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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 李士傑(Shyh-Jye Lee) | |
dc.contributor.author | Meng-Ju Lin | en |
dc.contributor.author | 林孟儒 | zh_TW |
dc.date.accessioned | 2021-06-16T09:57:31Z | - |
dc.date.available | 2017-02-08 | |
dc.date.copyright | 2017-02-08 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-12-16 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60122 | - |
dc.description.abstract | 細胞分裂與細胞分化往往需要精準分配彼此的比例來確保胚胎發育過程中適切的成長與神經特化。藉由鑽研斑馬魚腦部發育,我觀察到一個微管(microtubule)降解基因,stathmin-like 4 (stmn4)特別且專一地表現在背側中腦(dorsal midbrain)區域。而且該表現區域能與另一個泛神經分化代表基因,elavl3,交錯表現在發育中腦部,暗示stmn4於神經發育的角色。經由morpholino oligonucleotides (MOs)抑制部分蛋白質生成或是給予外加持續活化形式的Stmn4蛋白質,我發現不論是降低(knockdown)或是提高(overexpression)都導致神經分化訊號提早出現於背側中腦。同時,利用CRISPR/Cas9基因修改技術,我也製作出stmn4突變魚(mutant)。但是在其純合子(homozygous)突變幼魚裡卻只觀察到少於10%之前在降低表現幼魚(morphants)所看到的現象,這或許是因為stmn1b增加的補償作用來撫平stmn4的缺失。此外,在縮時攝影當中我發現到在morphants神經前驅細胞分裂與分化的差異。再者,morphants的細胞週期中明顯增長的G2相也能被我用外加的Cdc25a降回正常。最後,把Wnt訊號抑制更能將stmn4轉錄產物給剃除。這些結果都告訴我在神經龍骨時期(neural keel),Wnt訊息傳遞能藉由調控Stmn4在斑馬魚背側中腦的動態平衡來控制細胞週期中G2相長度,進而確保中腦免於過早進入神經分化。 | zh_TW |
dc.description.abstract | A delicate balance between proliferating and differentiating signals is necessary to ensure proper growth and neuronal specification. By studying the developing zebrafish brain, we observed a specific and dynamic expression of a microtubule destabilizer gene, stathmin-like 4 (stmn4), in the dorsal midbrain region. The expression of stmn4 was mutually exclusive to a pan-neuronal marker, elavl3 that indicates its role in regulating neurogenesis. We showed the knockdown or overexpression of stmn4 resulted in premature neuronal differentiation in dorsal midbrain. We also generated stmn4 maternal-zygotic knockout zebrafish by the CRISPR/Cas9 system. Unexpectedly, only less than 10% of stmn4 mutants showed similar phenotypes observed in that of stmn4 morphants. It might be due to the complementation of the increased stmn1b expression observed in stmn4 mutants. In addition, time-lapse recordings revealed the changes in cellular proliferation and differentiation in stmn4 morphants. Stmn4 morphants displayed a longer G2 phase that could be rescued by Cdc25a. Furthermore, the inhibition of Wnt could reduce stmn4 transcripts. These results suggest that the Wnt-mediated Stmn4 homeostasis is crucial for preventing dorsal midbrain from premature differentiation via the G2 phase control during neural keel stage. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T09:57:31Z (GMT). No. of bitstreams: 1 ntu-105-F96b41021-1.pdf: 3052826 bytes, checksum: ec39a8d4b7b44dd1a597c782c7a70c6e (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | ACKNOWLEDGEMENTS 致謝 ii
Contents iii 中文摘要 1 ABSTRACT 2 INTRODUCTION 3 Materials and methods 9 Ethics Statement 9 Zebrafish 9 Plasmid constructions and Site-directed mutagenesis 9 Cell transfection and co-localization analysis 11 Embryo microinjections 11 Generation of stmn4 knockout lines by CRISPR/Cas9 system 12 Whole-mount in situ hybridization and immunohistochemistry 13 Cell proliferation and G2 phase length analyses 15 Confocal microscopy imaging, time-lapse recording and data analysis 15 Quantitative real-time PCR 16 Statistical analysis 17 Results 18 stmn4 is highly expressed before neuronal