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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王致恬(Chih-Tien Wang) | |
| dc.contributor.author | Yu-Chieh Chen | en |
| dc.contributor.author | 陳鈺杰 | zh_TW |
| dc.date.accessioned | 2021-06-17T00:41:55Z | - |
| dc.date.available | 2017-02-16 | |
| dc.date.copyright | 2012-02-16 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-01-16 | |
| dc.identifier.citation | Alexiades MR, Cepko C. 1996. Quantitative analysis of proliferation and cell cycle length during development of the rat retina. Dev Dyn 205:293-307
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Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med 4:594-600 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66546 | - |
| dc.description.abstract | Synaptotagmin (Syt) 為一種鈣離子的感應蛋白家族，至今具有十七種 isoforms。Syt 可透過受鈣離子調節的胞吐作用來影響神經傳導物質的釋放。先前研究指出，早期大鼠視網膜發育過程中的自發性放電現象 (視網膜波) 對於視神經節細胞的軸突投射至腦區並進行視野分離的過程扮演重要的角色。然而其調控的機制仍不清楚。由於視網膜波是利用突觸傳遞所引發，因此我們認為 Syt 很可能參與調控視網膜波或視野分離的過程。由於目前對於 Syt isoforms 的時空表現情形仍不是很清楚，因此在本論文中，我們針對在大鼠視網膜發育早期，研究不同種 Syt isoforms 的表現情形。首先，我們利用單細胞反轉錄聚合酶連鎖反應來研究 Syt I-IV mRNA 在視網膜神經節細胞 (視網膜中唯一會投射至腦區的神經元) 內的表現情形。實驗結果發現，Syt III mRNA只在出生後第四天到第八天的特定時期表現，而此時間點恰好是視神經節細胞的軸突投射至腦區並進行視野分離的關鍵時期；Syt I 以及 Syt IV mRNA 則在出生後第二天到第九天都能夠偵測到；Syt II mRNA則不能在出生後第二天到第九天的視神經節細胞內偵測到，推測其可能未參與視神經節細胞的發育過程。此外，我們針對整個視網膜進行反轉錄聚合酶連鎖反應來研究 Syt I 以及 Syt IV mRNA 的表現情形。結果發現，在大鼠出生後到第十一天這段期間，此兩種 Syt isoforms mRNA 表現量都上升，暗示著Syt III 在視神經節細胞以及視網膜內其他種類的細胞中分別具有不同的表現情形。此外，我們藉由定序 Syt III cDNA 序列發現 Syt III 蛋白質序列在 287 以及 448 這兩個位置分別是絲氨酸 (serine) 和精氨酸 (arginine)；這個結果不同於先前研究的Syt III 蛋白質序列：此兩個位置分別是蘇氨酸 (threonine) 和賴氨酸 (lysine)。接著，我們利用免疫螢光染色的方式研究 Syt I 和 Syt III 在發育中視網膜表現的位置。研究結果顯示，Syt I 主要表現於突觸的位置，亦即視網膜的內叢狀層以及外叢狀層。此結果符合先前研究指出 Syt I 在突觸傳導中的角色。Syt III 則主要表現在視神經節細胞層、內叢狀層以及神經母細胞層。此結果暗示著 Syt III 可能對於神經細胞的分化以及遷移也扮演重要的角色。此外，為了更進一步了解 Syt III 在胞內的位置，我們將發育中的視網膜細胞分離後進行免疫螢光染色。研究結果發現 Syt III 大量表現在Brn3+ 和 Thy1.1+ 的視神經節細胞內，並且位於大小囊泡、早期核內體、溶小體以及軸突生長點。最後，利用即時定量反轉錄聚合酶連鎖反應來研究不同 Syt isoforms 的基因表現是否會受到視網膜發育中自發性放電現象 (視網膜波) 所影響。結果發現只有 Syt IV 的基因表現會受到視網膜波的活動所調控；而Syt III 的基因表現則不會。藉由以上的實驗結果，我們得知在視網膜發育過程中，Syt III 相對於 Syt I 而言，在視神經節細胞具有一個特別的時空表現模式。此結果將提供我們一些線索以及基礎來研究 Syt III 對於視網膜發育所扮演的角色。 | zh_TW |
| dc.description.abstract | Synaptotagmin (Syt) constitutes a large protein family with at least seventeen isoforms, many of which serve as calcium sensors and regulate neurotransmitter releases through calcium-regulated exocytosis. In early developing rat retina, the patterned spontaneous activities (retinal waves) play an important role in regulating eye-specific segregation of retinal inputs in the dorsal lateral geniculate nucleus (dLGN). However, the underlying mechanism is still unclear. Since the retinal waves are driven by synaptic transmission, we then think Syt be involved in regulating retinal waves and/or eye-specific segregation of retinal ganglion cell (RGC) axons. In this study, we investigated the expression profiles of Syt isoforms in the developing rat retina since the lack of information about the spatiotemporal expression patterns of Syt isoforms. First, we applied the single-cell RT-qPCR (sc-RT-qPCR) to screen the expression of Syt I-IV transcripts in developing retinal ganglion cells (RGCs), the retinal output neurons projecting to central brain targets. We found that Syt III mRNA can be detected in the RGCs from the postnatal day 4 to 8 (P4-P8), which is the critical period for eye-specific segregation of the retinotopic map. In contrast, Syt I and IV mRNAs were consistently expressed in the RGCs from P2 to P9. Syt II mRNA was not detectable in the RGCs from P2 to P9, which may not be involved in RGCs development. Furthermore, by qPCR from the whole retinal lysates, we detected an increasing mRNA expression level for both Syt I and Syt III from P0 to P11. The different expression profiles of Syt III mRNA between RGCs and whole retinas suggest different roles of Syt III in output neurons (RGCs) and other types of cells (e.g. interneurons) in the developing retina. Moreover, from the P0-P11 rat retinal cDNAs, we identified that Syt III harbored S287 and R448, distinct from the previous reports (T287 and