在中樞神經系統的發育過程中，神經幹細胞會先經歷神經發生 (neuro¬genesis)產生神經元，然後再通過神經膠質細胞發生 (gliogenesis)製造神經膠質細胞。這個時間順序是所有神經幹細胞分化過程中的固定模式。 Sox9是屬於一類具有 HMG (high mobility group)特徵性結構域的轉錄因子家族。以往轉基因小鼠研究證明， Sox9在脊髓和視網膜神經建構過程中，是引發神經膠質細胞新生程式的決定性主控基因。但是在小腦發育過程中，製造神經膠質細胞的調節機制仍未被界定。 / 在小鼠小腦發育過程中，室區 (ventricular zone)的神經祖細胞豐富表達 Sox9基因。因此，本實驗試圖利用條件基因剔除技術，研究 Sox9基因在小腦形成過程中的功能。結果顯示， Sox9基因在小腦被剔除後會導致包括蒲金耶氏細胞 (Purkinje cells)及 γ-氨基丁酸能中間神經元 (GABAergic interneurons)等室區衍生神經元大幅增加。與此同時，一些神經膠質細胞標記的表達亦受到影響。值得留意的是這些缺陷表型在胚胎發育後期才發生，與神經膠質細胞發生開始的時間框架一致。由於神經元和神經膠質細胞都是於共同的神經祖細胞池分化而成， Sox9基因的失活顯然影響了祖細胞池由製造神經元切換到神經膠質細胞生成的過程。進一步的微陣列基因晶片及半定量 RT-PCR分析顯示，數個參與細胞增殖、分化及細胞命運決定的基因表達量在 Sox9轉基因小鼠小腦中起了明顯的變化，而這些基因很可能與 Sox9共同調控神經膠質細胞發生的始初過程。 / 另一方面，我利用條件性 Sox9高效表達的小鼠作為動物模型及分析其表徵,希望更全面地了解 Sox9在小腦發育過程中的角色。於胚胎發育期間， Sox9基因高效表達並沒有擾亂小腦的發育；但由產後第 15周起，在小腦中持續性的 Sox9基因異位表達卻導致小鼠出現明顯的運動協調及身體平衡能力缺失。從 24 周 Sox9高效表達小鼠小腦組織分析顯示，其小腦中的貝格曼神經膠質細胞 (Bergmann glia)和蒲金耶氏細胞均出現缺陷表型，而這兩類細胞的異常變化很可能是導致條件性 Sox9高效表達小鼠運動協調缺失的主因。 / 在探究 Sox9如何調節小腦發育的同時，我發現負責分泌腦脊液及形成血腦屏障的脈絡叢 (choroid plexus)亦發生異常變化。初步分析顯示， Sox9的失活導致脈絡叢上皮細胞的凋亡率上升，而這亦解釋了為何顱內出血的情況在 Sox9基因剔除小鼠中較常見。 / 總括而言，這項研究的結果顯示 Sox9在小鼠小腦發育過程中扮演決定神經祖細胞命運的角色，在中樞神經系統發育中起著守恒的作用。而 Sox9基因的高效表達則會造成成年小鼠的運動功能障礙。此外，Sox9亦可能通過調控脈絡叢的發育和功能，以維持血腦屏障的完整性。我們需要更深入及全面的研究以了解 Sox9在小鼠小腦和脈絡叢發育中的作用及其分子機制。 / In the developing central nervous system (CNS), neural stem cells undergo a stereotypic pattern of temporal differentiation characterized by an initial wave of neurogenesis which then ceases to give way for a subsequent period of gliogenesis. Sox9 belongs to the highly conserved family of high mobility group (HMG) transcription factors, and has been shown to be the master regulator mediating the switch to the gliogenic program in several neuronal tissues including the spinal cord and the retina. While in the cerebellum, genetic control of such a developmental interval has remained poorly defined. / In the developing cerebellum, Sox9 is expressed abundantly in neural progenitors of the ventricular zone (VZ). Here, I analyzed cerebellar development of mice in which Sox9 is specifically inactivated in the cerebellum by the Cre/loxp recombination system. These mice exhibited an increased number of neuronal phenotypes, including the Purkinje cells (PCs) and GABAergic interneurons, while the expressions of several glial markers are compromised. These phenotypes occur only at late embryonic stage, a time frame which is consistent with the initiation of gliogenesis. Because neurons and glia share a common origin, the ablation of Sox9 apparently causes the progenitor pool to continue to produce neurons instead of switching to generate glial cells. Subsequent microarray and semi-quantitative RT-PCR analyses identified expression level changes in genes that have been previously implied in regulating cell fate decision and cell proliferation during development, which may possibly function in collaboration with Sox9 during the initiation of gliogenesis. / On the other hand, to comprehensively interrogate the role of Sox9 in cerebellar development, a conditional Sox9 overexpression mutant was characterized. While the ectopic expression of Sox9 did not perturb cerebellar development during embryogenesis, the