臺灣博碩士論文加值系統

本實驗室早期選殖到一斑馬魚KLF8 cDNA，全長為1763 bp，可轉譯出348 個胺基酸，經胺基酸序列比對顯示與人類的KLF8有63%的相似性。且斑馬魚KLF8靠近C端有3個krüpple-type zinc finger domain，而N端有一個repressor motif (PVALS/T)。當以antisense oligomer morpholino knockdown實驗來分析KLF8的功能時，在morphant 胚胎可觀察到圍心膜腫大及心臟形態不正常的情形，因此本論文的主要目的在研究斑馬魚KLF8基因在心臟發育所扮演的功能。利用不同KLF8反意寡核酸抑制KLF8蛋白質之形成，造成24 hpf (hour post fertilization) morphant腦部中後腦交界結構凌亂及中腦腦室區域縮小，而在28 hpf明顯觀察到心管變短，48 hpf心臟looping出現問題，72 hpf心臟looping不完全，心室與心房維持anterior-posterior方向，分為D-looping但不完全型態、L-looping、no-looping三種型態。同時本實驗發現斑馬魚胚胎注射KLF8-MO1濃度越高，造成心臟looping缺損、腹部有血液堆積的比例越高，導致心室與心房之腔體明顯變小，具有劑量相關(dose-dependent)的特性。進一步以全覆式原位雜合反應檢視心房或心室的標識基因，amhc、cmlc2在KLF8基因表現被抑制之胚胎中變化情形，結果顯示amhc及cmlc2表現量有增加的情形並確認心臟有D-looping但不完全型態、L-looping及no-looping三種型態。另外亦檢視與心臟發育相關的基因的變化情形，nkx2.5在24 hpf morphant心管表現量有增加的情形，且心管的jogging亦呈現左右不對稱的情形。而BMP2b在gastrula morphant的表現量則不受影響。由於KLF8 mRNA並不表現在心臟，且從學長的實驗結果顯示KLF8-MO1的morphant其肝、腸及外分泌胰臟有左右顛倒現象。脊椎動物左右側方向的決定會影響到臟器正常的發育過程。由文獻中得知若脊椎動物左右軸(L/R axis)不對稱的建立受到改變則會產生一些先天性的心臟疾病。因此我亦檢視與左右軸不對稱性建立相關的基因，主要觀察midline的結構及Nodal signaling pathways下游相關基因在KLF8-MO1 morphant中是否受到影響。結果顯示flh、ntl在脊索的表現量不受影響，而gsc在索前板表現量及情形與野生種胚胎一樣。cyc在Polster、索前板以及神經外胚層部位的表現量與野生種同期胚胎一樣。oep在索前板、脊索表現量與野生種同期胚胎一樣。lefty1在索前板表現量有增加情形，而在體節形成的後期，left1並沒表現在左側間腦以及左側側板中胚層。而lefty2在索前板以及底板表現量與野生種相同，但在體節形成的後期，lefty2並沒表現在左側側板中胚層。且pitx2和lefty1一樣沒有表現在左側間腦。綜合以上的結果，斑馬魚KLF8會調控與左右軸不對稱建立有關的基因進而影響心臟的發育。此外，我們並製作KLF8多株抗體來確認KLF8-MO的專一性，Western blot實驗結果顯示，KLF8-MO1和KLF8-MO2會抑制斑馬魚胚胎內KLF8的轉譯作用，而KLF8- 4 mm MO1則無法抑制KLF8的轉譯作用，達到MO專一性的抑制結果。

Previously, we have isolated KLF8 cDNA from zebrafish and it encodes 348 amino acids that sharing high (63%) amino acid sequence similarity with human KLF8. Zebrafish KLF8 protein has three tandem Cys2His2 zinc-finger motifs at the carboxyl terminus. In addition, a repressor motif (PVALS/T) is located at the N-terminus of KLF8 protein. To explore the function of KLF8 on zebrafish development, we used KLF8 specific antisense morpholino oligomer to knock-down KLF8 expression and detected abnormal heart morphology and swollen pericardiac chamber in KLF8 morphant embryos. Therefore, the main goal of this thesis is to investigate function of KLF8 in zebrafish heart development. In different KLF8-MOs injected embryos, we found defects in midbrain/hindbrain boundary formation, reduction of midbrain ventricle in 24 hour post fertilization (hpf) morphant embryos. Heart tube was shorter in 28 hpf morphant as compared with wild type. Defect in heart looping was observed in 48 hpf morphant embryos, their atriums and ventricles remained in an anterior-posterior orientation, which containing different looping defects including D-looping with defect, No-looping and L-looping in 72 hpf morphant embryos. Higher percentage of heart looping defect, pericardia effusion and dropsy of ventral position were found when higher dose of KLF8-MOs were used for injection, and there is a dose-dependent manner. To further study these phenotypes, we probed these embryos with different marker genes for heart development. In KLF8-MO1 injected embryos, expression levels of amhc, cmlc2 and nkx2.5 in the heart were significantly increased in KLF8 morphants. From the expression pattern of these marker genes, we also found defective heart looping morphogenesis including D-looping with defect, No-looping, and L-looping in KLF8 morphants. Therefore, cardiac jogging and looping polarity were affected in KLF8-MO1 injected embryos. We also examined expression level of BMP2b, which is important for heart development and found its expression pattern was not altered in KLF8 90% epiboly morphants as compared with wild type. Since KLF8 is not expressed in the heart, previous results showing defects in L-R axis asymmetry of different digestive organs in KLF8 morphant embryos, and the information regarding influence of L-R axis asymmetry on mammalian heart development, we further analyzed effects on genes involved in left-right axis asymmetry in KLF8 morphant embryos. Expression levels of flh and ntl in the notochord, and expression level of gsc in the prechordal plate were not altered in KLF8 gastrula morphants as compared with wild type. Thus, axial midline structures develop normally in KLF8 MO1-injected embryos. Expression levels of cyc and oep in the prechordal plate were not altered in KLF8 gastrula morphants. Expression level of lefty 1 in the prechordal late was increased, while lefty2 expression was not changed in KLF8 gastrula morphants when compared with wild type. Expression levels of lefty1 and pitx2 in the left diencephalon and that of lefty2 in the heart were significantly decreased in KLF8 22s stage morphants. Overall, these experimental observations suggest that KLF8 may regulate heart jogging and looping processes by modulating expressions of members of Nodal signaling pathway. In order to demonstrate the specificity of KLF8–MOs, We raised an anti-KLF8 polyclonal antibody. Western blot analyses showed reduction in putative KLF8 protein levels when embryos that have been respectively injected with either KLF8-MO1 or KLF8-MO2, while KLF8 protein level was not altered in embryos injected with KLF8-4mm MO1. These results demonstrate the specificity of KLF8-MO1 and KLF8-MO2 used in this study.

目錄
頁次
誌謝……………………………………………………i
中文摘要………………………………………………ii
英文摘要………………………………………………v
目錄……………………………………………………viii
實驗方法目錄…………………………………………ix
表目錄…………………………………………………xi
圖目錄…………………………………………………xii
導論……………………………………………………1
實驗方法………………………………………………21
結果……………………………………………………47
討論……………………………………………………59
參考文獻………………………………………………70
圖表……………………………………………………83
附錄……………………………………………………106

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