人類先天性巨結腸症（HSCR）主要表現為結腸末端的神經節缺失或稀少。 結腸末端的神經節來源於迷走源性神經脊細胞和骶源性神經脊細胞。迷走源性神經脊細胞是腸神經系統的主要來源，已被廣泛研究，而關於哺乳類包括人類的骶源性神經脊細胞的研究卻相當稀少。小鼠胚胎的骶源性神經脊細胞遷移途徑近期已被闡釋，其由神經管背側遷出並向腹側移動，於後腸附近聚集成旁神經節然後進入腸內。本研究運用一系列實驗鑒定了對小鼠骶源性神經脊細胞由旁神經節遷移至腸內過程有影響作用的因素。 / 研究發現骶源性神經脊細胞是沿著神經纖維並向口端方向遷移進腸內，同時由於迷走源性神經脊細胞於腸內向尾端遷移，它們在後腸末端相遇並相互作用，我們首先研究了這種相互作用以瞭解迷走源性神經脊細胞如何影響骶源性神經脊細胞的遷移。利用帶綠色螢光的小鼠骶源性神經脊細胞和可變螢光的小鼠迷走源性神經脊細胞（鐳射激發下由綠色變為紅色），以及鐳射共聚焦顯微鏡活細胞成像術，我們觀察了這兩種細胞在腸內相遇時的行為。當骶源性神經脊細胞和迷走源性神經脊細胞在神經纖維上相遇是，它們都停止了移動，不能前行。培養3天以後，骶源性神經脊細胞和迷走源性神經脊細胞在後腸中共同形成了神經細胞網路，其中骶源性神經脊細胞相對只占了小部分細胞。 / 由於骶源性神經脊細胞沿著由旁神經節發出的神經纖維遷移至腸內並且當在神經纖維上遇到迷走性源性神經脊細胞時便停止移動，我們進而研究了神經纖維在骶源性神經脊細胞遷移中的地位。活細胞成像術和免疫組化實驗表明旁神經節發出的神經纖維對骶源性神經脊細胞的遷移是非常重要的，它有助於細胞遷移同時，但當細胞到達纖維末端時它便也限制了細胞的遷移。儘管如此，體外實驗表明在一定培養條件下，骶源性神經脊細胞的遷移並不需要這些神經纖維。 / 由於有報導表明腸內微環境能影響腸神經脊細胞的遷移，我們利用2D電泳和質譜檢測了12.5天（骶源性神經脊細胞進入後腸之前）和13.5天（骶源性神經脊細胞進入後腸）胎鼠後腸中的蛋白表達情況。大多數鑒定到有差異表達的蛋白都與蛋白折疊、細胞生長和細胞骨架組織有關。我們選取了與細胞粘附和肌肉收縮有關的鈣離子依賴膜結合蛋白Anxa6，並結合腸內的平滑肌發育進行了進一步的研究。結果顯示在13.5天胎鼠中，旁神經節的口端方向有一段約600微米的腸的腹側的平滑肌還沒有發育，可能與腸神經脊細胞的遷移有關。但腸內平滑肌發育是否及如何影響腸神經脊細胞的遷移還需要進一步的研究。 / 綜上所述，骶源性神經脊細胞的遷移是一個複雜的過程，迷走源性神經脊細胞，神經纖維和腸內微環境都參與並能影響這個遷移過程。 / Hirschsprung’s disease (HSCR) in humans is characterized by the absence or reduction of enteric ganglia in the distal part of the colon. It is known that all enteric ganglia in the distal colon originate from neural crest cells (NCCs) at both vagal and sacral levels during embryonic development. Vagal NCCs have been well characterized as the main cellular source of the enteric nervous system (ENS), but however, information on the mammalian, including human, sacral NCCs is still scarce. Sacral NCCs in mouse embryos have been recently identified to be able to migrate from the dorsal neural tube to the mesenchyme, aggregate as pelvic ganglia adjacent to the hindgut and then enter the distal hindgut. In the present study, a series of experiments were performed to determine the factors that were involved in modulating their entry to the hindgut and their migration within the distal hindgut, using mouse embryos. / Having entered the hindgut, sacral NCCs migrated along nerve fibers in a caudal-to-rostral direction while vagal NCCs were colonizing the hindgut in a reverse, rostral-to-caudal direction. The migratory behaviors of the vagal and sacral NCCs were examined at the time when these two populations of NCCs met each other with live cell confocal imaging in the distal hindgut using GFP-expressing sacral NCCs (green fluorescent) and Ednrb-Kikume labeled vagal NCCs (red fluorescent) from transgenic mice. The rostral migration of sacral NCCs was observed to be temporarily affected when they met vagal NCCs on the nerve fiber. However, after 3 days in organotypic culture, sacral and vagal NCCs were found to intermingle with each other to form an interconnected cellular network in the hindgut with much greater cellular contribution from vagal NCCs than sacral NCCs. Hence, vagal NCCs were able to affect the migration and thus the final location of sacral NCCs within the hindgut. / Since sacral NCCs have been observed to enter the hindgut by migrating on the nerve fibers extending from pelvic ganglia, the role the nerve fibers in migration was then examined. Results obtained from time-lapse confocal live cell imaging and immunohistochemical localization indicated that nerve fibers extending from pelvic ganglia were very important for sacral NCCs migration. It was found that these nerve fibers could both assist in sacral NCC migration and also restrain their migration once the cells reached the distal tip of the fibers. However, under specific in vitro conditions, sacral NCCs were still able to migrate without the presence of nerve fibers. / The gut microenvironment surrounding the migrating NCCs has also been reported to affect NCCs migration. Therefore, protein molecules with differential expression levels prior to and after the entry of sacral NCCs to the distal hindgut between E12.5 to E13.5 were examined with 2-dimensional gel electrophoresis and mass spectrometry. The proteins identified with significant changes of expression (more than 1.5 folds) were grouped according to their predicted biological functions and involved in protein folding, cell growth and cytoskeletal organization. Among them, Anxa6, a calcium-dependent membrane binding protein related to cell adhesion and muscle contraction, was further examined for