探討斑馬魚ttbk2a和ttbk2b的功能;Investigation of ttbk2a and ttbk2b function in the zebrafish

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题名:	探討斑馬魚ttbk2a和ttbk2b的功能;Investigation of ttbk2a and ttbk2b function in the zebrafish
作者:	王又婷;Wang, Yu-Ting
贡献者:	生命科學系
关键词:	ttbk2a;ttbk2b;斑馬魚;腦;纖毛;ttbk2a;ttbk2b;zebrafish;brain;cilia
日期:	2018-07-31
上传时间:	2018-08-31 11:37:03 (UTC+8)
出版者:	國立中央大學
摘要:	初級纖毛在脊椎動物的神經系統發育中扮演非常重要的角色，然而，目前對於初級纖毛是如何去影響神經系統的機制仍然不是很清楚。Tau tubulin kinase 2 (TTBK2) 是一個表現在腦中用來調節初級纖毛形成的重要因子，TTBK2的突變將會造成脊髓小腦萎縮症，患者的小腦會有萎縮的現象。由此可知，TTBK2對於腦的發育形成非常重要，但其中的機制依然不是很了解。所以，在本篇研究中，我選擇利用斑馬魚研究ttbk2a以及ttbk2b的表現與功能。我利用RT-PCR及原位雜交法觀察ttbk2a及ttbk2b的mRNA表現，我發現在斑馬魚出生後第三天，ttbk2a及ttbk2b都開始有表現，但其中只有ttbk2b在出生的時候就開始表現到出生5.25小時候表現下降。從原位雜交法中，我發現ttbk2a及ttbk2b的mRNA表現在許多腦區，例如: ventral telencephalic area, medial zone of dorsal telencephalic area, periventricular grey zone of optic tectum, lateral division of valvula cerebelli, and corpus cerebelli。另外，為了探討ttbk2基因的功能，我利用CRISPRC-Cas9系統去做基因編輯，進而去模仿人類的疾病模式以及破壞掉ttbk2中最重要的區域。目前，已經建立出一系列的突變魚。初步我看到了一些突變魚的游泳能力下降。利用這些魚我們可以進一步探討ttbk2以及初級纖毛在腦中扮演的角色。;The primary cilium plays a critical role in the development of vertebrate nervous system. However, the function of primary cilia in brain remains unclear. Tau tubulin kinase 2 (TTBK2) is a regulator required for primary cilium formation, and its transcript can be detected in the brain. In human, mutations in TTBK2 serine rich domain cause spinocerebellar ataxia type 11 (SCA11) and the cerebellum will undergo degeneration. TTBK2 is essential for brain development, yet the mechanism is still elusive. Therefore, I focus on ttbk2a and ttbk2b in zebrafish. Zebrafish is a powerful model for the study of human disease. In this study, I characterized ttbk2a and ttbk2b RNA expression pattern during embryogenesis and in the adult tissues using RT-PCR and in situ hybridization. The ttbk2a and ttbk2b transripts started to be expressed after 3 dpf, and ttbk2b was also expressed transiently from the 1-cell stage to 50％ epiboly. Both ttbk2a and ttbk2b transcripts were detected in several adult fish brain regions, such as ventral telencephalic area (Vd), medial zone of dorsal telencephalic area (Dm), periventricular grey zone of optic tectum (PGZ), lateral division of valvula cerebelli (Val), and corpus cerebelli (Cce). To investigate ttbk2 function, I generated ttbk2 mutants by CRISPR-Cas9 system to create mutations mimicking human patients and disrupting the most important domains of ttbk2. I already obtained 5 lines of F1 fish. One G0 fish had reduced ability to swim in the swim tunnel assay, indicating that ttbk2 mutation may affect zebrafish swimming behavior. These mutant fish will be a useful tool to investigate the function and mechanism of ttbk2 and primary cilia.
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