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研究生:許宏宜
研究生(外文):Hsu, Hong-Yi
論文名稱:斑馬魚Gbx1andGbx2在胚胎基因調控網路之系統性分析
論文名稱(外文):Systematic analysis of Gbx1 and Gbx2 in Zebrafish Embryonic Gene Regulatory Network
指導教授:莊永仁喻秋華
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:英文
論文頁數:57
中文關鍵詞:基因調控網路斑馬魚胚胎
外文關鍵詞:Gene regulatory networkZebrafish embryo
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在後基因體時代,分子生物學的研究已經延伸到基因之間的相互作用關係,而基因體調控網路(genomic regulatory network)則是研究轉錄因子及訊息傳遞物間的作用而建立起一種結構性的網路,基因體條控網路的研究,不但可以仔細闡述發育的機制,也被用以解釋各種細胞內調控,細胞的分化,甚至生物的演化,也因此基因調控網路在近年來變的如此重要,目前已經有許多基因調控網路發表有關海膽內中胚層、爪蟾中胚層以及果蠅背腹部特化上。
Gbx 型轉錄因子會調控腦神經元的細胞命運而影響斑馬魚後腦分化。其中Gbx2比較著名在中後腦疆界(midbrain-hindbrain boundary, MHB)的研究上,此一生物現象是發生在兩個轉錄因子Gbx2以及Otx2之間,透過這兩基因調控產生地峽訊息中心(isthmus organizer)而控制周邊腦幹的分化。過去的研究證明Gbx1是作用在Gbx2上游的基因,並且被認為對於建立中後腦疆界扮演重要的角色。然而,目前還不清楚後腦如何分化?又是甚麼樣的訊號調控Gbx家族使腦幹分化?為了更了解這些機制,我們應用基因調控網路(gene regulatory network)的概念去廣泛地建立許多轉錄因子以及訊號傳導物之間的關係,透過建立這些關係可以構築成小的從屬網路,進而能解釋腦幹分化的詳細機制。在這份研究中,我們為了尋找被Gbx家族所調控的下游基因,利用RT-QPCR去大量檢測在Gbx被下調的斑馬魚胚胎中,其他的基因表現量,同一時間,我們實驗室也下調斑馬魚胚胎其他早期的重要轉錄因子並搜尋是否影響到Gbx家族。
為了更進一步想了解Gbx1以及Gbx2是如何被調控的,我們比較了七種物種(zebrafish, fugu, tetraodon, xenopus, opossum, mouse and human)的Gbx1及Gbx2 DNA序列,並且發現了許多高度保守性的序列有可能扮演調控子的角色,並發現已知能調控Gbx1以及Gbx2的轉錄因子Pbx2、Sox17以及Otx2之結合位置。我們現在正進行活體報導表現分析(in vivo reporter assay)去找尋調控子以及轉錄因子在直接作用在Gbx1以及Gbx2上的結合位置。我們對Gbx1以及Gbx2調控子的分析將讓我們找出具有重要功能調控Gbx1以及Gbx2轉錄因子的結合位置,更進一步能將基因調控網路連結到調控子上。我們得到的這些訊息將會有助於了解Gbx1以及Gbx2如何被調控錄因子的機制,以及它們又如何調控別的轉錄因子去影響腦發育的命運。
In the post genomic era, the study of molecular biology has expanded into the study of interactions between genes and molecules. Genomic regulatory networks concerns the transcription factors as well as signal transduction components and the interaction between them, those interaction forms a networks like architecture which explain fully the detail mechanism underlying the embryogenesis. The gene regulatory networks for early development have become so important in recent study; the GRN has been published for endomesoderm in sea urchin, mesoderm in Xenopus and dorsal-ventral specification in Drosophila.
Gbx class transcriptional factors regulate the cell fate of cephalic primordium to cause neuromeres segmentation in zebrafish hindbrain. Previous study had demonstrated midbrain-hindbrain boundary (MHB) as well as isthmus organizer is controlled by the interaction between Gbx2 and Otx2. Recently, Gbx1 had been proved to act on the upstream genes of Gbx2 and might play a crucial role in establishment of MHB. However, it is still unclear about how the hindbrain patterns and what signals activate Gbx family to promote brain regionalization. To gain a detail understanding about the mechanism, we apply gene regulatory network (GRN) to broadly constitute the relationship between transcriptional factors and signal transduction component, those interaction forms a sub-network which explain the detail mechanism about how brain develop into several segments. We search the downstream outputs of Gbx family by systematically screening the Gbx morphants coupling with RT-QPCR. At the same time, we also knock down several endomesoderm genes to screen if they act on Gbx family. Our data coupling with literature search can provide many outputs of Gbx family and some inputs acting on them.