its relationship with the muscle development in the hindgut at E12.5 to E14.5. The results showed that a segment of the hindgut (about 600μm) rostral to pelvic ganglia exhibited an incomplete layer of smooth muscle at E13.5. Whether Anxa6 and the smooth muscle are involved in the sacral NCC migration is worth further investigations. / In summary, sacral NCCs migration is a complex process regulated by their interactions with nerve fibers, vagal neural crest cells and possibly molecules in the hindgut microenvironment through which they migrate. / 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. / Chen, Jielin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 201-214). / Abstract also in Chinese. / Abstract --- p.I / 摘要 --- p.IV / Acknowledgements --- p.VI / Table of contents --- p.XII / Abbreviation --- p.XIII / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The enteric nervous system (ENS) --- p.1 / Chapter 1.1.1 --- Embryonic origin and development of the ENS --- p.2 / Chapter 1.1.2 --- Hirschsprung’s disease (HSCR) --- p.5 / Chapter 1.2 --- Enteric neural crest cells (ENCCs) --- p.7 / Chapter 1.2.1 --- Vagal neural crest cells (NCCs) --- p.8 / Chapter 1.2.2 --- Sacral neural crest cells (NCCs) and pelvic ganglia --- p.10 / Chapter 1.2.3 --- Interactions between neural crest cells (NCCs) --- p.14 / Chapter 1.3 --- Microenvironment within the gut --- p.16 / Chapter 1.3.1 --- Effect of molecules on ENCCs migration --- p.16 / Chapter 1.3.2 --- Effect of tissue age on ENCC migration --- p.19 / Chapter 1.3.3 --- Absence of ENCCs facilitates ENCC colonization --- p.20 / Chapter 1.4 --- Objectives of the present study --- p.22 / Chapter Figures and Legends --- p.25 / Chapter Chapter 2 --- Migratory behaviors of sacral and vagal neural crest cells in the distal hindgut --- p.32 / Chapter 2.1 --- Introduction --- p.32 / Chapter 2.2 --- Materials and methods --- p.36 / Chapter 2.2.1 --- Mouse strains --- p.36 / Chapter 2.2.2 --- Isolation of gut tubes and pelvic ganglia --- p.36 / Chapter 2.2.3 --- Photo-conversion of Ednrb-kikume labeled neural crest cells within the gut --- p.37 / Chapter 2.2.4 --- Preparation of general culture medium and organ culture agarose gel --- p.38 / Chapter 2.2.5 --- Organotypic culture --- p.39 / Chapter 2.2.6 --- Time-lapse live cell confocal microscopic imaging --- p.39 / Chapter 2.2.7 --- Whole mount gut preparations for immunohistochemical staining --- p.41 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Conversion of green fluorescent, Ednrb-kikume labeled vagal NCCs into red fluorescent --- p.42 / Chapter 2.3.2 --- Ednrb-kikume labeled cells were Sox10 immunorecative --- p.42 / Chapter 2.3.3 --- No discernible photo-toxicity after photo-conversion --- p.43 / Chapter 2.3.4 --- Sacral NCCs migration was hindered by vagal NCCs when they met on the nerve fiber --- p.43 / Chapter 2.3.5 --- Vagal NCCs migrated toward each other to form an interconnected network --- p.45 / Chapter 2.3.6 --- Sacral NCCs contributed much fewer cells than vagal NCCs in the terminal hindgut --- p.46 / Chapter 2.4 --- Discussion --- p.47 / Chapter 2.4.1 --- Ednrb-kikume mouse is a potentially ideal animal model for studies of NCCs migratory behaviors --- p.47 / Chapter 2.4.2 --- Migration of sacral NCCs in the hindgut was affected by vagal NCCs --- p.49 / Chapter 2.4.3 --- Migratory behaviors of vagal NCCs --- p.52 / Chapter 2.4.4 --- Vagal NCCs potentially preferred to move on nerve fibers --- p.53 / Chapter 2.4.5 --- Sacral NCCs contributed much less to the cellular network than vagal NCCs --- p.53 / Chapter 2.5 --- Summary --- p.55 / Chapter Table 2-1 --- Primary and secondary antibodies used in the experiments --- p.56 / Chapter Figures and Legends --- p.57 / Chapter Chapter 3 --- The relationship between nerve fiber extension and sacral neural crest cell migration in vitro --- p.81 / Chapter 3.1 --- Introduction --- p.81 / Chapter 3.2 --- Materials and methods --- p.86 / Chapter 3.2.1 --- Mouse strains --- p.86 / Chapter 3.2.2 --- Preparation of fibronectin (FN) coated coverslips and confocal dishes --- p.86 / Chapter 3.2.3 --- Preparation of media --- p.87 / Chapter 3.2.4 --- Isolation of pelvic ganglia --- p.87 / Chapter 3.2.5 --- In vitro culture of pelvic ganglia in 4-well plates or confocal dishes --- p.88 / Chapter 3.2.6 --- Live cell imaging