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Wang, Chengdong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 113-134). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Dong Ming. / Thesis submitted in: December 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 118-152). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgement --- p.iv / Table of Contents --- p.v / Abbreviation list --- p.xi / Chapter Chapter 1 --- General introduction / Chapter 1.1 --- Preamble --- p.1 / Chapter 1.2 --- Neural Crest Cells (NCCs) --- p.2 / Chapter 1.3 --- Enteric Nervous System (ENS) and Vagal Neural Crest Cells (Vagal NCCs) --- p.4 / Chapter 1.4 --- Sacral Neural Crest Cells (Sacral NCCs) --- p.7 / Chapter 1.5 --- Signalling Mechanisms of Sacral Neural Crest Cells --- p.17 / Chapter 1.6 --- Hirschsprung's Disease (HSCR) --- p.20 / Chapter 1.7 --- Objective of the Study and Contents of the Following Chapters --- p.21 / Chapter Chapter 2 --- Migration from the dorsal neural tube to the pelvic mesenchyme / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and Methods --- p.32 / Chapter 2.2.1 --- Animal --- p.32 / Chapter 2.2.2 --- Isolation of embryos from pregnant mice at E9.5 to --- p.32 / Chapter 2.2.3 --- Histological preparation of the caudal segments --- p.33 / Chapter 2.2.4 --- p75 immunohistochemical staining --- p.33 / Chapter 2.2.5 --- Preparation of rat serum --- p.34 / Chapter 2.2.6 --- Preparation of the culture medium --- p.34 / Chapter 2.2.7 --- Preparation of wheat germ agglutinin-gold conjugates (WGA-Au) --- p.35 / Chapter 2.2.8 --- Preparation of CMFDA --- p.35 / Chapter 2.2.9 --- Preparation of DiI --- p.36 / Chapter 2.2.10 --- "Microinjection of WGA-Au, DiI and CMFDA" --- p.36 / Chapter 2.2.11 --- Whole embryo culture --- p.37 / Chapter 2.2.12 --- Examination of cultured embryos --- p.37 / Chapter 2.2.13 --- Histological preparation of WGA-Au labelled embryos --- p.38 / Chapter 2.2.14 --- Silver enhancement staining of the sections of WGA-Au labelled embryos --- p.39 / Chapter 2.2.15 --- Cryosectioning of the embryos labelled with DiI --- p.39 / Chapter 2.2.16 --- p75 immunohistochemical staining of DiI-labelled cells --- p.40 / Chapter 2.3 --- Results --- p.41 / Chapter 2.3.1 --- Observations on embryos developed in vivo --- p.41 / Chapter 2.3.2 --- Closed yolk sac culture vs open yolk sac culture --- p.42 / Chapter 2.3.3 --- Neural crest cell labelling in the caudal part of embryos --- p.43 / Chapter 2.3.4 --- Neural crest cell labelling with DiI in the caudal part of the neural tube followed by in vitro culture from E9.5 to E11.0 --- p.45 / Chapter 2.3.5 --- Neural crest labelling with DiI in the caudal part of the neural tube followed by in vitro culture from E10.5 to E11.5 --- p.46 / Chapter 2.3.6 --- Focal labelling at the levels of the 26th and 29th somites followed by in vitro culture --- p.48 / Chapter 2.3.7 --- p75 immunohistochemical staining on the caudal part of the embryo at E10.5 --- p.49 / Chapter 2.3.8 --- p75 immunohistochemical staining on embryos labelled with DiI --- p.50 / Chapter 2.4 --- Discussion --- p.51 / Chapter 2.4.1 --- Embryos at E9.5 cultured with an intact yolk sac membrane grew better than those with the yolk sac membrane cut open --- p.52 / Chapter 2.4.2 --- Migration at the levels of the 24th to 28th somite --- p.53 / Chapter 2.4.3 --- Migration at the levels of the 29th to 33th somite --- p.58 / Chapter 2.4.4 --- Sacral NCCs migrate along a straight dorsolateral pathway --- p.60 / Chapter 2.4.5 --- "Most of the DiI positive cells are p75 positive, but not all of the p75 positive cells are DiI positive" --- p.62 / Chapter Chapter 3 --- Migration from the pelvic mesenchyme to the hindgut / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.2 --- Materials and Methods --- p.73 / Chapter 3.2.1 --- Isolation of hindguts with or without adjacent tissues from embryos at E10.5 to E13.5 --- p.73 / Chapter 3.2.2 --- Microinjection of DiI into the pelvic mesenchymal tissue of the h indguts --- p.74 / Chapter 3.2.3 --- Preparation of the culture medium --- p.74 / Chapter 3.2.4 --- Preparation of the culture dish --- p.74 / Chapter 3.2.5 --- Gut culture --- p.75 / Chapter 3.2.6 --- Cryosections of the hindguts after in vitro culture --- p.75 / Chapter 3.3 --- Results --- p.76 / Chapter 3.3.1 --- "Hindguts isolated from embryos at E10.5, E11.5, E12.5 and E14.5" --- p.76 / Chapter 3.3.2 --- p75 immunohistochemical staining of the serial sections through the caudal part of the embryos --- p.78 / Chapter 3.3.3 --- Observations on hindgut without pelvic plexus cultured from E11.5 to E14.5 --- p.81 / Chapter 3.3.4 --- "Culture of hindguts with pelvic mesenchyme cultured from E11.5 to E14.25, E14.5 and E15.5" --- p.82 / Chapter 3.3.5 --- Culture of the whole length of the gut tube without pelvic mesenchyme from E11.5 to E15.5 --- p.84 / Chapter 3.3.6 --- Culture of the whole length of the gut tube with pelvic mesenchyme from E11.5 to E15.5 --- p.84 / Chapter 3.3.7 --- "Culture of hindguts with Dil labelling in the pelvic mesenchyme from E11.5 to E14.0, E14.2 5, E14.5 and" --- p.85 / Chapter 3.3.8 --- Culture of the whole length of the gut tube with DiI labelling in the pelvic mesenchyme from E11.5 to E14.5 --- p.86 / Chapter 3.4 --- Discussion --- p.88 / Chapter 3.4.1 --- Development of the hindgut and the urogenital system from E10.5 to E14.5 --- p.88 / Chapter 3.4.2 --- No p75 positive cells were found in the hindgut before E13.5 --- p.89 / Chapter 3.4.3 --- The sacral neural crest cells migrate into the hindgut at around E14.5 --- p.91 / Chapter 3.4.4 --- "The sacral neural crest cells migrated in the serosa, and entered the myenteric plexus prior to populating the submucosal plexus" --- p.95 / Chapter 3.4.5 --- Most of DiI labelled sacral neural crest cells in the hindgut also expressed p75 --- p.98 / Chapter Chapter 4 --- Migration from the neural tube to the hindgut / Chapter 4.1 --- Introduction --- p.101 / Chapter 4.2 --- Materials and Methods --- p.104 / Chapter 4.3 --- Results --- p.106 / Chapter 4.3.1 --- Morphology observations on the hindguts isolated from DiI-labelled embryos --- p.106 / Chapter 4.3.2 --- Distribution of the DiI-labelled cells and p75 positive cells before the culture of the hindgut explant --- p.106 / Chapter 4.3.3 --- Culture of hindgut explanted from Dil-labelled embyos for 3.5 days from E11.0 to E14.5 --- p.107 / Chapter 4.4 --- Discussion --- p.109 / Chapter Chapter 5 --- General discussion and conclusions --- p.113 / References --- p.118 / Figures and Legends --- p.153 / Tables and Graphs --- p.203 / Appendix --- p.209

For the purpose of developing an effective therapeutic strategy for HSCR, the enteric neural crest stem cells were investigated firstly which were isolated from the E14.5 mouse embryonic gut, cultured as neurospheres and characterized by multiple immunofluorescence and reverse transcription-PCR, population doubling time, frequency of forming secondary neurospheres and limited dilution assay. In the differentiation culture medium, several types of cells were induced to form from the neurospheres derived from single cells. Hence the putative enteric neural crest stern cells, which were isolated from the embryonic mouse gut tube and cultured as neurospheres for many passages ex vivo with the demonstrated capacity of proliferation, self-renewal and differentiation, showed properties of stem cells. / Hirschsprung's disease (HSCR) is caused by the absence of the enteric neural crest-derived neurons at the distal region of the gut. Cell-based therapy using stem cells or progenitors gives the potential to supplement these missing enteric neurons in the gut. Enteric neural crest stem cells isolated from the human or rodent gut can give rise to neurons and glia after they are transplanted into the recipient guts of the mouse or rat. However, numbers of issues are unresolved about the basic biology of the enteric nervous system, the characteristics of the stem cells isolated from the enteric nervous system and the biological significance of these cells in prenatal and postnatal periods. In this study, the characteristics and therapeutic potential on HSCR of the enteric neural crest stem cells were explored. / In addition to the above, a recombination organotypic gut culture ex vivo showed that the colonization of enteric neural crest-derived cells in the recipient gut was influenced not only by the genotypes of enteric neural crest-derived cells themselves but also the microenvironment of the gut through which enteric neural crest-derived cells migrated. For instance, the developmental stage of the recipient gut and also the presence of endogenous enteric neural crest-derived cells along the migratory pathway of neural crest-derived cells both affected the extent of the migration and colonization of exogenous enteric neural crest-derived cells and stem cells. The gradual maturation and differentiation of the neighboring structures, such as the smooth muscle layer, during the time period of the enteric neural crest cells migration, might also suggest that these neighboring tissues may have a role in regulating the neural crest-derived cells migration. / In conclusion, enteric neural crest stem cells isolated from the embryonic mouse gut tube showed properties of stem cells, and had the potential to compensate missing enteric neural crest-derived cells both ex vivo and in vivo. However, the colonization of enteric neural crest-derived cells in the developing gut was affected cell-autonomously and also by the microenvironment of the gut and the presence of existing enteric neural crest-derived cells. / Their potential applications in the transplantation experiments were shown by transplantation of the neurospheres isolated to the gut tube maintained in an organotypic culture or to the descending colon of neonates at postnatal day 7. The development of the enteric neural crest stern cells from the neurospheres was found to be compatible to endogenous enteric neural crest-derived cells in the recipient gut as evidenced by the formation of interconnected cellular networks of donor stem cells and endogenous neural crest-derived cells. The enteric neural crest stem cells also possess the potential to compensate the loss of enteric neural crest-derived cells ex vivo and in vivo in recipient prenatal and postnatal guts. / Bao, Lihua. / Adviser: Wood Yee Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 208-228). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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