斑馬魚Otx2在胚胎基因調控網路之系統性分析｜國立清華大學博碩士論文庫

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研究生：	黃昶斌 Huang, Chang-Ben
論文名稱：	斑馬魚Otx2在胚胎基因調控網路之系統性分析 Systematic analysis of Otx2 in Zebrafish Embryonic Gene Regulatory Network
指導教授：	莊永仁
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2007
畢業學年度：	95
語文別：	中文
論文頁數：	54
中文關鍵詞：	內中胚層、神經外胚層、班馬魚、基因調控網路、順式調控元件
相關次數：	點閱：2 下載：0
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胚胎的發育主要決定在調控轉錄因子基因和細胞訊息途徑基因的表現，這整個計劃的執行是由轉錄因子(反式因子)結合到基因調控子(順式調控元件)上來控制基因的表現，在發育上許多這種調控的輸入和功能的輸出控制基因的表現形成像網路一般的結構。Otx基因家族帶有pair type homeobox轉錄因子在所有脊椎動物中是高度保守的（conserved），Otx基因家族在發育過程中的角色功能有二個，其中一個功能是在早期胚胎形成過程、另外一個是在頭部發育中扮演著重要的角色，二者都依賴Otx2在時間與空間上正確的表現。
為了研究以Otx2為中心的基因調控網路，首先我們利用反義寡核醣核酸morpholino顯微注射的方式，抑制了Otx2轉譯作用下調Otx2的表現量，並用real time Q-PCR來篩選36個轉錄因子在Otx2 morphants的表現。從Q-PCR的結果上我們發現在50% epiboly時期Otx2正調控5個中胚層特有的轉錄因子Gata5, Foxa2, Gata6, Foxa3和Sox17，在 segmentation時期，Otx2調控許多表現在神經外胚層的轉錄因子，其中表現在中腦區域的POU1, Brn1.2和POU12時間與空間上和Otx2的表現是重疊的且被Otx2所正調控，另外Otx2負調控一些表現在鄰近Otx2表現區域的其它腦區的轉錄因子Six3a, Six3b, Foxh1, Sox4, Pax2a和Gbx2，這些轉錄因子表現在不同腦區負責不同的腦區的特化，Otx2與這些轉錄因子作用是為了腦的特化與區域化。
其次我們比較不同物種Otx2的基因體序列來搜尋演化上的順式保留的元件(elements) 並建構順式基因調控子驅使綠螢光蛋白表現的建構物用來研究Otx2是如何被調控的。我們在斑馬魚的Otx2基因組中找到許多演化上保留的區域，其中5個是非常高度保留的分別命名為模組16, 27, 28, 29和36。模組27, 28, 29正好位於Otx2基因轉錄起始位置上游。在我們起動子分析的實驗結果中發現，在胚胎受精後5小時，第27和29號元件單獨均可以驅動綠螢光蛋白表現在原生內中胚層世系，在75% epiboly和體節形成的早期表現在前端神經外胚層，第29號元件更可驅動綠螢光蛋白表現在晚期的前腦。位於Otx2基因上游25Kb的模組16號可調控模組27和模組29表現在前腦和中腦。我們確定在斑馬魚的Otx2致少含有二個起動子(模組27和模組29)和模組16號可調控它們在前腦和中腦的表現。進一步搜尋第27號元件上的轉錄因子結合位置，我們找到了2個TCF結合位置﹐一個Foxh1結合位置和一個SOX結合位置，我們接下來會用定點突變來分析這些轉錄因子結合位置的角色。

The genomic program for development operates primarily by the regulated expression of genes encoding transcription factors and components of cell signaling pathways. This program is executed by cis-regulatory DNAs (e.g., enhancers and silencers) that control gene expression and the trans-factors that bind on them. The regulatory inputs and functional outputs of developmental control genes constitute network-like architectures. Otx gene family contains pair type homeobox transcription factors that are highly conserved in all vertebrates. The common consider Otx gene has two roles, one is playing function during early embryogenesis and another is playing the crucial role in head development that depend on precisely regulated the temporal and spatial expression of Otx2.
To investigate the gene regulatory subnetwork center by Otx2. First, we knockdown the expression of Otx2 by Morpholino and then used the real time Q-PCR to screen the expression of 36 transcription factors in Otx2 morphant. From our Q-PCR data, we found Otx2 positively regulate five endoderm specific transcription factors Gata5, Foxa2, Gata6, Foxa3 and Sox17 at the 50% epiboly stage. At the segmentation stage, Otx2 regulated several transcription factors that expressed in the neuroectoderm. POU1, Brn1.2 and POU12 expressed overlap with Otx2 in the midbrain were been positively regulated, Six3a, Six3b, Foxh1, Sox4, Pax2a and Gbx2 expressed in the other brain region surrounding midbrain were been negatively regulated. All of them had specification function in the different brain regions. The interaction of Otx2 with them is for brain specification and regionalization.
Secondary, we compared genome sequence of Otx2 across several species to search the evolutionary conserved elements and construction of the regulatory elements driven GFP reporter constructs to study how the Otx2 has been regulated. Five of them are very highly conserved which we named them: Module 16, 27, 28, 29, and 36. Module 27, Module 28 and Module 29 are just immediate upstream of the 5’ transcription start site. In vivo promoter assay results suggest that Module 27 and Module 29 alone can drive GFP reporter gene in dorsal side as early as 5 hours post fertilization and anterior neuroectoderm at 75% epiboly to early somite stage. Module 29 also drives GFP later in the forebrain. Module 16 is 25 kb upstream of the Otx2 gene, which can regulate Module 27, and Module 29 expressed in the forebrain and midbrain. We are sure zebrafish Otx2 have two promoters and Module 16 can regulate both of them express in forebrain and midbrain. Search the transcription factor binding sites in the Module 29, we find 2 TCF binding sites 1 Foxh1 binding site and 1 SOX binding site. We are now performing mutation analysis to study the role of these transcription factor-binding sites.

