Study for oligodendrocyte differentiation in the spinal cord of zebrafish
- 주제(키워드) zebrafish + oilgodendrocyte
- 발행기관 고려대학교 대학원
- 지도교수 박해철
- 발행년도 2011
- 학위수여년월 2011. 2
- 학위구분 박사
- 학과 일반대학원 의학과
- 원문페이지 109 p
- 실제URI http://www.dcollection.net/handler/korea/000000024990
- 본문언어 영어
- 제출원본 000045635164
초록/요약
Oligodedrocytes are glial cells that assemble myelin sheath around axons in the central nervous system. Myelin sheath is essential for the insulation of the axon and for the saltatory conduction of electric impulses. Therefore, defects in oligodendrocyte differentiation have been implicated in multiple neurological disorders, like seizure, ataxia and multiple sclerosis. However, little is known about the mechanism of oligodendrocyte differentiation and myelination. In the present study, we investigated the mechanisms of oligodendrocyte differentiation using the forward and reverse genetic studies with zebrafish model animal. First, we investigated the function of Frizzled 8a (Fz8a) mediated Wnt signaling in the differentiation of oligodendrocytes in zebrafish. We show that Fz8a plays a critical role in the specification and maturation of oligodendrocyte progenitor cells (OPCs) in the ventral spinal cord. Loss of Fz8a function perturbed the proliferation and organization of radial glial cells that give rise to OPCs in the ventral precursor region of spinal cord. In addition, we demonstrate that Wnt signaling activation after the specification of OPCs blocks the formation of mature oligodendrocytes and results in the elimination of OPCs Next, to investigate the mechanisms underlying myelination in vivo, we generated transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under the control of the mbp promoter. This transgenic fish displayed faithful EGFP expression in oligodendrocytes and Schwann cells in embryonic and adult zebrafish. Our results suggest that this transgenic zebrafish could be a valuable animal model to study oligodendrocyte differentiation and myelination in vivo. Last, we analyzed zebrafish mutant with defects in differentiation of oligodendrocyte. Leo1 is a component of evolutionarily conserved PAF1 complex, which is a RNA polymerase II-associated factor 1 complex and regulates overall processes of transcription. Here we report the Leo1 function in oligodendrocyte development using the zebrafish dalmuri (dalknu6) mutant conferring a mutation in Leo1. The dal mutant embryos specified OPCs normally but after that, OPCs failed to differentiate into mature, myelinating oligodendrocytes and are eliminated by apoptotic cell death. Cell cycle arrest by the treatment of Cdk inhibitor, FVP, and ectopic expression of cdkn1c rescued most of the distinct mutant phenotype in dal mutant embryos. Our data clearly demonstrate that Leo1 function is required for the expression of high levels of Cdkn1c protein, which is responsible for the differentiation of OPCs into myelinating oligodendrocytes.
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CONTENTS
ABSTRACT 4
CONTENTS 7
LIST OF FIGURES 12
Chapter1. Wnt signaling in oligodendrocyte development 14
Ⅰ. Abstract 14
Ⅱ. Introduction 15
Ⅲ. Materials and methods 18
1.Fish Lines 18
2.in situ hybridization 18
3.Bromodeoxyuridine (BrdU) Labeling and Immunohistochemistry 18
4.Morpholino injections 19
5.Lithium Chloride (LiCl) and 6-Bromoindirubin-3’-Oxime(BIO) Treatment 20
Ⅳ. Results 21
1.fz8a is expressed in neural precursors in the ventral spinal cord of zebrafish embryos 21
2.Fz8a function is not required for dorsoventral patterning of the spinal cord 23
3.Fz8a function is required for oligodendrocyte specification 27
4.Fz8a function is required for the maintaining radial glial proliferation and spatial organization 29
5.Down-regulation of Wnt signaling is required for the survival and maturation of OPCs 32
Ⅴ. Discussion 34
1.Fz8a function is required for OPC specification in the ventral spinal cord 34
2.Down-regulation of Wnt signaling is required for the maturation of OPCs 38
Chapter2. Visualization of myelination in vivo in transgenic zebrafish 41
Ⅰ. Abstract 41
Ⅱ. Introduction 42
Ⅲ. Materials and methods 45
1.Plasmid construction 45
2.Generation of Tg(mbp:egfp) Zebrafish 45
3.in situ hybridization 45
4.Immunohistochemistry 46
Ⅳ. Results 47
1.The mbp promoter drives EGFP expression in zebrafish oligodendrocyte myelination 47
2.Tg(mbp:egfp) transgenic fish express EGFP specifically in the oligodendrocyte population 51
3.Continuous myelination occurs in the adult zebrafish 54
4.The axon bundles were surrounded by thick EGFP+ processes 55
Ⅴ. Discussion 58
1.Visualization of myelin processes in living zebrafish 58
2.Continuous axon myelination in the postembryonic spinal cord 59
Chapter3. Identification of Leo1 function in oligodendrocyte differentiation 62
Ⅰ. Abstract 62
Ⅱ. Introduction 63
Ⅲ. Materials and methods 67
1.Fish Lines 67
2.immunohistochemistry 67
3.in situ hybridization 67
4.Bromodeoxyuridine (BrdU) Labeling and Immunohistochemistry 68
5.Chemical treatment 68
6.Morpholino injections 68
7.RNA preparation and Quantitative RT-PCR analysis 69
Ⅳ. Results 70
1.Impaired oligodendrocyte differentiation in the zebrafish dal mutant 70
2.Impaired OPC differentiation is caused by failure of cell cycle exit in the neural precursors of dal mutant embryos 74
3.Cell cycle arrest by FVP and cdknlc rescue dal mutant phenotypes 79
Ⅴ. Discussion 85
1.Mutation in leo1 caused impaired oligodendrocyte development in the zebrafish CNS 85
2.Leo1-regulated Cdkn1c function is required for the oligodendrocyte specification and maturation 86
References 90
국문초록 108
