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Graduate School of Ajou University Department of Neuroscience and Technology Course 3. Theses(Master)

신경세포 분화과정에서 Disabled-1과 Sonic Hedgehog의 역할

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dc.contributor.advisorYoung-Don Lee-
dc.contributor.author권일선-
dc.date.accessioned2019-10-21T06:47:58Z-
dc.date.available2019-10-21T06:47:58Z-
dc.date.issued2006-02-
dc.identifier.other1203-
dc.identifier.urihttps://dspace.ajou.ac.kr/handle/2018.oak/16839-
dc.description학위논문(박사)--아주대학교 일반대학원 :신경과학기술과정,2006. 2-
dc.description.tableofcontentsTABLE OF CONTENTS ● PART I ABSTRACT ------------------------------------------ i TABLE OF CONTENTS ------------------------------- iii LIST OF FIGURES------------------------------------- v LIST OF TABLES ------------------------------------- vii I. INTRODUCTION ------------------------------------ 1 II. MATERIALS AND METHODS ------------------------ 9 A.MATERIALS -------------------------------------- 9 B.METHOSDS -------------------------------------- 10 1. Animal and genotyping -------------------------- 10 2. Culture and differentiation of neurospheres -------- 11 3. Reverse transcriptase polymerase chain reaction (RT-PCR) --------------------------------------- 13 4. Western blot ------------------------------------- 13 5. Immunostaining ---------------------------------- 14 6. 5-bromo-2’-deoxyuridine (BrdU) – incorporation assay ------------------------------------------------- 16 7. Statistics ---------------------------------------- 16 III. RESULTS ------------------------------------------ 18 IV. DISCUSSION -------------------------------------- 51 V. CONCLUSION -------------------------------------- 60 REFERECES ------------------------------------------ 61 ● PART II I. INTRODUCTION ------------------------------------- 70 II. MATERIALS AND METHODS ------------------------- 73 A. MATERIALS -------------------------------------- 73 B. METHODS --------------------------------------- 74 1. Cloning of the human ShhN --------------------- 74 2. Tyrosine hydroxylase (TH) promoter assay ------ 75 3. Reverse transcriptase-polymerase chain reaction (RT-PCR) -------------------------------------- 75 4. Immunocytochemistry -------------------------- 76 III. RESULTS ----------------------------------------- 77 IV. DISCUSSION -------------------------------------- 85 V. CONCLUSION ------------------------------------- 89 REFERENCES ---------------------------------------- 90 국문요약 --------------------------------------------- 94| LIST OF FIGURES ● PART I Fig. 1. A potentially novel Reelin-Dab1 signal transduction ----------------------------------------------- 3 Fig. 2. Glial differentiation through Jak-STAT pathway ----------------------------------------------- 7 Fig. 3. Method for neurosphere culture ----------------- 12 Fig. 4. Verification of mouse embryo genotype by PCR -- 18 Fig. 5. Formation of Neurosphere and low nestin expression by Dab1 deficiency ----------------------------- 21 Fig. 6. Increase in differentiation into GFAP-positive cells from Dab1-/- neurospehres --------------------- 24 Fig. 7. Reduced expression of neuronal markers in Dab-/- cells during differentiation ----------------------- 28 Fig. 8. Increase of vimentin- or GFAP-positive cells in brain of Dab1-/- mouse, yotari ----------------------- 31 Fig. 9. Decreased number of neurons in yotari mice ----- 35 Fig. 10. Reduced growth of MAP2-positive dendrites in cortical plate of yotari cerebellum --------------- 38 Fig. 11. Enhanced expression of phosphorylated STAT3 (pSTAT3) in Dab1-/- cells during differentiation -- 40 Fig. 12. Increasing of PDGFRα expression in both Dab1-/- cells differentiated and mouse brain lysate ------ 42 Fig. 13. Reduction of proliferating cells in Dab1-/- cells and cortical plate of yotari mouse brain --------- 44 Fig. 14. Impairment of glial fibers in Dab1-/- cells and in cerebellum and hippocampus of postnatal yotari mice ----------------------------------------- 49 Fig. 15. Schematic presentation of crosstalk of Reelin-Dab1 and Jak-STAT pathway in the period of cell fate determination -------------------------------- 55 ● PART II Fig. 1. ShhN increases TH expression in uncommitted cells, P19 -------------------------------------- 81 Fig. 2. ShhN represses TH expression in TH-expressing neuroblastoma cells, PC12 ---------------------- 82 Fig. 3. ShhN interferes with the PKA/Cα –CREB pathway in neural stem cells ------------------------------- 83| LIST OF TABLES Table 1. PCR primer sequences and condition for RT-PCR --------------------------------------------- 13 Table 2. Antibodies used in this study ----------------- 15-
dc.language.isoeng-
dc.publisherThe Graduate School, Ajou University-
dc.rights아주대학교 논문은 저작권에 의해 보호받습니다.-
dc.title신경세포 분화과정에서 Disabled-1과 Sonic Hedgehog의 역할-
dc.title.alternativeIl-Sun Kwon-
dc.typeThesis-
dc.contributor.affiliation아주대학교 일반대학원-
dc.contributor.alternativeNameIl-Sun Kwon-
dc.contributor.department일반대학원 신경과학기술과정-
dc.date.awarded2006. 2-
dc.description.degreeMaster-
dc.identifier.localId565214-
dc.identifier.urlhttp://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000001203-
dc.description.alternativeAbstractPART I: Disabled-1 (Dab1) has been known to be involved in the transduction of Reelin-initiated signaling pathway that controls the migration and positioning of mature neurons during corticogenesis. Here I examined the function of Dab1 in differentiation of neural stem cells that were isolated from embryonic forebrain of Dab1-/- mouse, yotari. Upon differentiation, the cells from yotari mice exhibited significant expression of GFAP, an astrocyte marker, at the expense of lower expression of neuronal marker proteins. GFAP- and vimentin-immunoreactive cells were increased in developing cerebral cortical plate of yotari mouse embryos. In contrast, yotari mice showed the decreased number of neurons and altered formation of dendritic length and complexity. To elucidate the mechanism for enhanced astroglial differentiation, the activation of STAT3 was examined. The phosphorylated STAT3, an active form, was elevated in Dab1-/- cells after differentiation. Expressions of GFAP induced by Dab1 deficiency were diminished by AG490, specific inhibitor of Jak-STAT signaling pathway. In addition, the expression of NeuroD transcript, as transcription factor for neuronal cell determination, was lower in Dab1-/- cells than wild-type under differentiation. Formation of radial glia fibers was impaired in the cerebellum and hippocampus of yotari mice. In conclusion, I unravel novel roles of Dab1 necessary for neuronal differentiation and glial fiber formation and arrangement. PART II: In addition to vertebrate development, sonic hedgehog (Shh) functions as a ventralizing signaling molecule to induce the floor plate in early stages and motor neurons in spinal cord and dopaminergic neurons in midbrain in later stages of development. The 20 kilodalton fragment of the N-terminus of Shh (ShhN) is known to mediate the biological functions of Shh. In the present study, I examined the role of ShhN in regulating the tyrosine hydroxylase (TH) gene, a hallmark of dopaminergic neurons, in various cell lines which corresponded to various stages of neural development. Overexpression of ShhN in uncommitted mouse embryonal carcinoma P19 cells increased the TH gene expression, when these cells were induced to differentiate into neuronal cells in vitro. In contrast, ShhN repressed the expression of the endogenous TH gene in PC12 cells. Promoter analysis in neural stem cells is revealed that ShhN repressed the TH gene expression by antagonizing cAMP-dependent protein kinase A (PKA) signaling. The results suggest that ShhN may induce or facilitate differentiation of dopaminergic neurons in early stages but antagonize the PKA signaling to reduce the CREB-mediated TH gene expression in late born neurons-
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