using Nikon live cell imaging system --- p.88 / Chapter 3.2.7 --- WGA treatments on the pelvic ganglia culture --- p.89 / Chapter 3.2.8 --- Effect of embryonic cell proliferation medium and stem cell proliferation medium on pelvic ganglia growth in vitro --- p.90 / Chapter 3.2.9 --- Immunohistochemical staining --- p.90 / Chapter 3.3 --- Results --- p.92 / Chapter 3.3.1 --- Sacral NCCs and nerve fibers from the pelvic ganglia were in close association in vitro --- p.92 / Chapter 3.3.2 --- Migratory behaviors of sacral NCCs on the nerve fiber in vitro --- p.92 / Chapter 3.3.3 --- WGA treatments affected the growth of nerve fibers and sacral NCCs migration in vitro --- p.93 / Chapter 3.3.4 --- Sacral NCCs migrated without nerve fibers when cultured in proliferation media --- p.95 / Chapter 3.4 --- Discussion --- p.97 / Chapter 3.4.1 --- In vitro culture of pelvic ganglion --- p.97 / Chapter 3.4.2 --- Migratory behaviors of sacral NCCs in vitro --- p.98 / Chapter 3.4.3 --- Sacral NCCs migration was affected by the extension of nerve fibers from pelvic ganglia in vitro --- p.101 / Chapter 3.4.4 --- Nerve fibers from the pelvic ganglia were not necessary for sacral NCCs migration in vitro --- p.103 / Chapter 3.5 --- Summary --- p.106 / Chapter Figures and Legends --- p.107 / Chapter Chapter 4 --- Differentially expressed protein molecules in the distal hindgut before and after the entry of sacral neural crest cells --- p.128 / Chapter 4.1 --- Introduction --- p.128 / Chapter 4.2 --- Materials and methods --- p.132 / Chapter 4.2.1 --- Mouse strain --- p.132 / Chapter 4.2.2 --- Preparation of solutions for 2-dimensional (2D) gel electrophoresis --- p.132 / Chapter 4.2.3 --- Preparation of solutions for mass spectrometry --- p.133 / Chapter 4.2.4 --- Isolation of the distal hindgut and protein extraction --- p.133 / Chapter 4.2.5 --- Measurement of protein concentration --- p.134 / Chapter 4.2.6 --- 2D gel electrophoresis --- p.135 / Chapter 4.2.7 --- Mass spectrometry --- p.138 / Chapter 4.2.8 --- SDS-PAGE and Western blot --- p.139 / Chapter 4.2.9 --- Immunohistochemical staining of gut tubes and embryos --- p.140 / Chapter 4.2.10 --- Distal hindgut model reconstruction --- p.141 / Chapter 4.3 --- Results --- p.143 / Chapter 4.3.1 --- E13.5 was the critical stage at which sacral NCCs started to enter the hindgut --- p.143 / Chapter 4.3.2 --- Protein molecules identified by 2D electrophoresis and mass spectrometry --- p.144 / Chapter 4.3.3 --- Western blot analysis and immunostaining confirmed expression levels of Anxa6 --- p.145 / Chapter 4.3.4 --- Smooth muscle actin (SMA) and Anxa6 partially co-localized within the E13.5 hindgut --- p.146 / Chapter 4.3.5 --- Expression of SMA and Anxa6 before and after sacral NCC entry to the distal hindgut --- p.146 / Chapter 4.3.6 --- Reconstruction of distal hindgut images from serial sections with SMA and Anxa6 immunoreactivities --- p.147 / Chapter 4.4 --- Discussion --- p.149 / Chapter 4.4.1 --- Tissue age affected enteric NCC colonization --- p.149 / Chapter 4.4.2 --- Proteomics used in modern biological research --- p.150 / Chapter 4.4.3 --- Molecules differentially expressed in the distal hindgut at E12.5 and E13.5 --- p.151 / Chapter 4.4.4 --- Anxa6 and SMA expression in the distal hindgut --- p.153 / Chapter 4.4.5 --- The role of the smooth muscle development in sacral NCC entry into the hindgut --- p.155 / Chapter 4.5 --- Summary --- p.157 / Chapter Table 4-1 --- Identification of proteins by MALDI-TOF analysis and the MASCOT search program --- p.158 / Chapter Table 4-2 --- Differentially expressed proteins identified by 2-D electro-phoresis and MALDI-TOF/TOF and their predicted biological functions --- p.159 / Figures and Legends --- p.160 / Chapter Chapter 5 --- Conclusions and discussion --- p.182 / Chapter 5.1 --- Vagal NCCs hindered sacral NCC migration when they coalesced on the nerve fiber --- p.182 / Chapter 5.2 --- Nerve fibers from pelvic ganglia were important but not necessary for sacral NCCs migration in vitro --- p.186 / Chapter 5.3 --- Possible involvement of smooth muscle development in modulating sacral NCCs migration --- p.189 / Chapter 5.4 --- Future prospects --- p.191 / Chapter 5.4.1 --- Interactions of vagal and sacral NCCs within the hindgut of mouse embryos --- p.191 / Chapter 5.4.2 --- Role of nerve fibers for sacral NCCs migration ex vivo --- p.192 / Chapter 5.4.3 --- Role of Anxa6 in muscle development of the gut and NCCs migration --- p.193 / Chapter Appendix I --- Solutions used in 2-D electrophoresis --- p.195 / Chapter Appendix II --- Solutions for Colloidal Coomassie staining --- p.198 / Chapter Appendix III --- Procedures for embryo processing --- p.199 / Chapter Appendix IV --- Other solutions --- p.200 / References --- p.201