L448). Second, by immunofluorescence stainings, we found that Syt I was mainly expressed in the synaptic layers [both inner plexiform layer (IPL) and outer plexiform layer (OPL)], consistent with its role in synaptic transmission. However, Syt III showed a dot-like expression pattern in the ganglion cell layer (GCL), IPL, and neuroblast layer (NBL), implying that Syt III might be also involved in cell differentiation and maturation. Third, Syt III was largely expressed in the Brn3+ and Thy1.1+ RGCs and localized to two vesicles [small vesicles (SVs) and large dense-core vesicles (LDCVs)], early endosome/lysosome compartments and the growth cone. Finally, unlike Syt IV, we found the expression of Syt III was retinal wave activity-independent. To sum up, the unique spatiotemporal expression pattern of Syt III may provide us with some clues to investigate its possible role in the developing rat retina. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T00:41:55Z (GMT). No. of bitstreams: 1 ntu-101-R99b43008-1.pdf: 7576624 bytes, checksum: df18996070c7e5dc4cd34203f248c63f (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
Acknowledgement ii 中文摘要 iii Abstract v Abbreviations vii Contents x Chapter I Introduction 1 1.1 Neural development 1 1.2 Organization of visual system 1 1.2.1 The laminar structure of the retina 1 1.2.2 Visual transduction and visual pathway 2 1.3 Retinal development 3 1.4 Spontaneous synaptic neural activities in the early developing retina 3 1.4.1 Synaptic transmission 3 1.4.2 Retinal waves 4 1.5 Eye-specific segregation 4 1.6 Molecular mechanism of calcium-regulated exocytosis 5 1.7 Synaptotagmin (Syt) 6 1.8 Synaptotagmin III (Syt III) 8 1.8.1 Sequence and structure of Syt III 8 1.8.2 Locations of Syt III 9 1.8.3 Functions of Syt III 9 1.9 Objectives of the study 10 Chapter II Materials and Methods 12 2.1 Animals 12 2.2 Plasmid construction and subcloning 12 2.3 Retinal dissection 15 2.4 Sc-RT-PCR 15 2.5 Total RNA extraction of whole retina 17 2.6 RT-qPCR 18 2.7 Tissue fixation and cryosection 19 2.8 Antibodies 20 2.9 Immunohistochemistry 21 2.10 Immunocytochemistry 23 2.11 Preparation of cell lysates and immunoblotting (this work was done by Yu-Tze Chang in Dr. Juu-Chin Lu’s lab) 24 2.12 Dissociated retinal cell cultures 26 2.13 Retinal explant culture 27 2.14 Transient transfection of retinal explants 28 2.15 Statistics 29 Chapter III Results 30 3.1 Reference gene selection for qPCR analysis 30 3.2 The mRNA expression levels of different Syt isoforms were screened in the developing RGCs 30 3.3 The unique expression pattern of Syt III mRNA existed in the developing rat retina 31 3.4 The rat Syt III cDNA sequence in the developing retina contained S287 and R448 rather than T287 and L448 32 3.5 The specificity of homemade rabbit polyclonal anti-Syt III antibody was tested by immunoblotting and immunofluorescence staining in both cell lines and retinal tissues 32 3.6 Syt I and Syt III were expressed in different layers in the developing rat retina 35 3.7 Syt III was expressed mainly in the Brn3+ and Thy1.1+ RGCs in the retinal cross-sections and the P6 dissociated rat retinal cells 37 3.8 HA-Syt III was partially colocalized with Syp in PC12 cells 37 3.9 Endogenous Syt III was colocalized with two types of vesicle and early endosome/lysosome compartments in the P6 dissociated rat retinal cells 39 3.10 Syt III was expressed in the growth cone of RGCs in the P6 dissociated retinal cells 40 3.11 The expression of Syt III was unlikely affected by retinal waves 40 Chapter IV Discussion 42 4.1 The expression of Syt isoforms in the retina 42 4.2 The expression of Syt isoforms in the developing RGCs is examined by sc-RT-qPCR 43 4.3 The comparison between sc-RT-qPCR and immunofluorescence staining results 44 4.4 Controversial expression, sequence, and subcellualr localization of Syt III 45 4.5 A possible role of Syt III in regulating eye-specific segregation during early retinal development 47 4.6 The relationship between Syt isofroms and retinal waves during early retinal development 49 Chapter V Conclusion 51 References 52 List of Figures Figure 1 Structure of the