指導教授：張俊哲臺灣大學：昆蟲學研究所鍾成侑Chung, Chen-yoChen-yoChung2014-11-262018-06-292014-11-262018-06-292014http://ntur.lib.ntu.edu.tw//handle/246246/262109豌豆蚜為半翅目昆蟲中第一個被全基因體解序的昆蟲，由於其複雜生活史中的非遺傳多型性以及不同型式的胚胎發育使之成為發育生物學中獨特的模式物種，為更進一步瞭解豌豆蚜胚胎發育過程，解開發育基因之表現型式為不可或缺的方法，為達到此一目的，分子工具以及基因表現分析皆同等重要。早先，全體原位雜合反應已被發展並運用於監測基因於豌豆蚜卵巢的表現，但此一技術在同時偵測兩基因表現及建立三維空間表現上有其極限，因此，我發展了可信賴的全體螢光原位雜合程序來解決這些問題，利用不同螢光染色原理的優勢，我成功偵測到基因在不同組織間—如胚外以及胚體—的表現，此外，結合不同方式所進行的雙色螢光原位雜合也成功讓我同時觀察到兩個基因於胚胎中的表現，此螢光原位雜合程序也幫助我在接下來的研究中，解析發育基因的表現。在 2010 年由本實驗室所發表的報告中，我們發現 Aphb 信使核糖核酸 (messenger RNA) 除表現在早期胚胎前端，也會表現在中、晚期體節以及中央神經系統 (CNS) 中，顯示 Aphb 擁有保守的表現位置。在本研究中，我發現 Aphb 有一新的表現在生殖細胞，此生殖細胞的表現最早出現自原始生殖細胞 (primordial germ cell, PGC)，並且持續到胚胎最晚期，甚至也在形成中的生殖原區及其產生的卵母細胞前端表現。這樣的表現顯示Aphb 在早期可能取代了 bicoid 的角色，以及接下來時期可能協助生殖細胞的形成。為推測 Nanos (Nos) 及 Pumilio (Pum) 複合體是否可能抑制後端 Aphb 的轉譯，我分析了 Appum 蛋白質的結構以及信使核糖核酸表現，高度保守的 PUM-HD 顯示 ApPum 能夠抑制 Aphb 的轉譯，雖然沒有偵測到不對稱性的信使核糖核酸表現。將此結果與過去 Aphb, Appum, and ApNos表現做比較，我發現豌豆蚜在後端體軸決定是倚賴 Nos 及 Pum 複合體，但前端則是被 Aphb 決定。The pea aphid Acyrthosiphon pisum is a genomic model insect and a unique model for polyphenism due to its developmental plasticity in response to environmental cues. To uncover the relation of embryonic development and gene regulations, reliable expression protocols and functional tools are required. Whole-mount in situ hybridization (WISH) we previously reported can be used to monitor gene expressions during embryogenesis, however chromogenic signals are defective in double detection of genes and construction of three-dimensional image. I therefore developed a fluorescent in situ hybridization (FISH) protocol to overcome these defects. By means of different advantages of four FISH methods, I successfully detected gene expressions in somatic and extraembryonic tissues. The combination of FISH methods also allowed the double detection of genes in somatic cells, germ cells, or both in one preparation. This FISH protocol further aids me in revealing the expression of developmental genes. In our previous findings, mRNA expression of A. pisum hunchback (Aphb), a Drosophila homolog of hunchback, was found in the segments and central nervous system of mid/late stages apart from the anterior pole of early stages, implicating its conserved roles among arthropods and lower organisms. Here I discovered a novel expression pattern of Aphb in germ cells of the pea aphid. Germline expression of Aphb initiates while primordial germ cells formed, and maintains throughout developmental stages. In late embryos, Aphb is also expressed in maturing germaria as well as the protruding oocytes. These findings implicate that the homolog of hb in aphids replaces the role of bicoid in anterior determination and, moreover, has the roles in formation of germ cells. To reveal whether the complex of Nanos (Nos) and Pumilio (Pum) is required to repress the translation of anterior-localized Aphb in the posterior, I analyzed the structure of A. pisum Pum (ApPum) protein and the expression patterns of Appum mRNA. The highly conserved protein structure indicates the ApPum can repress the translation of Aphb, though the asymmetric expression of Appum mRNA, like Drosophila pum, was not found. Together with the known expression patterns of Aphb, Appum, and A. pisum Nos (ApNos), it appears that posterior determination of the pea aphid relies on the ApNos/ApPum complex and the anterior is determined by Aphb.