by Poon Lit Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 97-113). / Title page --- p.i / Acknowledgements --- p.ii / Table of contents --- p.iii / List of tables --- p.vii / List of figures --- p.viii / Abbreviations --- p.x / Abstract --- p.xi / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- Sacral agenesis --- p.2 / Chapter 1.1.1 --- Skeletal anomalies --- p.3 / Chapter 1.1.2 --- Neurological anomalies --- p.4 / Chapter 1.1.3 --- Other anomalies --- p.5 / Chapter 1.1.4 --- Etiology --- p.5 / Chapter 1.1.5 --- Pathogenetic mechanism of SA --- p.7 / Chapter 1.2 --- Retinoids --- p.8 / Chapter 1.2.1 --- RA in embryonic development --- p.9 / Chapter 1.2.2 --- Teratogenic effect of RA in embryonic development --- p.10 / Chapter 1.3 --- Strategy of the thesis --- p.13 / Chapter Chapter 2: --- General Materials and Methods --- p.15 / Chapter 2.1 --- Mouse maintenance and mating method --- p.16 / Chapter 2.2 --- All-trans RA preparation and injection --- p.16 / Chapter 2.3 --- Dissection of embryos --- p.16 / Chapter 2.4 --- Preparation of histological sections --- p.17 / Chapter 2.4.1 --- Dehydration and embedding --- p.17 / Chapter 2.4.2 --- Sectioning --- p.18 / Chapter 2.4.3 --- Haematoxylin and eosin staining --- p.18 / Chapter 2.5 --- Plasmid preparation --- p.19 / Chapter 2.5.1 --- Competent cells preparation --- p.19 / Chapter 2.5.2 --- Bacterial transformation --- p.19 / Chapter 2.5.3 --- Mini-scale preparation of plasmid DNA --- p.20 / Chapter 2.6 --- In situ hybridization --- p.21 / Chapter 2.6.1 --- Sample preparation --- p.21 / Chapter 2.6.2 --- Probe synthesis --- p.21 / Chapter 2.6.3 --- Hybridization --- p.23 / Chapter 2.6.4 --- Post-hybridization wash and antibody labeling --- p.24 / Chapter 2.6.5 --- Post-antibody wash and colour development --- p.25 / Chapter 2.6.6 --- Embryo powder preparation --- p.25 / Chapter Chapter 3: --- Time and Dose Responses to RA --- p.26 / Chapter 3.1 --- Introduction --- p.27 / Chapter 3.1.1 --- Time response --- p.27 / Chapter 3.1.2 --- Dose response --- p.29 / Chapter 3.2 --- Materials and methods --- p.31 / Chapter 3.2.1 --- Dose response --- p.31 / Chapter 3.2.2 --- Time response --- p.31 / Chapter 3.2.3 --- Skeletal preparations and staining --- p.31 / Chapter 3.2.4 --- Early teratogenic responses to RA --- p.32 / Chapter 3.3 --- Results --- p.33 / Chapter 3.3.1 --- Dose response --- p.33 / Chapter 3.3.1.1 --- Tail length --- p.33 / Chapter 3.3.1.2 --- Vertebral pattern --- p.33 / Chapter 3.3.2 --- Time response --- p.36 / Chapter 3.3.2.1 --- Tail length --- p.36 / Chapter 3.3.2.2 --- Vertebral pattern --- p.36 / Chapter 3.3.2.3 --- Other anomalies associated with human SA --- p.40 / Chapter 3.3.3 --- Early teratogenic responses in RA-treated embryos --- p.41 / Chapter 3.4 --- Discussion --- p.44 / Chapter Chapter 4: --- RA-induced Cell Death in Tail Bud --- p.51 / Chapter 4.1. --- Introduction --- p.52 / Chapter 4.1.1 --- Cell death --- p.52 / Chapter 4.1.2 --- Methods in cell death identification --- p.54 / Chapter 4.2 --- Materials and methods --- p.57 / Chapter 4.2.1 --- Histology of tail bud region --- p.57 / Chapter 4.2.2 --- Detection of internucleosomal DNA fragmentation --- p.57 / Chapter 4.2.2.1 --- Sample collection --- p.57 / Chapter 4.2.2.2 --- DNA extraction --- p.58 / Chapter 4.2.2.3 --- Analysis of DNA fragmentation pattern by ethidium bromide staining --- p.59 / Chapter 4.2.2.4 --- Analysis of DNA fragmentation pattern by autoradiography --- p.59 / Chapter 4.2.3 --- TUNEL --- p.61 / Chapter 4.2.3.1 --- Sample collection / Chapter 57 4.2.3.2 --- In situ end-labeling --- p.62 / Chapter 4.2.4 --- In situ hybridization of Wnt-5A --- p.63 / Chapter 4.2.4.1 --- Sample collection --- p.63 / Chapter 4.2.4.2 --- Probe preparation --- p.64 / Chapter 4.3 --- Results --- p.65 / Chapter 4.3.1 --- Histological examination of cell death in tail bud --- p.65 / Chapter 4.3.2 --- Analysis of DNA fragmentation of tail bud cells --- p.66 / Chapter 4.3.3 --- TUNEL --- p.67 / Chapter 4.3.4 --- Wnt-5A gene expression --- p.68 / Chapter 4.4 --- Discussion --- p.71 / Chapter Chapter 5: --- Retinoic Acid Receptors (RARs) --- p.75 / Chapter 5.1 --- Introduction --- p.76 / Chapter 5.1.1 --- RARs and their isoforms --- p.76 / Chapter 5.1.2 --- Expression pattern of RARs during embryogenesis --- p.77 / Chapter 5.1.3 --- RAR mutants and functional redundancy of RARs --- p.78 / Chapter 5.1.4 --- RARs and SA --- p.79 / Chapter 5.2 --- Materials and methods --- p.80 / Chapter 5.2.1 --- In situ hybridization of RAR-α --- p.80 / Chapter 5.2.1.1 --- Sample collection --- p.80 / Chapter 5.2.1.2 --- Probe preparation --- p.80 / Chapter 5.2.2 --- In situ hybridization of RAR-β --- p.81 / Chapter 5.2.2.1 --- Sample collection --- p.81 / Chapter 5.2.2.2 --- Probe preparation --- p.81 / Chapter 5.2.3 --- In situ hybridization of RAR-γ --- p.82 / Chapter 5.2.3.1 --- Sample collection --- p.82 / Chapter 5.2.3.2 --- Probe preparation --- p.82 / Chapter 5.3 --- Results --- p.83 / Chapter 5.3.1 --- RAR-α gene expression --- p.83 / Chapter 5.3.2 --- RAR-β gene expression --- p.84 / Chapter 5.3.3 --- RAR-γ gene expression --- p.86 / Chapter 5.4 --- Discussion --- p.88 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.92 / Chapter 6.1 --- Conclusion and future perspectives --- p.93 / References --- p.97 / Appendices --- p.114 / Figures --- p.118

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