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Study on the mitotic machinery using zebrafish embryogenesis : 제브라피시 배발생과정을 활용한 세포분열기작 연구

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dc.contributor.advisor이현숙-
dc.contributor.author전희연-
dc.date.accessioned2017-07-14T00:50:34Z-
dc.date.available2017-07-14T00:50:34Z-
dc.date.issued2015-08-
dc.identifier.other000000067126-
dc.identifier.urihttps://hdl.handle.net/10371/121427-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2015. 8. 이현숙.-
dc.description.abstractCell division culminates with mitosis in which duplicated chromosomes are equally segregated to two daughter cells to maintain genomic integrity. To finely regulate the mitotic procedures, many mitotic kinases, phosphatases, and motor proteins are involved. In this study, I aim to discover the conserved function of mitotic machineries such as Aurora-A kinase and Kif18a motor protein using zebrafish embryogenesis as a model system.
Aurora-A is a serine/threonine kinase, which regulates many intricate processes during mitosis in a phosphorylation-dependent manner. Despite the emerging interest for clinical applications, our understanding on Aurora-A function at the organism level is still limited. Here, I have discovered the function of Aurora-A in zebrafish embryo. Using morpholino (MO) technology, I showed that Aurora-A morphant zebrafishes were unhealthy displaying cell death and growth retardation with short and bended trunks. The developmental defects might be attributable to the abnormal mitotic progression, manifested by monopolar and/or disorganized spindle formation and mitotic arrest, accompanying spindle assembly checkpoint activation. Cell death in the absence of Aurora-A was partially rescued by p53 MO co-injection. Taken together, I elucidate that zebrafish Aurora-A is essential for mitotic progression and its depletion induces p53-dependent cell death, which can provide insight into the molecular mechanism of anti-cancer drug targeted for Aurora-A.
Chromosome movements in mitosis are directed by microtubules which are regulated by motor proteins. Kif18a is a kinesin-8 family motor protein, which has been identified as a suppressor of chromosome movements. It is known to regulate microtubule dynamics such as pre-anaphase chromosome oscillations and chromosome congression through two regulation modules: N-terminal catalytic motor domain and C-terminal tail domain. Tail domain in kinesin-8 is known to have controversies whether it has microtubule depolymerase activity or not. Despite of the important role of Kif18a in in vitro and cell-line data, there are still limited knowledge on the physiological role of Kif18a. Therefore, I attempted to dissect the role of two different Kif18a knockout zebrafishes: the C-terminus disrupted one using ZFN (zinc finger nuclease) technique and the motor targeted one using CRISPR/Cas9 technique. As Kif18a C-terminal domain is not required for the viability, fertility, and mitosis, I am now generating motor domain targeted Kif18a mutant zebrafish using a CRISPR/Cas9 knockout method. I suggest that this study, using zebrafish, provides the knowledge on the evolutionary conserved role and regulation mechanism of mitotic machineries.
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dc.description.tableofcontentsABSTRACT…………………………………………………..…i
TABLE OF CONTENTS………………………………...…….v
LIST OF FIGURES…………………………………….….…viii

I. INTRODUCTION………………………………………….1
I-1. Cancer, mitosis and mitotic machinery…………………….1
I-2. Zebrafish as a model for studying on mitotic machineries...5
I-3. Aurora-A mitotic kinase………………………….…….…..7
I-4. Kif18a mitotic motor protein………………….….……...10

II. MATERIALS AND METHODS…………………………13
II-1. Fish maintenance………………………………………....13
II-2. Cloning of zebrafish Aurora-A, Mad2, and Kif18a……….13
II-3. RNA extraction and RT-PCR…………………………….14
II-4. In situ hybridization…………………………………...….15
II-5. Microinjection of mropholinos (MOs) and sense RNAs….15
II-6. Antibody generation in mice and western blotting……….16
II-7. Immunofluorescence assay in whole embryos…………....17
II-8. Flow cytometry………………………………………......18
II-9. Time-lapse microscopy of live embryos…………………18
II-10. Analysis of the spindle assembly checkpoint activation by Mad2 localization……………………………………………..19
II-11. TUNEL assay…………………………………………….20
II-12. Genotyping of Kif18a C-terminus deleted zebrafish……..20


III. RESULTS………………………….……………………22

Part 1. Depletion of Aurora-A in zebrafish causes growth retardation due to mitotic delay and p53-dependent cell death
III-1. Zebrafish Aurora-A is critically required for embryonic proliferation during development……………………………..22
III-2. Knockdown expression of Aurora-A leads to growth retardation and cell death………………………….…………..26
III-3. Knockdown of Aurora-A induces defects in mitotic progression…..….…………………………………………….33
III-4. Aurora-A morphants show delayed mitotic timing which may be due to the SAC (spindle assembly checkpoint) activation……………………………………………………...42
III-5. Cell death in Aurora-A morphant embryos is p53-dependent…………..…………………………………………46

Part 2. Dissecting the role of Kif18a in zebrafish: whether Kif18a is a motor or a depolymerase
III-6. Zebrafish Kif18a is conserved and its expression pattern suggests its importance during embryogenesis………………..55
III-7. Zebrafish Kif18a is important during embryogenesis…….62
III-8. Zebrafish Kif18a C-terminal domain is dispensable for viability and fertility………………………………………….66
III-9. Kif18a C-terminal domain is not required for proper chromosome segregation…………………………………..…74


IV. DISCUSSION………………………………………...…..78
IV-1. Zebrafish, as a model system to study mitosis and development, mirroring tumorigenesis…………….…………78
IV-2. Safeguarding of genomic integrity in normal embryogenesis………………………………………………..79
IV-3. The functions of Aurora-A in zebrafish system…….……80
IV-4. Underlying molecular mechanism of anti-cancer drug targeting Aurora-A……………………………….…………...81
IV-5. Dissecting the roles between N-terminal Motor and C-terminus of Kif18a……………………………………………83
IV-6. Implications for the conserved roles of Kinesin-8 family proteins………………………………………….……………84
IV-7. Perspectives in cancer therapy……………….…………...85

V. REFERENCES……………………………………………90

국문초록………………………………………….…………100
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dc.formatapplication/pdf-
dc.format.extent4130234 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectZebrafish-
dc.subjectembryogenesis-
dc.subjectmitotic machinery-
dc.subjectAurora-A kinase-
dc.subjectKif18a motor protein-
dc.subject.ddc570-
dc.titleStudy on the mitotic machinery using zebrafish embryogenesis-
dc.title.alternative제브라피시 배발생과정을 활용한 세포분열기작 연구-
dc.typeThesis-
dc.contributor.AlternativeAuthorStudy on the mitotic machinery using zebrafish embryogenesis-
dc.description.degreeDoctor-
dc.citation.pagesiv, 102-
dc.contributor.affiliation자연과학대학 생명과학부-
dc.date.awarded2015-08-
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