摘要 i Abstract iii Table of Contents v List of Tables ix List of Figures x Abbreviations xii Chapter 1: Introduction 1 1 1.1 The role of hunchback among insects 2 Segmentation in Drosophila 2 The role and expression patterns of Drosophila hb 3 The role and expression patterns of hb in other insects 4 Regulation of the hb mRNA expression 7 The expression of hb in the asexual viviparous pea aphid Acyrthosiphon pisum 7 1.2 The role of pumilio in Drosophila and other insect models 8 The role of translational regulation in developmental biology 8 Translational regulation in the development of Drosophila 9 Translational regulation in the development of other insects 10 The function of pumilio 11 The structure of PUF protein 11 The mechanism of translational regulation of pumilio 12 The anteroposterior axis formation of the pea aphid 12 1.3 The pea aphid as a genome model and a research model 14 Pea aphid as a model organism 14 Essential criteria for a model organism 15 The development of in situ hybridization (ISH) 15 The use of WISH in different organisms 16 Common steps of chromogenic in situ hybridization (CISH) in model organisms 17 Improvement of chromogenic WISH in the pea aphid 18 The development of fluorescent in situ hybridization (FISH) 19 The application of whole-mount FISH in Drosophila 21 The application of FISH in other organisms 21 Chapter 2: Materials and methods 24 2.1 The culture of asexual pea aphid 25 2.2 Synthesis of riboprobes for the whole-mount FISH study 25 2.3 Cloning and sequence analysis of Appum 27 2.4 Riboprobe synthesis of Appum 27 2.5 Cloning and sequence analysis of hb ortholog in A. pisum (Aphb) and M. persicae (Mphb) 28 2.6 Riboprobe synthesis of Aphb and Mphb 29 2.7 Dissection of asexual ovaries 30 2.8 Detection of endogenous fluorescence 30 2.9 Riboprobe hybridization 31 2.10 Color development of chromogenic in situ hybridization (CISH) and substrate fluorescence-fluorescent in situ hybridization (SF-FISH) 32 2.11 Direct fluorescent antibody staining 33 2.12 TSA (Plus) systems 33 2.13 Double whole-mount FISH with combination of SF and TSA 35 2.14 Microscopy 36 Chapter 3: Results 37 3.1 Germline expression of Aphb during embryogenesis of the asexual viviparous pea aphid 38 Molecular cloning and identification of Myzus persicae hunchback 38 The initiation of germline Aphb expression 40 Germline expression of Aphb throughout the mid and late embryonic stages 40 Aphb expression in the forming germarium 41 Germline expression of M. persicae ortholog of Aphb 43 3.2 The presumptive roles of A. pisum homolog of Drosophila pumilio in anteroposterior formation of the pea aphid 44 Cloning and identification of Acyrthosiphon pisum homolog of pumilio 44 Appum expression during early development of telotrophic ovariole 45 The mRNA expression of Appum before katatrepsis 46 Pre-expression of Appum in maturing germaria of asexual embryos 47 Identification of expression patterns of Appum isoform 1-3 47 Transcriptional expression of Appum during oogenesis of sexual ovariole 48 3.3 Development of whole-mount FISH in the pea aphid 49 Disturbance of endogenous fluorescence 49 Detection of gene expression using different FISH methods 51 Detecting extraembryonic and somatic gene expression 53 Double FISH using SF/TSA, TSA/TSA, or SF/TSA Plus in the whole-mount viviparous embryo 55 Application of FISH to sexual ovaries using TSA system 57 Chapter 4: Discussion 58 4.1 Presumptive roles of MpHb 59 4.2 Anterior localization of hb in A. pisum and M. persicae 59 4.3 The presumable translational repression of Mphb by M. persicae Nos 60 4.4 The conservation of Aphb expression in CNS 60 4.5 The presumptive roles of the non-canonical expression of the Drosophila gap gene: the germline hb expression in aphids 61 4.6 The existence of Appum in oviparous and viviparous ovaries 62 4.7 The putative roles of Appum 63 4.8 The presumptive regulation in anteroposterior axis formation of A. pisum 63 4.9 The putative roles of Appum isoforms 65 4.10 Performance of whole-mount WISH and FISH in an viviparous ovary 66 4.11 The efficiency of TSA 67 4.12 The key factors that affect the sensitivity in FISH 67 References 70 Tables 81 Figures 92 Additional pages 132 博士班口試問與答記錄 14235179589 bytesapplication/pdf論文使用權限：不同意授權豌豆蚜前後體軸全體螢光原位雜合生殖細胞hunchbackpumilio[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/262109/1/ntu-103-D96632003-1.pdf