continued aberrant expression of Sox9 in the cerebellum led to noticeable locomotor deficits in adult mice from 15 weeks onwards. Histological examinations at 24 weeks revealed abnormalities in both the Bergmann glia and PCs, which possibly accounted for the motor defects observed in the mutant mice. / In the course of studying the role of Sox9 in cerebellar development, noticeable abnormalities were also observed in the choroid plexus (ChP), a neurovascular tissue responsible in setting up the blood-brain barrier. Initial analysis showed that the ablation of Sox9 induced apoptosis in the ChP epithelium, which possibly explained the higher frequency of intracranial hemorrhage observed in the mutant. / In summary, the findings from this study suggest that Sox9 plays a conserved role in the developing CNS as a key molecular component in determining the neuron-glial fate choice during cerebellar development, while the ectopic expression of Sox9 could induce locomotor dysfunction in adult mice. In addition, Sox9 may also contribute to the maintenance of vascular integrity by regulating ChP development and functionality. More comprehensive investigation is required to understand the molecular mechanisms of Sox9 action during mouse cerebellar and ChP development. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Leung, Kit Ying Crystal. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 166-184). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Chapter Declaration --- p.i / Chapter Abstract --- p.iii / Chapter Abstract in Chinese --- p.v / Chapter Acknowledgements --- p.vii / Chapter Table of Contents --- p.ix / Chapter List of Figures --- p.xiii / Chapter List of Tables --- p.xv / Chapter List of Abbreviations --- p.xvi / Chapter CHAPTER 1 --- General Introduction / Chapter 1.1 --- Preface: The developing central nervous system - Why it matters --- p.1 / Chapter 1.2 --- Development of the Mammalian Central Nervous System: An Overview --- p.3 / Chapter 1.2.1 --- Neural induction, neurulation and the formation of the neural tube --- p.3 / Chapter 1.2.2 --- Regionalization of the rostral neural tube and formation of brain vesicles --- p.4 / Chapter 1.3 --- The Cerebellum --- p.7 / Chapter 1.3.1 --- Functions of the cerebellum --- p.7 / Chapter 1.3.2 --- Disorders of the cerebellum --- p.8 / Chapter 1.3.3 --- Gross anatomy and organization of the cerebellum --- p.11 / Chapter 1.3.4 --- Cellular constituents of the cerebellum - diversity and biochemistry --- p.15 / Chapter 1.3.5 --- Neuronal circuitry of the mature cerebellum --- p.16 / Chapter 1.4 --- Development of the Cerebellum --- p.20 / Chapter 1.4.1 --- Overview of mouse early cerebellar development --- p.20 / Chapter 1.4.2 --- Germinal matrices of the cerebellar primordium --- p.22 / Chapter 1.4.3 --- Timeline of the birth of cerebellar neurons and glial cells --- p.25 / Chapter 1.4.4 --- Postnatal development of the cerebellum --- p.27 / Chapter 1.4.5 --- Genetic regulation of cerebellar development --- p.30 / Chapter 1.5 --- SOX9 and the SOX Family of Transcription Factors --- p.33 / Chapter 1.5.1 --- SOX9 as a transcription factor --- p.33 / Chapter 1.5.2 --- Molecular regulation of SOX9 action --- p.36 / Chapter 1.5.3 --- SOX9 in development and disease --- p.38 / Chapter 1.6 --- Scope of the Thesis --- p.45 / Chapter CHAPTER 2 --- Characterization of a Mouse Model with Sox9 Conditional Knockout / Chapter 2.1 --- Chapter Summary --- p.47 / Chapter 2.2 --- Introduction --- p.49 / Chapter 2.3 --- Materials and Methods --- p.54 / Chapter 2.3.1 --- Animal husbandry --- p.54 / Chapter 2.3.2 --- Breeding strategy for the generation of Sox9 conditional knockout mutants --- p.54 / Chapter 2.3.3 --- DNA extraction and genotyping --- p.55 / Chapter 2.3.4 --- Histological examination of the cerebellum --- p.57 / Chapter 2.3.5 --- β-Galactosidase staining of embryos --- p.59 / Chapter 2.3.6 --- Microarray analysis --- p.59 / Chapter 2.3.7 --- Validation of microarray data by semi-quantitative RT-PCR --- p.60 / Chapter 2.3.8 --- In situ hybridization --- p.61 / Chapter 2.3.9 --- Image acquisition and photo editing --- p.65 / Chapter 2.3.10 --- Statistical analysis --- p.66 / Chapter 2.4 --- Results -- Part I: En1[superscript Cre]- driven Sox9 Conditional Knockout --- p.67 / Chapter 2.4.1 --- Expression of Sox9 during mouse embryonic development --- p.67 / Chapter 2.4.2 --- Effective ablation of Sox9 in the cerebellum of En1[superscript Cre/]⁺; Sox9[superscript fx/fx] mutant --- p.68 / Chapter 2.4.3 --- Deficiency of Sox9 did not cause cerebellar developmental abnormalities in the Sox9 CKO mutants --- p.71 / Chapter 2.5 --- Results -- Part II: Pax2[superscript Cre]-driven Sox9 Conditional Knockout --- p.76 / Chapter 2.5.1 --- Effective ablation of Sox9 in the cerebellum of Pax2[superscript Cre/]⁺; Sox9[superscript fx/fx] mutant --- p.76 / Chapter 2.5.2 --- Sox9 deletion resulted in cerebellar malformation at late embryonic stage --- p.78 / Chapter 2.5.3 --- Loss of Sox9 caused an increased neuronal production from the ventricular zone of the Pax2[superscript Cre/]⁺; Sox9[superscript fx/fx] mutant --- p.80 / Chapter 2.5.4 --- Sox9 deletion did not alter rhombic lip-derived neurons --- p.84 / Chapter 2.5.5 --- Expression of glial markers were compromised in the Sox9 CKO mutant at late embryonic stages --- p.84 / Chapter 2.5.6 --- Comparison of cerebellar gene expression profiles between the Sox9 CKO mutant and control --- p.90 / Chapter 2.5.7 --- Expression analysis of the proto-oncogene transcription factor Prdm16 in the mouse brain --- p.93 / Chapter 2.6 --- Results -- Part III: Sox9 and the Development of the Choroid Plexus --- p.95 / Chapter 2.6.1 --- Partial loss of Sox9 in the Pax2[superscript Cre]; Sox9[superscript fx/fx] CKO mutant induced choroid plexus abnormalities and increased susceptibility to intracranial hemorrhage --- p.95 / Chapter 2.6.2 --- The mutant choroid plexus was non-cancerous --- p.98 / Chapter 2.6.3 --- Increased apoptosis in the Sox9 CKO mutant choroid plexus --- p.100 / Chapter 2.7 --- Discussion --- p.102 / Chapter 2.7.1 --- Sox9 plays an essential role in determining the neuron-glial fate choice in the developing cerebellum --- p.102 / Chapter 2.7.2 --- Potential influence of genetic background on Sox9 CKO mutant phenotypes --- p.104 / Chapter 2.7.3 --- Prdm16 