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Title: | 以神經發育的觀點來探索RBFOX3 (NeuN)在小鼠視覺與成體海馬迴中神經新生所扮演的角色 Investigating the Roles of RBFOX3 (NeuN) in Mouse Vision and Adult Hippocampal Neurogenesis ⎯ from Neurodevelopmental Point of View |
Authors: | Yi-Sian Lin 林宜賢 |
Advisor: | 黃憲松(Hsien-Sung Huang) |
Keyword: | RBFOX3,視網膜,視覺,神經發育,成體海馬迴神經新生, RBFOX3 (NeuN),Retina,Vision,Neural development,Adult hippocampal neurogenesis, |
Publication Year : | 2017 |
Degree: | 碩士 |
Abstract: | RBFOX3是RNA結合蛋白FOX剪切因子家族的一個成員,功能為調控脊椎動物神經系統中前信使RNA(pre-mRNA)的選擇性剪切。已知RBFOX3的抗原即為NeuN,它是一種神經細胞的標記,且會表現在大部份類型的成熟神經元中。然而,RBFOX3在生理上的功能仍然很不清楚。因此,我們利用了Rbfox3基因剔除小鼠來尋找RBFOX3在大腦中所扮演的角色。我們過去的研究發現RBFOX3參與了海馬迴的神經傳遞及突觸可塑性,以及調控海馬迴的突觸生成。此外,缺少Rbfox3容易引發癲癇症狀,空間學習、物體識別(novel object recognition)能力、焦慮程度亦都受到影響。在水迷宮實驗中,我們也發現Rbfox3基因剔除鼠的視覺學習能力有缺損。由於尚未有研究指出RBFOX3與視覺間的關聯性,且確認Rbfox3基因剔除鼠是否具有正常的視覺功能對於進一步的行為實驗至關重要,因此RBFOX3在視覺中所扮演的角色值得探索。在論文的第一部份,我量測了RBFOX3在小鼠視網膜中時間與空間上的表現情形,並發現RBFOX3表現在含有BRN3A的視網膜神經節細胞(ganglion cells)和含有Calbindin的無長突細胞(amacrine cells)。為了探查RBFOX3是否對細胞型態而言是重要的,我利用免疫螢光染色法及H&E染色法分別計算了Rbfox3基因剔除小鼠與相對應的野生型小鼠其視網膜中的細胞數目及視網膜各層的厚度。結果顯示RBFOX3對於維持正常視網膜內叢狀層的結構以及鼻側與下側視網膜神經節細胞層中表現Calbindin的無長突細胞是重要的。此外,利用霍亂毒素B亞基追蹤法發現RBFOX3缺失並不影響視網膜神經節細胞的軸突投射到大腦中的目標區域。在行為的層次上,透過瞳孔光感反射(pupillary light reflex)及視動反應(optomotor response)實驗發現Rbfox3基因剔除鼠具有正常的視覺功能。整體而言,儘管缺少Rbfox3會造成視網膜形態上的缺陷,但不影響小鼠的視力。
在論文的第二部分,因為已知Rbfox3跟一些人類的神經發育疾病有關聯,因此我量測了RBFOX3在不同腦區發育過程的表現量來看RBFOX3在大腦發育過程中是如何受到調控。我發現在不同腦區的RBFOX3表現情形都不一樣。此外,利用Ki67和DCX當作標記來標定齒狀回中不同發育階段的新生細胞,我發現RBFOX3會調控成體海馬迴中的神經新生。這個發現讓我們對於RBFOX3在神經新生及神經發育中的角色有新的見解。 RBFOX3 is an RNA binding protein, belongs to the FOX family that regulate alternative splicing of pre-mRNA in the vertebrate nervous system. The epitope of RBFOX3 has later been identified as NeuN, a neuronal marker that is expressed in most types of mature neurons. Yet, the physiological roles of RBFOX3 remain poorly understood. Therefore, we have generated Rbfox3 knockout mice to investigate roles of RBFOX3 in the brain. Previous findings from our lab show that RBFOX3 is involved in hippocampal neurotransmission and synaptic plasticity, as well as synaptogenesis regulation. The deletion of Rbfox3 increased seizure susceptibility, and impaired spatial learning, novel object recognition, and anxiety. During the water maze task, deficits were observed as well in visual learning for Rbfox3 knockout mice. Since no study regarding the relationship between RBFXO3 and vision has been reported and whether Rbfox3-/- mice have normal visual function is critical for further behavioral tests, it is worth investigating the roles that RBFOX3 plays in vision. In the first part of my thesis, I profiled the temporal and spatial expression of RBFOX3 in the mouse retina and found out that RBFOX3 is expressed in BRN3A-positive ganglion cells and Calbindin-positive amacrine cells. To examine whether RBFOX3 is critical for cell morphology, I performed immunofluorescence staining and H&E staining to quantify the cell number and retinal layer thickness in the retina of Rbfox3-/- mice and their wild-type counterparts. My results showed that RBFOX3 is required for maintaining normal structure of the IPL and Calbindin-positive amacrine cell number in the GCL of the nasal and inferior retina. Furthermore, RBFOX3 is dispensable for ganglion cell axon innervation to targeted brain regions through CTB tracing examination. In the behavioral level, Rbfox3 knockout mice showed normal visual function, which is tested through pupillary light reflex and optomotor response assessments. Taken together, despite the fact that Rbfox3 deletion causes deficits in retinal morphology, it does not affect a mouse’s vision. In the second part of my thesis, knowing that Rbfox3 is associated with human neurodevelopmental disorders, I profiled the RBFOX3 developmental expression pattern in distinct brain regions, in order to examine how RBFOX3 is regulated during brain development. I found out RBFOX3 expression is distinct in different developing brain regions. Furthermore, using Ki67 and DCX as markers to label newborn cells of distinct developmental stages in the dentate gyrus, I found the role that RBFOX3 plays in regulating adult hippocampal neurogenesis. Therefore, this finding provides a new insight on roles of RBFOX3 in neurogenesis and neurodevelopment. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68915 |
DOI: | 10.6342/NTU201703600 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 腦與心智科學研究所 |
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