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Title: | Rasd1在斑馬魚幼魚內之功能及表現調控 Function and Expression Regulation Control of Rasd1 in Zebrafish Larvae |
Authors: | He-Yen Pan 潘和彥 |
Advisor: | 管永恕 |
Keyword: | rasd1,rab6bb,dexamethasone,CRISPR/Cas9,斑馬魚, rasd1,rab6bb,dexamethasone,CRISPR/Cas9,zebrafish, |
Publication Year : | 2016 |
Degree: | 碩士 |
Abstract: | Neuropilins是Class III Semaphorins的穿膜受體蛋白,並且在軸突導向中扮演著重要的角色。Class III Semaphorins (Sema3s)通常參與在排斥性軸突導向,並且其機制已經有被許多研究報導過。然而,Sema3s也同時參與在吸引性軸突導向,其機制目前仍尚未明朗。Sema3D為一種Sema3s,並且表現在3到4天斑馬魚的腳間核(Interpeduncular nucleus)。同個時間點,Sema3D的受體Nrp1a表現在斑馬魚左韁核(Left habenula)。抑制Nrp1a表現將造成左韁核的軸突被導向腳間核的量減少,其肇因於缺少Nrp1a的軸突無法被Sema3D所吸引。過去的實驗室成員發現rasd1以及rab6bb兩個small GTPases與nrp1a共同表現在第4天斑馬魚左韁核內。各別以Morpholino knock down兩個small GTPases將造成與nrp1a 的Morphants相似的軸突導向缺失。我接收了rasd1突變種並且製造了rab6bb突變種,兩個突變種都是經由CRISPR/Cas9技術製造。我的分析資料顯示與野生種比較,rasd1突變種並沒有明顯的軸突導向缺失。此外,我發現Dexamethasone可引起特定組織表現rasd1,但韁核內生性rasd1表現並不受到Dexamethasone影響。除了rasd1突變種外,我使用CRISPR/Cas9製造了一隻在生殖細胞中帶有目標位置突變的rab6bb基因的斑馬魚。突變rab6bb基因的製造將可幫助我們了解蛋白質Rab6bb在細胞內中的的功能。 Neuropilins are transmembrane proteins and act as the receptors of Class III Semaphorins in axonal target finding process. Class III Semaphorins (Sema3s) usually play a role in repulsive axon guidance events and the mechanism has been well-studied. However, Sema3s also play roles in attractive axon guidance events with unknown mechanism. Sema3D is expressed in interpeduncular nucleus (IPN), and Nrp1a is expressed in left habenula (LHA) at 3dpf and 4dpf. Disruption of nrp1a displays LHA axon projection defect due to loss of Nrp1a-mediated axon attraction by Sema3D. Previously our laboratory had identified rasd1 and rab6bb that are coexpressed with nrp1a in zebrafish LHA at 4dpf. Their morphants also display similar LHA axonal projection defect as nrp1a morphants do. I have adopted rasd1 and generated rab6bb mutants that were created with the CRISPR/Cas9 system. My analyses indicated that there was no significant difference in volume and morphology between rasd1 mutants and WT. In addition, I found that tissue-specific ectopic rasd1 expression can be induced in choroid plexus in the hindbrain and hematocyte-like cells by dexamethasone treatment, but the rasd1 expression in HA neurons is not influenced. Besides to rasd1 knockout mutants, one F0 fish has been confirmed to carry rab6bb mutation in the germline cells at the targeted location. The generation of rab6bb mutant fish will be valuable to understand the roles of zygotic rab6bb beyond its basic cellular function as a protein. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78133 |
DOI: | 10.6342/NTU201602323 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生化科學研究所 |
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ntu-105-R02b46027-1.pdf Restricted Access | 1.86 MB | Adobe PDF |
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