Functional analysis of zebrafish KLF8 on left-right asymmetry and heart development

• Main Authors: Hsin-Chi Liao, 廖信吉
• Other Authors: Sheng-Ping L. Hwang
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/63267413765328714062

碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 94 === 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.