|Abstract: ||為了提供一個了解基因缺陷與人類疾病關係的研究平台，實驗室之前以DNA化學突變劑N-ethyl-N-nitrosourea (ENU) 處理具綠螢光心臟之Tg(BMP4:EGFP) 基因轉殖斑馬魚並篩選出一具心臟缺陷並為隱性致死突變斑馬魚H002。24 hpf H002 mutant其表現型為腦部出現混濁的情形；48 hpf H002 mutant 的腦部、retina 及全身體長變小，腦部血液循環受阻，部分H002 mutant伴隨著延遲或輕微心臟looping缺陷；72 hpf H002 mutant 腦部、retina及全身體長持續變小，部分H002 mutant心臟開始拉直變細長；96 hpf H002 mutant頭部、眼睛及全身體長持續變小，3/4以上的lens 裸露在retina外但仍由角膜包覆著，部分H002 mutant血液循環幾乎喪失，心臟拉直變細長且圍心腔腫大，通常在受精後第五天或第六天陸續死亡。TUNEL assay 結果顯示 24 hpf H002 mutant在腦部、retina、spinal cord及尾巴背側有大量的細胞凋亡產生。經由 Q-PCR 的分析，發現在34及74 hpf H002 mutant中，caspase 8、p53、△113p53 及 p53 所誘導的基因如 p21、PUMA、mdm2、cyclin G1 表現量均有顯著上升的情形。全覆式原位雜合反應結果顯示腦部標誌基因tbr1、eng3、krox20 在24 hpf H002 mutant 端腦及間腦、中腦後端及中後腦交界、菱腦原節 3 和 5 之表現情形與其 wild type sibling 沒有分別。同樣的CNS及retina 標誌基因 zic1 及 rx1結果顯示分別在 24及49 hpf H002 mutant 表現的位置與其wild type sibling 亦沒有分別。PH3 抗體免疫螢光染色結果顯示 24及34 hpf H002 mutant 增生細胞與其wild type sibling相比有減少的情形。相較於wild type sibling，流式細胞儀 DNA content分析結果顯示 24 hpf H002 mutant 其Sub-G1、4N及大於4N 之細胞族群有上升的情形，而H002 mutant 其G1及 S之細胞族群則有下降的情形。由γ-tubulin與α-tubulin之免疫螢光染色結果顯示 24 hpf H002 mutant 有絲分裂細胞之中心體數目與紡錘絲極性與其wild type sibling 沒有分別，但均比較大。由染色體抹片結果顯示 24 hpf H002 mutant 有多倍體的現象產生。由 Giemsa染色之細胞結果顯示24 hpf H002 mutant 其細胞可順利度過有絲分裂。藉由SSLP marker對H002 mutant carrier與定位雜交種所產生之胚胎進行PCR得到的連鎖圖譜顯示H002 mutant gene位於LG12上且突變基因坐落於 L12E5及Z26459兩 SSLP marker之間。本論文研究結果顯示位於 LG12 之 H002 突變基因對於細胞週期的調控扮演一不可或缺之重要角色。|
In order to investigate relationships between gene defects and human diseases, a recessive embryonic lethal H002 ENU mutant fish showing heart defect was previously obtained by ENU (N-ethyl-N-nitrosourea) treatment of Tg(BMP4:EGFP) transgenic fish. 24 hpf H002 mutant embryos possess cloudy brain. 48 hpf H002 mutants show decreased size of brain with circulation defect, reduced sizes of retinas and total body length. At the same time, some 48 hpf H002 mutants are accompanied with delayed or moderate heart looping defects. Sizes of brain, retinas and total body length of 72 hpf H002 mutants continuously decrease. Some 72 hpf H002 mutants have heart looping defects resulting linear heart. Sizes of brain, retinas, and total body length of 96 hpf H002 mutants continuously decrease and more than 3/4 lens of 96 hpf H002 mutants are outside of retinas but still wrapped by the cornea. Some of 96 hpf H002 mutants exhibit no circulation containing linear hearts with enlarged pericardial cavity. H002 mutants often die between 5 and 6 dpf. TUNEL assay reveal that 24 hpf H002 mutants have severe apoptosis in the brain, spinal cord, retinas and dorsal tail. Q-PCR results indicate significant up-regulation of caspase 8, p53, △113p53 as well as p53–regulated genes including p21, PUMA, mdm2, and cyclin G1. Whole mount in-situ hybridization results indicate that expression patterns of brain marker genes including tbr1, eng3, and krox20 in the telecephalon, diencephalon, posterior midbrain, midbrain/hindbrain boundary, and rhombomere 3 and 5 of 24 hpf H002 mutant are similar as those in wild type sibling. Expression patterns of CNS marker zic1 and retina marker rx1 in 24 and 49 hpf H002 mutants are also similar as those in wild type sibling. Immunofluorescence staining of PH3 reveals reduction in proliferative cell number in respective 24 and 34 hpf H002 mutant as compared with wild type sibling. Flow cytometry analysis show increase of respective Sub-G1, 4N, and > 4N cell populations and decrease of G1 and S cell populations in 24 hpf H002 mutant as compared with wild type sibling. Immunofluorescence staining of γ-tubulin and α-tubulin reveal that centrosome numbers and spindle polarity in mitotic cells of 24 hpf H002 mutants are the same as wild type sibling, however larger sizes of centrosomes and spindle are detected in 24 hpf H002 mutants as compared with wild type sibling. Metaphase spread results indicate that 24 hpf H002 mutant are polyploid. Gimsa stain reveals that cells of H002 mutants can go through complete mitosis. H002 mutant gene may be located between L12E5 and Z26459 SSLP markers on the LG12 analyzed by SSLP marker mapping to compare PCR banding patterns in embryos generated from H002 mutant carrier and mapping cross. Overall, results from this thesis indicate that H002 mutant gene may play essential roles in the regulation of cell cycle.