differentiation in dorsal midbrain 18 Characterization of Stmn4 19 Knockdown of stmn4 causes precocious neuronal differentiation in dorsal midbrain 20 Targeted mutation of stmn4 causes mild precocious neuronal differentiation in dorsal midbrain and the increase in stmn1b expression 22 Stmn4 homeostasis is essential for neural progenitor cell maintenance in dorsal midbrain 25 Stmn4-mediated precocious neuronal differentiation occurs during the neural keel stage 28 Knockdown of stmn4 prolongs G2 phase to trigger precocious neuronal differentiation in dorsal midbrain 30 Inhibition of Wnt signaling downregulates the expression of stmn4 31 Discussion 33 References 43 Acknowledgements 57 Tables 59 Table 1. List of gRNAs used. 59 Table 2. List of primers used. 60 Figures 61 Figure 1. Stmn4 prohibited elavl3 expression in the dorsal midbrain during early embryogenesis. 61 Figure 2. Zebrafish Stmn4 was localized to Golgi apparatus in a cysteine-dependent manner. 63 Figure 3. Ubiquitous expression of CA-Stmn4 disrupts patterning during gastrulation. 65 Figure 4. Overexpression of constitutively active stmn4 induced depolymerization of microtubules in zebrafish embryos. 67 Figure 5. Both two stmn4 translation-blocking morpholino oligonucleotides were designed and proved to owe high efficiency. 69 Figure 6. stmn4 knockdown resulted in premature expression of elavl3 in zebrafish embryos. 71 Figure 7. Establishment of stmn4 mutants by CRISPR/Cas9 technology. 73 Figure 8. Targeted mutation of stmn4 caused mild precocious neuronal differentiation in dorsal midbrain. 75 Figure 9. Elevated stmn1b expression in stmn4 mutant embryos partially contributed to the compensation mechanism which result in mild precocious neuronal differentiation in dorsal midbrain. 77 Figure 10. Overexpression of CA-Stmn4 or depletion of stmn4 by shRNA induced elavl3 precocious expression in dorsal midbrain. 79 Figure 11. Overexpression of CA-Stmn4 induced precocious expression of elavl3 in neuronal precursor cells during neural keel stage. 81 Figure 12. Deficiency of stmn4 prolonged the G2 phase in a Cdc25a-dependent manner. 83 Figure 13. Block of Wnt signaling inhibited the expression of stmn4. 85 Figure 14. A proposed model describing interaction between proliferation and differentiation through Wnt-Stmn4 regulation during early neurogenesis. 87 Video Captions 89 Video 1. Neuronal differentiation in the dorsal midbrain of untreated embryos 89 Video 2. Neuronal differentiation in the dorsal midbrain of ca-stmn4-expressing embryos 89 | |
dc.language.iso | en | |
dc.title | Stathmin-like 4是維持斑馬魚幼魚中腦內神經前驅細胞的關鍵因子 | zh_TW |
dc.title | Stathmin-like 4 is a critical factor for the maintenance of neural progenitor cells in dorsal midbrain of zebrafish larvae | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 黃聲蘋(Sheng-Ping Hwang),江運金(Yun-Jin Jiang),管永恕(Yung-Shu Kuan),鄭邑荃(Yi-Chuan Cheng) | |
dc.subject.keyword | stathmin-like 4,Wnt,Cdc25a,神經分化,神經分裂, | zh_TW |
dc.subject.keyword | stathmin-like 4,Wnt,Cdc25a,neuronal differentiation,proliferation, | en |
dc.relation.page | 89 | |
dc.identifier.doi | 10.6342/NTU201603764 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-12-16 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生命科學系 | zh_TW |
顯示於系所單位: | 生命科學系 |
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