CONTENTS........................................................................................................................................    1
LIST OF TABLES AND FIGURES................................................................................................ 3
INTRODUCTION
A. Gene Regulatory Network..........................................................................................................4
B. Development of zebrafish embryo..............................................................................................4
C. Otx2 and its role in endoderm and head formation.....................................................................5
D. Hypothesis, rationale and significance.......................................................................................6
MATERIALS AND METHODS
A.    Zebrafish maintenance and embryo collection..........................................................................    8
B.    Resource of genomic DNA........................................................................................................8
C.    DNA constructs: cloning and vector.........................................................................................    8
D.    Morpholino used in this study to knockdown genes’ expression..............................................    8
E.    Microinjection of Morpholino or DNA constructs into one cell stage embryo.........................    9
F.    RNA extraction.........................................................................................................................    9
G.    RT-PCR....................................................................................................................................    10
H.    Embryo observation and photography.....................................................................................    10
I.    Quantitative polymerase chain reaction (Q-PCR)...................................................................    10
J.    Computational identification of cis-regulatory elements.........................................................    12
RESULTS
A.    Morphology of Otx2 knockdown embryos..............................................................................    13
B.    The selected 36 candidate genes...............................................................................................13
C.    The transcription factors positively regulated by Otx2 at early stage in the endoderm territory....................................................................................................................................    14
D.    The transcription factors positively regulated by Otx2 on later neuroectoderm territory....................................................................................................................................    14
E.    The transcription factors negatively regulated by Otx2 on neuroectoderm, endoderm or mesoderm................................................................................................................................    14
F.    Upstream inputs of Otx2 from other researchers’ known down experiments.........................    15
G.    Construction of Otx2 sub-network based on perturbation experiments and literature search......................................................................................................................................    16
H.    Searching cis-regulatory modules and direct binding sites of Otx2 for possible transcription factors.....................................................................................................................................    17
I.    Construction of different GFP reporter constructs to demonstrate the function of each module....................................................................................................................................18
J.    Conserved modules drive GFP reporter gene express in primitive endomesoderm lineage and anterior neuroectoderm...........................................................................................................    18
K.    Conserved Module 16 drive two Otx2 promoters express in the forebrain and midbrain...................................................................................................................................    19
L.    Mutation analysis of conserved transcription factor binding sites..........................................    19
DISCUSSION
A. The role of Otx2 on endoderm..................................................................................................20
B. The role of Otx2 on neuroectoderm..........................................................................................    21
C. Search for more Otx2 downstream genes.................................................................................    22
D. Cis-regulatory modules regulated Otx2....................................................................................    22
E. Evolutionary implication of Otx2 in the endoderm formation..................................................24
REFERENCE...................................................................................................................................    47
APPENDIX
A1.  Primers used to amplify the regulatory regions........................................................................    49
A2.  Primers used for Q-PCR...........................................................................................................    50
B.   The example of Q-PCR data generated from standard with known concentration..................    52
C.   The formula for calculated molecules......................................................................................53
D. Establish Gene Regulatory Network by “BioTapestry” Interactive Network Viewer”...............................................................................................................................................54

LIST OF TABLES AND FIGURES
Table 1. Titration of the Otx2 morpholino causing the defect of embryos at 2 days post-fertilization.........................................................................................................................25
Table 2A: Genes affected by Otx2 at different time points: ........................................................26
Table 2B: Otx2 gene expression affected by others.......................................................................27
Table 3A. Expression patterns of Otx2-promoter-GFP constructs from 5hpf to24hpf.............28
Table 3B. Expression patterns of Otx2-Module 16-promoter-GFP constructs from 5hpf to 24hpf............................................................................................................................................29
Figure 1. Morphology of Otx2 morphants.....................................................................................30
Figure 2. Selected transcription factors used in this study...........................................................31
Figure 3: Otx2 activates presumptive endoderm genes: Gata5, Foxa2, Gata6, Foxa3 and Sox17 at early stage....................................................................................................................32
Figure 4: Otx2 activates neuroectoderm genes: POU1, Brn1.2, POU12 and Gbx1 at later somite stage.................................................................................................................................33
Figure 5: Otx2 represses early expressed genes: Otx2, Foxh1, Tbx16, Sox32 and Bon at later stage.............................................................................................................................................34
Figure 6: Otx2 repressed neuroectoderm, endoderm or mesoderm transcription factors at later somite stage........................................................................................................................36
Figure 7: Upstream inputs of Otx2.................................................................................................38
Figure 8. Gene regulatory sub-network of Otx2............................................................................39
Figure 9. Conservation of the genomic sequence of Otx2.............................................................40
Figure 10.Otx2-GFP reporter constructs and summary of in vivo reporter assay results........41
Figure 11. GFP expressional pattern of Module 29–GFP.............................................................42
Figure 12. GFP expressional pattern of Module 27–GFP.............................................................43
Figure 13. GFP expressional pattern of Module 16–Module 29–GFP.........................................44
Figure 14. GFP expressional pattern of Module 16– Module 27–GFP........................................45
Figure 15. Conserved cis-regulatory modules and transcription factor binding site predict on the Otx2.......................................................................................................................................46

                                

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