-knockdown morphant embryos by scanning electric microscope (SEM) and found significant defect in the olfactory pit comparing to the wild type embryos. Hence, we proposed that those genes may involved in olfactory organ development. In order to understand the detail regulatory mechanism, we have investigated the expression patterns of several olfactory relative marker genes, including omp, anxV, coe2, neuroD, emx1 and dlx3, by in situ hybridization. It appeared that the development of olfactory organs have been affected significantly in the early stages of the arnt2a/b/c morphant embryos. The repression of neural crest marker, foxd3, suggested that the neural crest cells are decreased in arnt2a/b/c morphant embryos. It was known that the cranial neural crest cells play important role in inducing olfactory placode development and a portion of them form the olfactory cartilage. This study revealed that arnt2a/b/c are very important in olfactory development. In contrast, there was no significant defect in hif1a+hif3a knockdown morphant embryos. It suggested that the hif1a+hif3a antisense morpholino affected the olfactory development indirectly. In addition to the olfactory-related markers, the expression patterns of a number of neural maker genes, such as ngn1, coe2, l1.1, were also affected in the arnt2a/b/c morphant embryos. It suggested that ARNT2A/B/C play essential roles in neural system development.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M91360021
http://ntour.ntou.edu.tw/ir/handle/987654321/17518
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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