Hou, Yonghui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 174-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Chan, Ka Ki Alex. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 245-263). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese abstract --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vii / Chapter Chapter One --- General introduction --- p.1 / Chapter 1.1 --- Structure and function of the enteric nervous system --- p.1 / Chapter 1.2 --- Neural crest cells (NCC) --- p.5 / Chapter 1.2.1 --- Vagal neural crest cells --- p.7 / Chapter 1.2.2 --- Sacral neural crest cells --- p.10 / Chapter 1.3 --- Prespecialization of the neural crest cells to form ENS --- p.15 / Chapter 1.4 --- Signaling pathways involved in ENS development --- p.19 / Chapter 1.4.1 --- Endothelin signaling pathway --- p.20 / Chapter 1.4.2 --- Ret signaling pathway: GDNF/Ret/GFRa1 --- p.22 / Chapter 1.4.3 --- Ret signaling pathway: NRTN/Ret/GFRa2 --- p.26 / Chapter 1.4.4 --- Phox2b --- p.28 / Chapter 1.4.5 --- Sox10 --- p.29 / Chapter 1.5 --- Hirschsprung's Disease (HSCR) --- p.31 / Chapter 1.6 --- Objective of studies --- p.32 / Figures and legends --- p.35 / Chapter Chapter Two --- The early migratory pathways of mouse sacral neural crest cells --- p.39 / Chapter 2.1 --- Introduction --- p.39 / Chapter 2.2 --- Materials and Methods --- p.46 / Chapter 2.2.1 --- Animals --- p.46 / Chapter 2.2.2 --- Isolation of the mouse embryos at E95 --- p.46 / Chapter 2.2.3 --- Preparation ofWGA-Au --- p.47 / Chapter 2.2.4 --- Preparation of Dil --- p.48 / Chapter 2.2.5 --- Microinjection ofWGA-Au or Dil --- p.48 / Chapter 2.2.6 --- Preparation of rat serum --- p.49 / Chapter 2.2.7 --- Preparation of culture medium --- p.50 / Chapter 2.2.8 --- in vitro whole embryo culture system --- p.50 / Chapter 2.2.9 --- Examination of embryo after culture --- p.51 / Chapter 2.2.10 --- Histological preparation of WGA-Au labelled embryos --- p.51 / Chapter 2.2.11 --- Silver enhancement staining on sections of WGA-Au labelled embryo --- p.52 / Chapter 2.2.12 --- Histological preparation of Dil labelled embryos --- p.53 / Chapter 2.2.13 --- Reconstruction of the mouse embryos --- p.53 / Chapter 2.2.14 --- Cell counting on labelled sacral NCC between the anterior and posterior halves of the somite --- p.54 / Chapter 2.2.15 --- Cell counting on migrating labelled sacral NCC for each somite at different developmental stages --- p.55 / Chapter 2.3 --- Results --- p.57 / Chapter 2.3.1 --- Development of E9.5 mouse embryo in vitro and in vivo --- p.57 / Chapter 2.3.2 --- Labelling of sacral neural crest cells by means of different cell markers --- p.58 / Chapter 2.3.3 --- Migration of sacral neural crest cells at different developmental stages --- p.59 / Chapter 2.3.3.1 --- Distribution of sacral NCC at the 26th somite stage --- p.60 / Chapter 2.3.3.2 --- Distribution of sacral NCC at the 28th somite stage --- p.61 / Chapter 2.3.3.3 --- Distribution of sacral NCC at the 30th somite stage --- p.61 / Chapter 2.3.3.4 --- Distribution of sacral NCC at the 32nd somite stage --- p.63 / Chapter 2.3.3.5 --- Distribution of sacral NCC at the 34th somite stage --- p.64 / Chapter 2.3.4 --- Defined migration pathways of the sacral neural crest cells --- p.65 / Chapter 2.3.5 --- Quantification of migrating sacral NCC at different somite axial levels at different developmental stages --- p.66 / Chapter 2.4 --- Discussion --- p.68 / Chapter 2.4.1 --- E9.5 mouse embryo grew normally in vitro using whole embryo culture --- p.69 / Chapter 2.4.2 --- Migration of sacral neural crest cells at 26th somite stage --- p.70 / Chapter 2.4.3 --- Migration of sacral neural crest cells at 28th somite stage --- p.72 / Chapter 2.4.4 --- Migration or sacral neural crest cells at 30th somite stage --- p.73 / Chapter 2.4.5 --- Migration of sacral neural crest cells at 32nd somite --- p.75 / Chapter 2.4.6 --- Migration