as a potential candidate in a Sox9-dependent transcriptional regulatory cascade during the initiation of gliogenesis --- p.105 / Chapter 2.7.4 --- Sox9 may be important in choroid plexus development --- p.107 / Chapter 2.7.5 --- Chapter conclusion --- p.108 / Chapter CHAPTER 3 --- Characterization of a Mouse Model with Sox9 Conditional Overexpression / Chapter 3.1 --- Chapter Summary --- p.116 / Chapter 3.2 --- Introduction --- p.118 / Chapter 3.3 --- Materials and Methods --- p.120 / Chapter 3.3.1 --- Animal husbandry --- p.120 / Chapter 3.3.2 --- Breeding strategy for the generation of Sox9 overexpression mutants --- p.120 / Chapter 3.3.3 --- Genotyping --- p.120 / Chapter 3.3.4 --- Histological examination of the cerebellum --- p.121 / Chapter 3.3.5 --- Behavioral tests --- p.122 / Chapter 3.3.6 --- Image and video acquisition --- p.123 / Chapter 3.3.7 --- Video processing --- p.124 / Chapter 3.3.8 --- Statistical analysis --- p.124 / Chapter 3.4 --- Results --- p.125 / Chapter 3.4.1 --- Sox9 was overexpressed in only a subset of cells in the mutant cerebellum --- p.125 / Chapter 3.4.2 --- Overexpression of Sox9 did not cause developmental abnormalities in the cerebellum of En1[superscript Cre/]⁺; Z/Sox9 mutant embryos --- p.127 / Chapter 3.4.3 --- En1[superscript Cre/]⁺; Z/Sox9 mutants manifested locomotion deficits during adulthood --- p.132 / Chapter 3.4.4 --- Abnormal Purkinje cell dendritic arborization and Bergmann glial scaffold in adult En1[superscript Cre/]⁺; Z/Sox9 mutants --- p.138 / Chapter 3.5 --- Discussion and Chapter Conclusion --- p.143 / Chapter CHAPTER 4 --- General Discussion, Future Works and Conclusions / Chapter 4.1 --- An Evolutionary Conserved Role of Sox9 in Determining the Neuron-glial Fate Choice during Vertebrate CNS Development --- p.147 / Chapter 4.2 --- Prdm16 may be important in the transcriptional cascade during the initiation of gliogenesis in mouse cerebellar development --- p.148 / Chapter 4.3 --- A Potential Neuroprotective Role of Sox9 in the Adult Cerebellum --- p.149 / Chapter 4.4 --- Future Works --- p.150 / Chapter 4.4.1 --- Dissecting the dual roles for Sox9 in neural stem cell maintenance and gliogenesis --- p.150 / Chapter 4.4.2 --- The contribution of glutamate toxicity to the cerebellar phenotypes observed in the Sox9 CKO mutant --- p.152 / Chapter 4.4.3 --- The involvement of Prdm16 and Notch signaling in cerebellar development --- p.153 / Chapter 4.4.4 --- The molecular mechanism of Sox9-dependent neurodegenerative phenotypes in the conditional overexpression mutant --- p.153 / Chapter 4.4.5 --- The importance of Sox9 in choroid plexus development --- p.154 / Chapter 4.4.6 --- Improving the specificity of Cre deleter mouse lines --- p.155 / Chapter 4.5 --- Conclusions --- p.155 / APPENDIX / Chapter I. --- Microarray Data --- p.157 / Chapter II. --- References --- p.166
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328291 |
| Date | January 2012 |
| Contributors | Leung, Kit Ying Crystal., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xviii, 184 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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