碩士 / 國立臺灣海洋大學 / 生物科技研究所 / 95 / Apoptosis is termed as programmed cell death, which plays a crucial role in the development of central nervous system (CNS). Some pro-apoptosis proteins such as DAPK family, Daxx and Par-4 play an important role in neuronal cell death. These proteins was found to interact with each other. To study the function of pro-apoptosis proteins in zebrafish development and neuronal cell death, we first cloned zebrafish ZIPK, Daxx and Par-4 cDNAs, and the zebrafish DAPK which was provided by Dr. Ruey-Hwa Chen. Through whole mount in situ hybridization we found that all four genes were expressed in the zebrafish brain. In order to understand the role of each gene in CNS development, we injected antisense oligonucleotide morpholino (MO) of zDAPK, zDaxx and zPar-4, respectively, to zebrafish embryos. Injection of zDaxx-MO and zPar-4-MO exihibit effect on embryos development; However, injection of zDAPK-MO induces the formation of smaller head and enlarged ventricle of hindbrain of zebrafish embryos. When using the brain marker genes, such as TROY, Pax2.1 and Pax6, and the genes of zDaxx and zPar-4 as probes for whole mount in situ hybridization with DAPK-MO injected zebrafish embryos, DAPK-MO injection affects the expression pattern of these genes. In addition, the lateral line development of zebrafish is regulated by central nervous system. After stainging and counting the neuromast number, we found that the number of neuromast decreased, suggesting that zDAPK may play an important role in early zebrafish development of CNS. In addition, we overexpressed zDaxx-HA-GFP and zPar-4-HA-GFP driven by neuronal specific HuC promoter and injected into zebrafish embryos at 1-cell stage. After injection, the green fluorescence signal from the fusion protein was observed at 24 hpf, while decreased at 48 hpf. The result suggests that the disappearance of the GFP signal may be related to cell death. On the other hand, when the mutant form of zPar-4-GFP without leucine zipper domain was injected into the zebrafish embryos, the GFP signal was observed in the neuronal cells and axons from 48 hpf to 96 hpf. This result suggests that the leucine zipper domain of zPar-4 was involved in the expression of the GFP. As for zZIPK, zZIPK-induced apoptosis in zebrafish was not observed, suggesting that the function of zZIPK in zebrfish is different from the mammalian homolog. Based on results, we suggest that zDAPK plays a crucial role in regulating neuronal cell death in zebrafish; perhaps zDAPK can interact with zDaxx and zPar-4 to regulate the apoptosis mechanism of zebrafish nervous system.
| Identifer | oai:union.ndltd.org:TW/095NTOU5111002 |
| Date | January 2007 |
| Creators | Shan Su, 蘇珊 |
| Contributors | Chang-Jen Huang, 黃銓珍 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 92 |
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