of sacral neural crest cells at 34th somite stage --- p.77 / Chapter 2.4.7 --- Majority of sacral neural crest cells migrate along the dorsomedial pathway --- p.80 / Figures and Legends --- p.82 / Tables --- p.136 / Chapter Chapter Three --- The early migratory pathways of Dom mouse sacral neural crest cells --- p.139 / Chapter 3.1 --- Introduction --- p.139 / Chapter 3.2 --- Materials and Methods --- p.145 / Chapter 3.2.1 --- Animals --- p.145 / Chapter 3.2.2 --- In vitro culture of Dom mouse embryos --- p.145 / Chapter 3.2.3 --- Genotyping by polymerase chain reaction (PCR) --- p.146 / Chapter 3.2.4 --- Treatment of the harvested Dom mouse embryos --- p.147 / Chapter 3.2.5 --- Reconstruction of images and cell counting --- p.148 / Chapter 3.2.6 --- Percentage of migrating sacral neural crest cells reduction in Dom mouse embryo --- p.148 / Chapter 3.3 --- Results --- p.150 / Chapter 3.3.1 --- Migration of sacral neural crest cells in Dom mouse embryos at different developmental stages --- p.150 / Chapter 3.3.1.1 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 26th somite stage --- p.150 / Chapter 3.3.1.2 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 28th somite stage --- p.151 / Chapter 3.3.1.3 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 30th somite stage --- p.152 / Chapter 3.3.1.4 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 32nd somite stage --- p.154 / Chapter 3.3.1.5 --- Distribution of sacral neural crest cells of Dom mouse embryos at the 34th somite stage --- p.156 / Chapter 3.3.2 --- Number of migrating sacral NCC of different genotypes of Dom mouse embryos at different developmental stage --- p.158 / Chapter 3.4 --- Discussion --- p.160 / Chapter 3.4.1 --- The use of Dom mouse model to study the etiology of Hirschsprung's disease (HSCR) --- p.161 / Chapter 3.4.2 --- Migration of sacral NCC in Dom mouse embryos --- p.164 / Figures and legends --- p.169 / Tables --- p.230 / Chapter Chapter Four --- General discussion and conclusions --- p.236 / Appendix --- p.241 / References --- p.245

Nogo is well established for its inhibitory action on axon regeneration in the adult central nervous system. It binds to the Nogo receptor (NgR) through an extracellular active site on the protein-Nogo-66. Although it is reported that Nogo is widely expressed in the developing brain, its exact function during development of the nervous system is unclear. / The contribution of Nogo on patterning the axon routing at the optic chiasm of mouse embryo was investigated in this thesis. Using immunocytochemical staining, Nogo protein was localized on the Miller glial cells in the retina and at the optic disk. A few migrating retinal neurons also expressed Nogo. In the chiasm, Nogo was localized exclusively on the radial glia, which generate a midline domain where turning of uncrossed axons occurs. In vitro study showed expression of NgR on retinal neurites and growth cones, and neurite outgrowth from both dorsal nasal (contralaterally projecting) and ventral temporal (ipsilaterally projecting) retina was inhibited by Nogo. In the pathway, NgR expression was regionally regulated. NgR was obvious in the optic stalk and the optic tract, but not in the chiasm. Blocking Nogo function with NEP1-40, a peptide antagonist of NgR, in brain slice culture of the pathway produced significant reduction in the uncrossed projection, but had no effect on axon crossing at the midline. Furthermore, the age related fiber arrangement in the optic tract was abolished after disturbing of Nogo function. Similar abnormalities were observed in slices treated with Nogo blocking antibody. In vitro studies showed that NEP1-40 rescued the inhibition of Nogo to the retinal neurites. The downregulation of NgR at the chiasm was supported by in vitro assays showing significant reduction of receptor expression on dorsal nasal but not ventral temporal growth cones when they encountered the chiasm, thus generating a differential inhibition to ventral temporal neurites. / These results provide evidences that Nogo is a guidance molecule during the development of CNS. Interaction of Nogo and its receptor plays important role for patterning the axon divergence in the mouse optic pathway and the age related fiber order in the optic tract. / Wang Jun. / "September 2006." / Adviser: Sun-On Chan. