Tam Wing-yip. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 91-113). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese Abstract --- p.iv / Acknowledgements --- p.vi / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Hirschsprung's disease --- p.1 / Chapter 1.2 --- Neural crest cells and enteric nervous system --- p.3 / Chapter 1.3 --- Genetics of Hirschsprun´gةs disease --- p.10 / Chapter 1.3.1 --- RET/GDNF/NTN signaling pathway --- p.10 / Chapter 1.3.2 --- EDNRB/EDN3/ECE-1 signaling pathway --- p.13 / Chapter 1.3.3 --- Dominant megacolon and Sox10 --- p.15 / Chapter 1.3.4 --- Other genes involved in intestinal aganglionosis --- p.16 / Chapter 1.4 --- Objectives of the present study --- p.19 / Chapter Chapter 2 --- Enteric Neural Crest Cells Migration in Dominant Megacolon Mouse Embryos --- p.21 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.26 / Chapter 2.2.1 --- Animal --- p.26 / Chapter 2.2.2 --- Preparation of rat serum --- p.26 / Chapter 2.2.3 --- Isolation of embryos from pregnant mice --- p.27 / Chapter 2.2.4 --- Preparation of wheat germ agglutinin-gold (WGA-Au) --- p.28 / Chapter 2.2.5 --- Microinjection of WGA-Au conjugate --- p.28 / Chapter 2.2.6 --- Whole embryo culture --- p.29 / Chapter 2.2.7 --- Examination of cultured embryos --- p.30 / Chapter 2.2.8 --- Histological preparation of WGA-Au injected embryos --- p.30 / Chapter 2.2.9 --- Silver enhancement staining and histological examination of the sections --- p.31 / Chapter 2.2.10 --- Genotyping by polymerase chain reaction --- p.32 / Chapter 2.2.11 --- TUNEL assays --- p.33 / Chapter 2.3 --- Results --- p.35 / Chapter 2.3.1 --- In vivo development of Dominant megacolon mouse embryos of different genotypes --- p.35 / Chapter 2.3.2 --- In vitro development of embryos in control and experimental groups --- p.35 / Chapter 2.3.3 --- Migration of vagal neural crest cells in Dom embryos --- p.36 / Chapter 2.3.4 --- Apoptotic cells detection at the vagal region by TUNEL assay --- p.37 / Chapter 2.3.5 --- Migration of sacral neural crest cells in Dom embryos --- p.37 / Chapter 2.3.6 --- Apoptotic cells detection at the sacral region by TUNEL assay --- p.38 / Figures and Tables / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.4.1 --- In vitro culture system supporting the normal development of mouse embryos --- p.40 / Chapter 2.4.2 --- WGA-Au as a cell marker for tracing the NCCs migration --- p.41 / Chapter 2.4.3 --- Vagal neural crest cells migration in Dom mouse embryos --- p.42 / Chapter 2.4.4 --- Apoptotic cell death does not contribute to the total aganglionosis in Dom homozygous embryos --- p.43 / Chapter 2.4.5 --- Sacral neural crest cells migration in Dom mouse embryos --- p.45 / Chapter 2.4.6 --- NCCs migration in zebrafish colourless mutant --- p.47 / Chapter 2.4.7 --- Limitation of the method used in this study --- p.49 / Chapter 2.4.8 --- Conclusions --- p.49 / Appendices / Chapter Chapter 3 --- Migration of Enteric Neural Crest-derived Cells in the Developing Gut of Dominant Megacolon Mouse Embryos --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Materials and Methods --- p.55 / Chapter 3.2.1 --- Isolation of the gut from Dom mouse embryos --- p.55 / Chapter 3.2.2 --- Whole mount immunohistochemistry --- p.55 / Chapter 3.3 --- Results --- p.57 / Chapter 3.3.1 --- PGP9.5 immunoreactivity in the 12.5 d.p.c. Dom embryos --- p.57 / Chapter 3.3.2 --- TH immunoreactivity in the 12.5 d.p.c. Dom embryos --- p.58 / Chapter 3.3.3 --- PGP9.5 immunoreactivity in the 14.5 d.p.c. Dom embryos --- p.59 / Figures and Tables / Chapter 3.4 --- Discussion --- p.61 / Chapter 3.4.1 --- The use of PGP9.5 and TH antibodies as markers for studying the migration of enteric neural crest-derived cells --- p.61 / Chapter 3.4.2 --- Incomplete migration of neural crest-derived cells within the gut of Dom heterozygous embryos --- p.62 / Chapter 3.4.3 --- Failure of sacral NCCs to invade the hindgut of Dom heterozygous embryos --- p.63 / Chapter 3.4.4 --- PGP9.5 and TH positive signals in the gut of Dom homozygous embryos --- p.64 / Chapter 3.4.5 --- Early differentiation of neural crest-derived cells into neurons due to haploinsufficiency of Sox10 --- p.65 / Chapter 3.4.6 --- Conclusions --- p.66 / Chapter Chapter 4 --- Localization of Interstitial Cells of Cajal in the Gut of Dominant Megacolon Mice --- p.67 / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2. --- Materials and Methods --- p.72 / Chapter 4.2.1 --- Isolation of the gut from mouse embryos and adult mice --- p.72 / Chapter 4.2.2 --- Cryosection and immunohistochemistry --- p.73 / Chapter 4.2.3 --- Whole-mount immunohistochemistry --- p.73 / Chapter 4.2.4 --- Total RNA extraction --- p.74 / Chapter 4.2.5 --- Reverse transcription for the first strand cDNA synthesis --- p.75 / Chapter 4.2.4 --- Reverse transcription-Polymerase chain reaction (RT-PCR) --- p.76 / Chapter 4.3 --- Results --- p.77 / Chapter 4.3.1 --- PGP9.5 and c-kit immunoreactivity in the Dom wild type colon --- p.77 / Chapter 4.3.2 --- c-kit immunoreactivity in the Dom heterozygous adult colon --- p.78 / Chapter 4.3.3 --- c-kit and SCF expression during gut development --- p.78 / Figures and Tables / Chapter 4.4 --- Discussion --- p.80 / Chapter 4.4.1 --- The importance in studying the development of ICCs in aganglionic gut --- p.80 / Chapter 4.4.2 --- ICCs development in Dominant megacolon mice --- p.81 / Chapter 4.4.3 --- The relationship between enteric neurons and ICCs development --- p.83 / Chapter 4.4.4 --- Advantages of using confocal microscopy and whole- mount preparations to study the ICCs development --- p.85 / Chapter 4.4.5 --- Conclusions --- p.86 / Chapter Chapter 5 --- General Discussion and Conclusions --- p.87 / References --- p.91