신경발생과정에서 Shh의 역활
Role of Sonic Hedgehog in Development of the CNS
- 주제(키워드) Sonic hedgehog , Neural induction , Differentiation , Nestin , N-myc , P19 cells , Pro-neural gene , Neurogenin1 , Mash1
- 발행기관 아주대학교
- 지도교수 서해영
- 발행년도 2011
- 학위수여년월 2011. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000011545
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Sonic hedgehog (Shh) functions as a morphogen to induce early dorso-ventral patterning of the central nervous system (CNS) and specifies cell fates of spinal motor neurons. Shh has also been known to promote cell cycle progression of embryonic stem cells as well as adult neural stem/progenitor cells. However, it has not been elucidated how Shh induces neural induction during early development of nervous system. To elucidate the role of Shh in neural induction, we used a pluripotent embryonal carcinoma cell line, P19 and investigated the effects of Shh on inducing the expression of nestin, the marker for neural stem/progenitor cells. In early stages, we found that Shh promoted the CNS-specific enhancer of the nestin gene through regulation of E-box on nestin 2nd intron 5’-region by Nmyc that is a target gene of Shh signaling. Shh also increased population and proliferation of neural stem cells in vitro or in vivo. In late stages, Shh induced specification of motor neurons as well as dopaminergic neurons. Simultaneously, Shh inhibited the expression of meso-endoderm lineage markers, Gsc, Sox17, and GATA4. The results suggest that Shh promotes neurogenesis in multiple ways: by inducing the neuroectodermal cells, by repressing non-neural cell fates, and by promoting differentiation of neurally committed progenitor cells into motor and dopaminergic neurons.
more초록/요약
Nestin is known as a specific marker for neural stem cells and regulated by the enhancer located on the nestin 2nd intron of nestin gene. Nestin 2nd intron is highly conserved among species and crucial for nestin expression in central nervous system (CNS) progenitor cells. The transgenic mice that constructed to express green fluorescence protein (GFP) with nestin enhancer have been developed and have great advantage to represent live neural stem cells during research in vivo and vitro. In this study, we characterized GFP expressing the neural stem cells or progenitors in vitro culture. We also investigated that Ngn1, which is a basic helix-loop-helix (bHLH) transcription factor that is expressed in neuronal precursors during development of the nervous system, can promote nestin expression by nestin 2nd intron enhancer. E-box 2 in 637bp of nestin 2nd intron were thought to be strong candidates for bHLH factors during the early neurogenesis. Ngn1 increased the E-box 2 mediated reporter gene activity. To evaluate the functional mechanism of E-box mediated nestin gene expression in vivo, we generated transgenic (Tg) mice with the 637bp fragment of nestin 2nd intron containing the wild or mutated E-box2. Wild type tg mice successfully exhibited CNS-specific LacZ expression. We also found that LacZ positive cells are proliferating neural progenitors or intermediate progenitors, which is expressed with Ki67 and Tbr2, and can differentiate into vGLUT2 positive glutamatergic neurons in developing cortex. These results indicate that pro-neural Ngn1 promotes nestin expression through nesitn 2nd intron E-box 2 and Ngns mediated LacZ positive cells can differentiate into glutamatergic neurons.
more목차
I. INTRODUCTION 1
PART I 1
A. Hh(hedgehog) 1
B. The role of Hh signaling in neural 2
C. Dorso-ventral patterning 4
D. Cell type specification 5
E. Cell proliferation in tumors 6
F. P19 embryonic carcinoma cells (EC) 7
G. Purpose 8
PART II 9
A. Neural stem cells 9
B. Nestin 9
C. basic helix-loop-helix (bHLH) transcription factor 10
II. MATERIALS AND METHODS 12
A. Materials 12
B. Mathods 13
1. Plasmids 13
2. P19 cell culture 13
3. Differentiation of P19 cells 14
3.1 Conventional differentiation 14
3.2 Current differentiation 14
4. Reverse Transcription Polymerase Chain Reaction (RT-PCR) 15
5. Western blotting 16
6. Luciferase assay 17
7. RNA interference 17
8. Immunocytochemistry 18
9. In utero injection and electroporation 18
10. Immunohistochemistry 19
11. Chromatin Immunoprecipitation (ChIP) assay 20
III. RESULTS 22
PART I 22
1. Shh induces neural lineage and blocks meso-endodermal line in P19 cells 22
2. Shh regulates CNS specific Nestin expression through Nestin 2nd intron in P19 cells 24
3. Shh directly promtes CNS specific Nestin expression in P19 cells 27
4. Shh promotes CNS specific Nestin expression through N-myc 29
5. Shh induces population of neural stem cells in vivo 34
6. Shh directly induces neural differentiation of P19 cells 38
7. Shh promotes differentiation of P19 cells into motor and dopaminergic neurons 40
8. Shh regulates the expression of Id and Hes1 during neural differentiation of P19 cells 42
9. Id expression correlates with stemness of NSCs 44
10. Id proteins increase the proliferating neural precursors in the developing brain 46
PART. II 51
11. Ngn1 regulates CNS specific nestin expression though the NE2 of Nestin 2nd intron3-region 51
12. Ngn1 promotes neural progenitor cells into glutamatergic neuron in vivo 57
IV. DISCUSSION and CONCLUSIONS 66
REFERENCES 72
국문요약 83
