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研究生:王宣堯
研究生(外文):Hsuan-Yao Wang
論文名稱:探討金屬磷酸酯酶MPPED1於小鼠大腦發育過程中表現區域之分析
論文名稱(外文):Distribution and Ontogeny of Metallophophoesterase Domain Containing Gene1, Mpped1, in the Mouse Brain
指導教授:陳俊銘陳俊銘引用關係
指導教授(外文):Chun-Ming Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:52
中文關鍵詞:金屬磷酸酯酶大腦新皮質大腦皮質發育孤兒受體蛋白COUP-TFI
外文關鍵詞:MPPED1neocortexcorticogenesisCOUP-TFIERK5cAMP hydrolysis
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MPPED1,全名為Metallophosphoesterase Doamin Containing Gene 1,此基因可以轉譯出一個在演化上高度保留但其功能仍未知的蛋白質。在人類當中,MPPED1的信使RNA(mRNA)已經證實會在成年大腦之中表現,也顯示在中樞神經系統當中MPPED1可能扮演重要的角色。為了要瞭解MPPED1可能具有的生物功能,我們首先系統性的去分析在小鼠大腦發育過程當中MPPED1表現的狀況。我們發現到MPPED1在胚胎而至成鼠時期主要表現在大腦新皮質以及海馬迴CA1的區域,而並不在視丘,脊髓及小腦表現,暗示著MPPED1可能參與在前腦的發育過程當中。我們實驗室進一步利用yeast two-hybrid的方式去找尋會與MPPED1共同作用的蛋白質以瞭解MPPED1可能的功能。其中包括了COUP-TFI以及ERK5還有許多其他的蛋白質已經被證實可以與MPPED1進行交互作用。COUP-TFI已經被報導是一個在大腦新皮質早期各區域的形成過程當中非常重要的轉錄因子。為了探討MPPED1是否也會參與在大腦新皮質各區域之決定的過程,我們以RNA whole-mount in situ hybridization的方法檢視MPPED1在大腦新皮質表面的表現情況。不同於COUP-TFI呈現漸層性或區域性的表現;MPPED1則是均勻的表現。此外,進一步檢視MPPED1與其作用蛋白ERK5,COUP-TFI在大腦發育過程中表現區域之分佈。我們發現到MPPED1,ERK5以及COUP-TFI在發育過程中都會表現在大腦新皮質以及海馬迴CA1區域。最後,我們利用基因突變鼠剔除beta-catenin或Sonic Hedgehog進而造成大腦新皮層不正常發育,並在此狀況下去瞭解MPPED1的表現是否產生變化。發現到在Emx1-Cre;beta-catfx/fx老鼠中,MPPED1在不正常發育的大腦新皮質中的表現量是下降的。總結這些結果,暗示著在前腦的發育過程中,MPPED1在大腦新皮層以及海馬迴當中可能具有某些重要的功能。
MPPED1, Metallophosphoesterase Doamin Containing Gene 1, encodes a novel protein with unknown function and is highly conserved from nematodes and insects to birds and mammals. Human MPPED1’s transcript has been identified in adult brain, indicating that MPPED1 may play an important role in central nervous system. To gain insight into biological functions of MPPED1, the expression of Mpped1 are systematically analyzed through mouse brain ontogeny. Mpped1 is highly expressed in the postmitotic region of the neocortex and CA1 region of the hippocampus from embryonic to adult stage, but not in the thalamus, the spinal cord and the cerebellum, suggesting that Mpped1 may participate in telencephalic development. To further investigate the function of MPPED1, several proteins have been identified to interact with MPPED1 through yeast two-hybrid screening in our lab, including COUP-TFI and ERK5. COUP-TFI (Chicken Ovalbumin Upstream Promoter Transcriptional Factor 1) is an important intrinsic factor for early regionalization of neocortex. To examine whether MPPED1 involves in the cortex arealization or not, whole mount in situ hybridization experiments are performed to identify the expression pattern of MPPED1 on the surface of neocortex. MPPED1 does not display expression gradient as like COUP-TFI exhibiting high-caudal to low-rostral expression gradient. In addtion, by performing in situ hybridization and immunofluorescence staining, MPPED1, COUP-TFI and ERK5 are found to show the same expression distribution in brain neocortex and hippocampal CA1. Finally, the expression patterns of MPPED1 are characterized in the beta-catenin and SHH mutant neocortex, and the expression level of Mpped1 is reduced in the Emx1-Cre;beta-catfx/fx malformed neocortex. In summary, these results indicate that MPPED1 may be responsible for certain functions in neocortex and hippocampus during telencephalic development.
Abbreviations............................................................................................................1
Abstract in Chinese................................................................................................3
Abstract in English.................................................................................................4
I. Introduction
I-1. Coritcogenesis in the mammalian neocortex.............................................5
I-2. The role of signaling morphogens in developing telencephalon..............6
I-3. Patterning of the mammalian neocortex...................................................8
I-4. Metallophosphoesterase Domain Containing Gene 1 (MPPED1)..........9
I-5. MPPED1-interacting proteins, COUP-TFI and ERK5...........................9
II. Specific Aims.....................................................................................................13
III. Materials and Methods
III-1. Plasmids.....................................................................................................14
III-2. Mice...........................................................................................................15
III-3. Brain preparation, tissue processing and tissue section........................17
III-4. Digoxigenin-labeled riboprobe preparation...........................................18
III-5. Hematoxylin and Eosin Staining (H&E staining)..................................18
III-6. In situ hybridization.................................................................................19
III-7. Immunofluorescence (IF).........................................................................20
III-8. Whole mount in situ hybridization.........................................................21
IV. Results
IV-1. Characterization of MPPED1 expression during brain development.23
IV-2. Identification of MPPED1 expression on the surface of neocortex......25
IV-3. Expression patterns of MPPED1, COUP-TFI and ERK5 during telencephalic development.......................................................................25
IV-4. The expression patterns of MPPED1 under 刍-catenin mutant background...............................................................................................26
IV-5. Characterization the expression patterns of MPPED1 under Sonic hedgehog (SHH) mutant background.....................................................28
V. Discussion
V-1. MPPED1 is a brain specific gene expressed from embryonic to adult stage in mice................................................................................................29
V-2. The distribution and dynamic expression of MPPED1 during telencephalic development.........................................................................29
V-3. The putative roles of MPPED1-COUP-TFI and MPPED1-ERK5 complexes in brain development...............................................................30
V-4. The expression patterns of MPPED1 in malformed neocortex..............32
V-5. The potential role of MPPED1 based on its biochemical function........33
VI. References.........................................................................................................36
VII. Figures..............................................................................................................41
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