碩士 / 國立陽明大學 / 生理學研究所 / 91 / Muscarinic acetylcholine receptors (mAChRs) wildly express in the central nervous system and peripheral tissues. There are five distinguished subtypes (M1-M5) of mAChRs according to molecular cloning experiments. The ACh neuronal and non-neural signaling and regulation of cell function are mediated through specific subtype of mAChR and the coupled G-proteins. The physiological roles of each mAChR subtype have been revealed in mAChR knockout mice. However, it is difficult to study the mAChR role in embryogenesis in mice, because the mice embryo develops in the uterus. Zebrafish embryo is a better system for studying development and genetics, because the zebrafish fertilizes and develops externally, the embryo is transparent, and the embryo can survive without blood supply for a few days. Moreover, an embryo gene can be specifically knocked-down by microinjection of a morpholino-modified antisense oligonucleotide (MO). Recently, we have cloned the zebrafish genes encoding the M2 and M5 mAChRs, respectively. We have adapted the gene knockdown techniques to demonstrate that the M2 mAChR mediates the ACh-induced bradycardia in the zebrafish embryo. In this study, we microinjected the M2 mAChR MO (M2MO) into the zebrafish one-cell embryos and traced the embryogenesis by tape-recording the embryos under a dissection
microscope. The M2MO-injected embryos showed abnormal phenotypes
including small eyes, small head, large pericardial cavity, blood colorless, shrunk muscle, pale skin, fuzzy somite boundary, etc. We have focused on the analysis of blood colorless phenotype first. The blood colorless phenotype is specific due to the M2MO, because microinjection of a four-base mismatch M2MO (M24mp) resulted in normal blood color. Furthermore, the M2MO effect on blood colorless phenotype is
dose-dependent, ranging from 1 to 0.01 mM. The M2MO effect on blood colorless phenotype is time-dependent, and most obvious at 48 hpf, and decreases gradually until 108 hpf. The blood colorless is due to reduced hemoglobin expression as shown by o-dianisidine staining of zebrafish embryos. In order to understand the physiological effect of blood colorless on embryo survival, we study the mortality rate of zebrafish
embryos from 48 to 108 hpf and found the mortality increased in 84 hpf embryos. Our studies provide evidences that M2 mAChR plays a role in zebrafish embryos hemoglobin synthesis.
| Identifer | oai:union.ndltd.org:TW/091YM000116011 |
| Date | January 2003 |
| Creators | Shu-Fen Hsu, 許淑芬 |
| Contributors | Ching-Fong Liao, Ph. D., 廖欽峰 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 48 |
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