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1474. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 130-142). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Li Mei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 182-202). / Abstracts in English and Chinese. / Abstract --- p.i / 中文摘要 --- p.iii / Acknowledgements --- p.v / List of Abbreviations --- p.vi / CONTENTS --- p.viii / Chapter Chapter 1 --- General introduction --- p.1 / Chapter 1.1 --- Alkaline phosphatase --- p.1 / Chapter 1.1.1 --- Distribution --- p.1 / Chapter 1.1.2 --- Molecular characteristics of alkaline phosphatase --- p.4 / Chapter 1.1.3 --- Properties of alkaline phosphatase --- p.8 / Chapter 1.1.4 --- Role of alkaline phosphatase --- p.10 / Chapter 1.2 --- Mouse embryonic brain development --- p.18 / Chapter 1.2.1 --- General developing process --- p.18 / Chapter 1.2.2 --- The crainal nerve nuclei in the embryonic mouse brainstem --- p.20 / Chapter 1.2.3 --- The process of neurogenesis in central nerve system --- p.22 / Chapter 1.3 --- Alkaline phosphatase expressed in developing neural tube --- p.26 / Chapter 1.4 --- Summary --- p.30 / Chapter 1.5 --- Objectives of study --- p.31 / Chapter Chapter 2 --- AP expression pattern in embryonic mouse brainstem --- p.33 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.1.1 --- AP expressed in developing neural tube --- p.33 / Chapter 2.1.2 --- Methods for alkaline phosphatase detection --- p.35 / Chapter 2.2 --- Materials and methods --- p.39 / Chapter 2.2.1 --- Animal and procedure --- p.39 / Chapter 2.2.2 --- Preparation of tissue sections and histochemistry --- p.39 / Chapter 2.2.3 --- Electron microscopy study of AP location --- p.41 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Histochemical demonstration of AP --- p.42 / Chapter 2.3.2 --- Stage-specificity and tissue-specificity of AP activity in the neural tube --- p.43 / Chapter 2.3.3 --- Cytochemical localization of AP activity --- p.46 / Chapter 2.3.4 --- Sencitivity to pH of the histochemical staining for AP --- p.46 / Chapter 2.3.5 --- Inactivation of AP activity --- p.47 / Chapter Chapter 3 --- Quantitative studies of AP activity in embryonic mouse brainstem --- p.48 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.1.1 --- Basic knowledge about enzyme kinetic study --- p.48 / Chapter 3.1.2 --- Enzyme assay for alkaline phosphatase --- p.50 / Chapter 3.2 --- Materials and methods --- p.52 / Chapter 3.2.1 --- Animals and sample preparation --- p.52 / Chapter 3.2.2 --- Measurement of AP activities --- p.53 / Chapter 3.2.3 --- Data analysis --- p.54 / Chapter 3.3 --- Results --- p.54 / Chapter 3.3.1 --- "Determination of reaction duration, initial velocity and Km of AP activity" --- p.54 / Chapter 3.3.2 --- Comparision of AP activity in the brainstem and cortex and at different stages --- p.55 / Chapter 3.3.3 --- Effects of physical and chemical factors on AP activity --- p.55 / Chapter Chapter 4 --- Electrophoresis study of AP activity --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Materials and methods --- p.60 / Chapter 4.2.1 --- AP extraction --- p.60 / Chapter 4.2.2 --- Polyacrylamide gel electrophoresis (PAGE) --- p.61 / Chapter 4.2.3 --- Detection of AP activity --- p.61 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- Demonstration of AP activity on the gels --- p.62 / Chapter 4.3.2 --- Comparison of AP from the brain at different stages --- p.62 / Chapter 4.3.3 --- "Comparison of AP in the embryonic brainstem with those in the adult mouse placenta, kidney, liver and intestine" --- p.63 / Chapter 4.3.4 --- Effect of heating and chemical factors on AP activity in the embryonic brainstem --- p.63 / Chapter Chapter 5 --- Study of the cell types expressing AP activity --- p.65 / Chapter 5.1 --- Introduction --- p.65 / Chapter 5.2 --- Materials and methods --- p.67 / Chapter 5.2.1 --- Materials --- p.67 / Chapter 5.2.2 --- Immunostaining of AP in the embryonic brainstem --- p.68 / Chapter 5.2.3 --- Double staining for AP and cells markers --- p.70 / Chapter 5.3 --- Results --- p.70 / Chapter 5.3.1 --- Effectiveness of anti-TNAP antibody on the embryonic mouse brain --- p.70 / Chapter 5.3.2 --- Expression pattern of different neural cell markers at E13.5 --- p.71 / Chapter 5.3.3 --- Co-localization of AP and specific cell markers in E13.5 brain --- p.72 / Chapter