To further analyze the Gbx1 and Gbx2 regulatory region, we compare the evolutionary conserved elements from seven different species ranged from fugu, tetraodon, xenopus, opossum, and mouse to human, and find several important cis-regulatory modules on zebrafish Gbx1 and Gbx2 genome. In the conserved regions, there are predicted binding sites of Pbx2, Sox 17 and Otx2, which are known to regulate Gbx2 and Gbx1 transcription. We are now performing in vivo reporter assay to find real cis-regulatory modules and direct transcription factor binding sites on Gbx1 and Gbx2 locus. This study of the regulatory elements on Gbx1 and Gbx2 will allow us to identify the important transcription factor binding sites involved in the regulation of Gbx1 and Gbx2, and further enable us to link the gene regulatory networks on the cis-regulatory elements. This information could be useful for establish the underlying mechanisms of how the Gbx1 and Gbx2 are regulated by other transcription factors and how they regulate other transcription factors in order to determine the brain fate.
PAGES OF CONTENTS
PAGES OF CONTENTS - 1 -
ABBREVIATION - 3 -
INTRODUCTION - 4 -
A. Gene regulatory network (GRN) - 4 -
B. The history and background of Gene Regulatory Networks - 4 -
C. Midbrain-hindbrain boundary controls the cephalic neuron primordium - 5 -
D. The expression profile of Gbx1 and Gbx2 - 7 -
E. Hypothesis, rationale and significance - 8 -
MATERIALS AND METHODS - 10 -
A. Zebrafish husbandry - 10 -
B. Resource of Genomic DNA - 10 -
C. Construction - 10 -
D. Microinjection - 11 -
i. Morpholino Antisense Oligos (MO) Microinjection - 11 -
ii. Microinjection of DNA constructs into one cell stage embryo - 11 -
E. RNA extraction - 12 -
F. RT-PCR - 12 -
G. Quantitative polymerase chain reaction (Q-PCR) - 13 -
H. Observation by microscope - 14 -
RESULTS - 15 -
A. Morphology of Gbx2 morphants - 15 -
B. Effects of Gbx2 knockdown on genes involved in midbrain-hindbrain boundary - 15 -
C. Morphology of Gbx1 morphants - 17 -
D. Effects of Gbx1 knockdown on genes on candidate transcription factors - 17 -
E. Upstream gene targets on Gbx family - 18 -
F. Role of Gbx1 and Gbx2 in Gene regulatory network of zebrafish embryonic development - 19 -
G. Searching for cis-regulatory modules and direct transcription factor binding sites on Gbx1 and Gbx2 genome - 19 -
Discussion - 22 -
A. Examined the gene expression of zebrafish detection by Q-PCR - 22 -
B. Gbx2 has multiple functions in zebrafish development - 22 -
C. Gbx1 plays a different role on hindbrain and spinal cord development - 24 -
D. Whole genome searching the other affected gene - 25 -
E. The upstream inputs on Gbx1 and Gbx2 combine the downstream outputs of Gbx to constitute complex network - 26 -
F. Cis-regulatory modules on Gbx1 and Gbx2 - 27 -
REFERENCES - 28 -
LIST OF FIGURES AND TABLES - 31 -
Table.1A: The titration of Gbx2 morpholino - 31 -
Table.1B: The titration of Gbx1 morpholino - 31 -
Table.2: The list of candidate genes - 32 -
Table.3A: The summery of the affected genes regulated by Gbx1 and Gbx2 - 33 -
Table.3B: The summery of upstream inputs on Gbx1 and Gbx2 - 34 -
Table.4: Regulatory activity of Cis-regulatory modules in Gbx2 - 35 -
Figure.1: Morphalogy of Gbx1 and Gbx2 - 36 -
Figure.2: The genes examined in Gbx2 morphants from previous study - 37 -
Figure.3A: Gbx2 regulates neuroectoderm genes in brain region - 38 -
Figure.3B: Gbx2 affected the genes in anterior hindbrain and forebrain - 39 -
Figure.4: Gbx2 also has effect on the genes in other layers - 40 -
Figure.5A: Gbx1 acts on the genes in brain region - 41 -
Figure.5B: Gbx1 regulates the genes in spinal cord region - 42 -
Figure.6: Gbx1 can activate the genes in endoderm - 43 -
Figure.7A: Gbx1 expression was regulated by upstream genes - 44 -
Figure.8: The gene regulatory network of Gbx1 and Gbx2 - 46 -
Figure.9A: Conserved modules of Gbx2 from UCSC genome browser - 47 -
Figure.9B: Gbx2 reporter constructs - 48 -
Figure.10A: Conserved modules of Gbx1 from UCSC genome browser - 49 -
Figure.10B: Gbx1 reporter construct - 49 -
Figure.11: GFP expressional pattern of conserved modules regulating Gbx - 50 -
Figure.12A: The predicted transcriptional factors binding sites on Gbx2 - 51 -
Figure.12B: The predicted transcriptional factors binding sites on Gbx1 - 52 -
Appendix.1 Primers used for this study - 53 -
A. Primers used to amplify the regulatory regions of Gbx2 - 53 -
B. Primers used to amplify the regulatory regions of Gbx - 53 -
C. Primers used for Q-PCR - 54 -
Appendix.2 pEGFP-N1 map - 56 -
Appendix.3 Molecule calculation from QPCR - 57 -
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