臺灣博碩士論文加值系統

Aeolosoma viride為一種淡水生環節動物，擁有強大的再生能力，在切除頭部後，A. viride可以在五天內完成頭部再生。本研究成功選殖出A. viride 體內Wnt signaling pathway 相關基因Avi-wnt4、 Avi-wnt8, Avi-β-catenin及Avi-notum，並研究它們在頭部再生過程中的表現及調控。結果顯示：在切除頭部後，avi-wnt4、avi-β-catenin及avi-notum會在再生中的blastema中表現。而blastema中的β-catenin在再生過程會進入細胞核開啟canonical Wnt pathway.。在Wnt pathway抑制劑XAV939處理後，A. viride的頭部再生會受到抑制；然而，在Wnt pathway促進劑azakenpaulone處理後，A. viride的頭部再生也會受到抑制，但是XAV939和azakenpaulone對再生的抑制效果會因為處裡藥物的時間點不同而產生不同的效力。因此，根據實驗結果，我們推論canonical Wnt/β-catenin Signaling pathway在調控A. viride的頭部再生過程中需要先關閉後開啟。

Aeolosoma viride, a fresh water annelid, has been used as an animal model for regeneration study. After decapitation, in which the anteriormost 4 segments including the brain were removed, A. viride can regenerate its lost parts within five days. Among many physiological pathways, the canonical Wnt signaling pathway is known to play roles not only in development and stem cell fate determination but also in regeneration. In this study, genes encoding four different components of Wnt signaling pathway, Avi-wnt4, Avi-wnt8, Avi-β-catenin and Avi-notum, have been cloned. All of them were expressed in the newly regenerating cell mass, the blastema. In addition, this pathway exerts its effect by nuclear import of β-catenin, functioning as a transcription co-activator, it was demonstrated that β-catenin indeed localizes into the nucleus in cells of blastema during regeneration. This result indicates that the pathway is in an on state during A. viride regeneration. Furthermore, after treated with XAV939, a Wnt pathway inhibitor, the anterior regeneration of A. viride was obviously inhibited. This result supports that Wnt pathway is indispensable for proper head regeneration in A. viride. However, after treated with azakenpaulone, a Wnt pathway activator, the anterior regeneration of A. viride was also obviously inhibited. However, the inhibitory effects of XAV939 and azakenpaulone took place at different time points. Therefore, it can be inferred that the canonical Wnt signaling pathway is regulated differently in the regeneration process of A. viride. That is, this pathway should be inactivated at the beginning, and then be activated at late during the anterior regeneration.

中文摘要 i
Abstract ii
Introduction 1
Regeneration 1
Wnt signaling pathway 1
The relationship among regeneration, body axis patterning and Wnt signaling pathway 2
Aeolosoma viride 3
Regeneration in A. viride. 4
Aim 4
Main finding in the thesis 4
Material and methods 6
Aeolosoma viride 6
Head amputation 6
Treatments during anterior regeneration 6
mRNA extraction 7
Reverse transcription 7
Gene cloning of Avi-wnt4, Avi,wnt8, AvI-β-catenin and Avi-notum 8
Synthesis of DIG-labled Riboprobes of Avi-wnt4, Avi-β-catenin and Avi-notum 11
Sample preparation for in situ hybridization or Immuno-fluorescence 11
Whole-mount In situ hybridization 12
Whole-mount Immuno-fluorescence 13
EdU labeling for proliferating cell 13
Statistical analysis 14
Results 15
Molecular cloning and phylogenetic analysis of the canonical Wnt signaling pathway genes 15
Gene expression patterns of the canonical Wnt signaling pathway related genes during anterior regeneration in A. viride 16
The effects of the canonical Wnt signaling related-drugs to anterior regeneration 17
Nuclear translocation of β-catenin during the anterior regeneration 18
Cell proliferation on the blastema during anterior regeneration 20
Discussion 21
Gene conservation 21
A/P body axis patterning 21
Anterior Regeneration in A. viride 22
An axis-pattern model for the anterior regeneration in A. viride 25
Other puzzles in the thesis 26
Future works 27
Reference 28
Table. 32
Figures 33
Supplement data 58

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