Chapter 6 --- Discussion --- p.74 / Chapter 6.1 --- Stage-dependence and tissue-specificity of AP expression in the developing mouse brainstem --- p.75 / Chapter 6.2 --- Possible molecular nature of AP expressed in the developing mouse brainstem --- p.80 / Chapter 6.3 --- The possible cell types that express the enzyme activity --- p.83 / "Figures, Tables, Graphs and Legends" --- p.87 / Appendices --- p.165 / Appendix A: Sources of materials --- p.165 / Appendix B: The process of sample for staining --- p.167 / Appendix C: Protocol of histochemical staining for AP --- p.170 / Appendix D: Protocol of electron microscopy study for AP activity --- p.172 / Appendix E: Protocol of enzyme assay for AP activity --- p.174 / Appendix F: Protocol of immunostaining (ABC method) --- p.175 / Appendix G: Protocol of double staining with fluorescent detection --- p.177 / Appendix H: Protocol of electrophoresis analysis for AP --- p.179 / References --- p.182

Chung Kit Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 90-104). / Abstracts in English and Chinese. / Chapter Chapter 1 --- General Introduction --- p.1-22 / Chapter Chapter 2 --- Expression of Chondroitin Sulfate Proteoglycans (CSPGs) in the Chiasm of Mouse Embryos / Introduction --- p.23-24 / Materials and Methods --- p.25 -27 / Results --- p.28-33 / Discussion --- p.34-40 / Figures --- p.41-45 / Chapter Chapter 3 --- Effects on Axon Routing after Removal of Chondroitin Sulfate Proteoglycans by Enzymatic Digestion / Introduction --- p.46 -47 / Materials and Methods --- p.48 -50 / Results --- p.57 / Discussion --- p.57-61 / Figures --- p.62-66 / Chapter Chapter 4 --- Immediate Effects of Prenatal Monocular Enucleation on the Cellular and Molecular Environment in the Development of Retinofugal Pathway / Introduction --- p.67-69 / Materials and Methods --- p.70-72 / Results --- p.73.77 / Discussion --- p.78-82 / Figures --- p.83-86 / Chapter Chapter 5 --- General Conclusion --- p.87-89 / References --- p.90 -104

by Cheung Chui Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 96-107 (2nd gp.)). / Abstracts in English and Chinese. / ABSTRACT (ENGLISH) --- p.i / ABSTRACT (CHINESE) --- p.iii / ACKNOWLEDGEMENTS --- p.v / TABLE OF CONTENT --- p.vii / Chapter CHAPTER ONE --- GENERAL INTRODUCTION / Chapter 1.1 --- Early development of the central nervous system --- p.1 / Chapter 1.2 --- Neural crest cells and Cardiac neural crest cells --- p.1 / Chapter 1.3 --- Role of neural crest cells in cardiovascular development --- p.4 / Chapter 1.4 --- Methods in tracing neural crest cells --- p.7 / Chapter 1.5 --- Neural crest-related defects --- p.14 / Chapter 1.6 --- Animal models for studying neural crest defects --- p.16 / Chapter 1.7 --- Recent studies on the migration of cardiac neural crest cells in mammals --- p.19 / Chapter 1.8 --- Objectives of the present study --- p.22 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.26 / Pregnant mice --- p.26 / Pregnant Splotch mice (Sp2H) --- p.26 / Preparation of the handling medium --- p.27 / Preparation of the culture medium --- p.27 / Gas mixtures for embryo culture --- p.30 / Preparation of wheat germ agglutinin-gold conjugates (WGA-Au) --- p.30 / Preparation of the fixative --- p.30 / DNA solution for genotyping of Splotch embryos --- p.31 / Primers used in PCR for genotyping of Splotch embryos --- p.31 / PCR reagent system --- p.32 / 10XTBE --- p.32 / Chapter 2.2 --- Methods --- p.33 / Isolation of embryos from pregnant mice --- p.33 / In situ labelling of exogenous dye --- p.34 / Orthotopical grafting of neural crest fragment --- p.36 / Whole embryo culture --- p.37 / Morphological examination of cultured embryos --- p.38 / Histological examination of cultured embryos --- p.38 / Examination of labelled cells in sectioned embryos --- p.39 / Genotyping of Splotch embryos by PCR --- p.40 / Gel electrophoresis --- p.41 / Chapter CHAPTER THREE --- RESULTS / Chapter 3. 