eyeball and the retina 61 Figure 2 Central projection targets of retinal ganglion cells (RGCs) 63 Figure 3 Illustration and mechanisms of retinal waves 64 Figure 4 C2 domains of Synaptotagmin (Syt) are crucial for calcium-regulated exocytosis 66 Figure 5 The process of harvesting a single RGC 67 Figure 6 Sc-RT-qPCR amplification plots of Syt I-IV mRNAs in the RGCs from P2 to P9 68 Figure 7 Syt I and Syt III mRNA expression profiles of a single RGC during early developing rat retina 69 Figure 8 Expression patterns of Syt III and Syt I mRNAs during early developing rat retina 70 Figure 9 Identification two sequence conflicts (S287 and R445) of Syt III cDNA in rat retina 71 Figure 10 The specificity of affinity purified anti-Syt III antibody was tested in PC12 cells and rat retinal neurons by immunofluorescence staining 73 Figure 11 The affinity purified anti-Syt III antibody could specifically recognize Syt III in PC12 cells overexpressing Syt III 75 Figure 12 Syt I was expressed in the inner plexiform layer (IPL) and outer plexiform layer (OPL) in the developing rat retina 76 Figure 13 Syt III was mainly expressed in the GCL, IPL, and NBL in the developing rat retina 78 Figure 14 Syt IV was expressed in the GCL, IPL, and OPL in the developing rat retina 80 Figure 15 Syt I and Syt III were localized to different layers in the developing rat retina 82 Figure 16 Syt III was expressed in the Brn3+ RGCs 84 Figure 17 Syt III was strongly expressed in the Brn3+ and Thy1.1+ RGCs of the P6 dissociated retinal cells 85 Figure 18 HA-Syt III fusion protein may partially target to SVs but not LDCVs in PC12 cells 87 Figure 19 Native form of Syt III may target to SVs in PC12 cells 88 Figure 20 Endogenous Syt III was localized to both types of vesicle (LDCVs and SVs) and early endosome/lysosome compartments in the P6 dissociated retinal neurons 91 Figure 21 Syt III was expressed in the growth cone of P6 RGCs 92 Figure 22 Syt III showed the retinal wave-independent gene expression 93 List of Tables Table 1 List of RT-qPCR primers used in this study 94 Table 2 List of primary antibodies used in this study 95 Table 3 List of secondary antibodies used in this study 96 Appendix List of Appendix Figures Appendix Figure 1 Reference gene selection for qPCR analysis 98 Appendix Figure 2 Specificity of anti-Syt III antiserum was tested in HEK 293T cells by immunoblotting 99 Appendix Figure 3 The specificity of affinity purified anti-Syt III antibody was tested in PC12 cells by immunoblotting 100 Appendix Figure 4 Syt III mRNA was expressed in wild-type PC12 cells 101 List of Appendix Tables Appendix Table 1 The eight candidates of reference genes for qPCR analysis 102 Appendix Figure 2 The expression profile of eight candidate reference genes in the RGCs from P2 to P9 by sc-RT-qPCR 103 The 2011 annual meeting of the Society for Neuroscience (Washington, D.C., U.S.A. 11/12-16/2011): abstract and poster 104 | |
| dc.language.iso | en | |
| dc.subject | 連鎖反應 | zh_TW |
| dc.subject | synaptotamin | zh_TW |
| dc.subject | 視網膜發育 | zh_TW |
| dc.subject | 視神經節細胞 | zh_TW |
| dc.subject | 視網膜波 | zh_TW |
| dc.subject | 視野分離 | zh_TW |
| dc.subject | 單細胞定量反轉錄聚合酶 | zh_TW |
| dc.subject | retinal ganglion cells | en |
| dc.subject | retinal development | en |
| dc.subject | synaptotagmin | en |
| dc.subject | single-cell RT-qPCR | en |
| dc.subject | eye-specific segregation | en |
| dc.subject | retinal waves | en |
| dc.title | Synaptotagmin isoforms 在發育中大鼠視網膜的時空表現模式 | zh_TW |
| dc.title | The spatiotemporal expression patterns of Synaptotagmin isoforms in the developing rat retina | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 焦傳金(Chuan-Chin Chiao),徐立中(Li-Chung Hsu),周申如(Shen-Ju Chou) | |
| dc.subject.keyword | synaptotamin,視網膜發育,視神經節細胞,視網膜波,視野分離,單細胞定量反轉錄聚合酶,連鎖反應, | zh_TW |
| dc.subject.keyword | synaptotagmin,retinal development,retinal ganglion cells,retinal waves,eye-specific segregation,single-cell RT-qPCR, | en |
| dc.relation.page | 103 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-01-16 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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