1 --- Initial migration of cardiac neural crest cells in normal ICR mouse embryos --- p.43 / Gross morphological examination of cultured embryos --- p.43 / Distribution of WGA-Au labelled cells in ICR normal mouse embryos --- p.45 / Chapter 3.2 --- Initial migration of cardiac neural crest cells in Splotch embryos --- p.50 / Genotyping --- p.50 / Morphological examination of Splotch mutant embryos --- p.50 / Morphological examination of in vivo Splotch embryos --- p.53 / Distribution of WGA-Au labelled cells in Splotch Embryos --- p.54 / Chapter 3.3 --- Transplantation of neural crest fragments in Splotch embryos --- p.60 / Morphological features of Splotch embryos after orthotopic grafting --- p.60 / Histological examination of Splotch embryos after grafting --- p.61 / Distribution of WGA-Au labelled cells in Splotch embryos after grafting --- p.62 / Chapter CHPATER FOUR --- DISCUSSION / Chapter 4.1 --- Development of embryos in vitro --- p.65 / Chapter 4.2 --- Methodology --- p.70 / In situ labelling of WGA-Au in embryos --- p.70 / Counting of labelled cells in Sploch embryos --- p.72 / Transplantation of neural crest fragments --- p.72 / Chapter 4.3 --- Initial migration of cardiac neural crest cells --- p.74 / Distribution of cardiac neural crest cellsin normal mouse embryos --- p.74 / Differences in the distribution of labelled neural crest cells In different genotypes of Splotch embryos --- p.78 / Distribution of cardiac neural crest cells in Splotch embryos After transplanting of neural crest fragments --- p.83 / Chapter 4.4 --- Factors in extracellular matrix affecting the migration of neural crest cells --- p.88 / Chapter CHAPTER FIVE --- CONCLUSION --- p.91 / REFERENCES --- p.96 / FIGURES AND LEGEND / TABLES / GRAPHS

Leung Kin Mei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 106-125). / Abstracts in English and Chinese. / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1-11 / Chapter CHAPTER 2 --- ENZYMATIC REMOVAL OF CHONDROITIN SULFATES ABOLISHES THE AGE-RELATED ORDER IN THE OPTIC TRACT OF MOUSE EMBRYOS / INTRODUCTION --- p.12-13 / MATERIALS AND METHODS --- p.13-18 / RESULTS --- p.18-24 / DISCUSSION --- p.24-29 / FIGURES --- p.30-39 / Chapter CHAPTER 3 --- EXPRESSION OF PHOSPHACAN AND NEUROCAN IN THE DEVELOPING MOUSE RETINOFUGAL PATHWAY / INTRODUCTION --- p.40-42 / MATERIALS AND METHODS --- p.42-43 / RESULTS --- p.44-49 / DISCUSSION --- p.49-55 / FIGURES --- p.56-61 / Chapter CHAPTER 4 --- HEPARAN SULFATE PROTEOGLYCAN EXPRESSION IN THE OPTIC CHIASM OF MOUSE EMBRYOS / INTRODUCTION --- p.62-63 / MATERIALS AND METHODS --- p.63-65 / RESULTS --- p.66-70 / DISCUSSION --- p.70-76 / FIGURES --- p.77-82 / Chapter CHAPTER 5 --- EXPRESSION OF NEURAL CELL ADHESION MOLECULES IN THE CHIASM OF MOUSE EMBRYOS / INTRODUCTION --- p.83-85 / MATERIALS AND METHODS --- p.85-88 / RESULTS --- p.88-92 / DISCUSSION --- p.92.95 / FIGURES --- p.96-102 / Chapter CHAPTER 6 --- GERNEAL CONCLUSION --- p.103-105 / REFERENCES --- p.106-125

During the growth phase of oogenesis, mammalian oocytes increase several hundred-fold in volume. Although it is known that ovarian granulosa cells send growth promoting signals, neither these external signals nor the transduction pathways that become activated in the oocyte are known. Therefore, the presence and the activity of candidate signaling pathways in growing murine oocytes were investigated. By immunoblotting, the MAP kinases, ERK1 and ERK2, as well as their activating kinase MEK, were detected in oocytes at all stages of growth. However, using a phospho-specific anti-ERK antibody, no immunoreactive species were detectable in isolated granulosa cells or oocytes at any stage of growth, except metaphase II. Phosphorylated ERK was also present, although in smaller quantities, in oocyte-granulosa cell complexes at the later stages of growth. Furthermore, when ovarian sections were stained with an anti-ERK antibody, the protein was found to be highly concentrated in the cytoplasm of oocytes at all stages of growth, with lower levels in the nucleus. Another member of the MAP kinase family, Jun kinase (JNK), was investigated. By immunoblotting, JNK was detected in growing oocytes. Experiments using an anti-JNK antibody on ovary sections revealed the protein to be uniformly distributed in non-growing and growing oocytes with no evidence of preferential nuclear localization. These results imply that an interaction between the oocyte and the granulosa cells may be required to generate phosphorylated ERK. They also imply that growth signals probably are not relayed through ERK, but do not exclude a role for Jun kinase in mediating oocyte growth.

Access to abstract restricted until 12/14/2014. / Access to thesis restricted until 12/14/2014. / Department of Biology