Pathogenesis of sacral agenesis studied using a mouse model.

by Poon Lit Man.Thesis (M.Phil.)--Chinese University of Hong Kong, 1996.Includes bibliographical references (leaves 97-113).Title page --- p.iAcknowledgements --- p.iiTable of contents --- p.iiiList of tables --- p.viiList of figures --- p.viiiAbbreviations --- p.xAbstract --- p.xiChapter Chapter 1: --- General Introduction --- p.1Chapter 1.1 --- Sacral agenesis --- p.2Chapter 1.1.1 --- Skeletal anomalies --- p.3Chapter 1.1.2 --- Neurological anomalies --- p.4Chapter 1.1.3 --- Other anomalies --- p.5Chapter 1.1.4 --- Etiology --- p.5Chapter 1.1.5 --- Pathogenetic mechanism of SA --- p.7Chapter 1.2 --- Retinoids --- p.8Chapter 1.2.1 --- RA in embryonic development --- p.9Chapter 1.2.2 --- Teratogenic effect of RA in embryonic development --- p.10Chapter 1.3 --- Strategy of the thesis --- p.13Chapter Chapter 2: --- General Materials and Methods --- p.15Chapter 2.1 --- Mouse maintenance and mating method --- p.16Chapter 2.2 --- All-trans RA preparation and injection --- p.16Chapter 2.3 --- Dissection of embryos --- p.16Chapter 2.4 --- Preparation of histological sections --- p.17Chapter 2.4.1 --- Dehydration and embedding --- p.17Chapter 2.4.2 --- Sectioning --- p.18Chapter 2.4.3 --- Haematoxylin and eosin staining --- p.18Chapter 2.5 --- Plasmid preparation --- p.19Chapter 2.5.1 --- Competent cells preparation --- p.19Chapter 2.5.2 --- Bacterial transformation --- p.19Chapter 2.5.3 --- Mini-scale preparation of plasmid DNA --- p.20Chapter 2.6 --- In situ hybridization --- p.21Chapter 2.6.1 --- Sample preparation --- p.21Chapter 2.6.2 --- Probe synthesis --- p.21Chapter 2.6.3 --- Hybridization --- p.23Chapter 2.6.4 --- Post-hybridization wash and antibody labeling --- p.24Chapter 2.6.5 --- Post-antibody wash and colour development --- p.25Chapter 2.6.6 --- Embryo powder preparation --- p.25Chapter Chapter 3: --- Time and Dose Responses to RA --- p.26Chapter 3.1 --- Introduction --- p.27Chapter 3.1.1 --- Time response --- p.27Chapter 3.1.2 --- Dose response --- p.29Chapter 3.2 --- Materials and methods --- p.31Chapter 3.2.1 --- Dose response --- p.31Chapter 3.2.2 --- Time response --- p.31Chapter 3.2.3 --- Skeletal preparations and staining --- p.31Chapter 3.2.4 --- Early teratogenic responses to RA --- p.32Chapter 3.3 --- Results --- p.33Chapter 3.3.1 --- Dose response --- p.33Chapter 3.3.1.1 --- Tail length --- p.33Chapter 3.3.1.2 --- Vertebral pattern --- p.33Chapter 3.3.2 --- Time response --- p.36Chapter 3.3.2.1 --- Tail length --- p.36Chapter 3.3.2.2 --- Vertebral pattern --- p.36Chapter 3.3.2.3 --- Other anomalies associated with human SA --- p.40Chapter 3.3.3 --- Early teratogenic responses in RA-treated embryos --- p.41Chapter 3.4 --- Discussion --- p.44Chapter Chapter 4: --- RA-induced Cell Death in Tail Bud --- p.51Chapter 4.1. --- Introduction --- p.52Chapter 4.1.1 --- Cell death --- p.52Chapter 4.1.2 --- Methods in cell death identification --- p.54Chapter 4.2 --- Materials and methods --- p.57Chapter 4.2.1 --- Histology of tail bud region --- p.57Chapter 4.2.2 --- Detection of internucleosomal DNA fragmentation --- p.57Chapter 4.2.2.1 --- Sample collection --- p.57Chapter 4.2.2.2 --- DNA extraction --- p.58Chapter 4.2.2.3 --- Analysis of DNA fragmentation pattern by ethidium bromide staining --- p.59Chapter 4.2.2.4 --- Analysis of DNA fragmentation pattern by autoradiography --- p.59Chapter 4.2.3 --- TUNEL --- p.61Chapter 4.2.3.1 --- Sample collectionChapter 57 4.2.3.2 --- In situ end-labeling --- p.62Chapter 4.2.4 --- In situ hybridization of Wnt-5A --- p.63Chapter 4.2.4.1 --- Sample collection --- p.63Chapter 4.2.4.2 --- Probe preparation --- p.64Chapter 4.3 --- Results --- p.65Chapter 4.3.1 --- Histological examination of cell death in tail bud --- p.65Chapter 4.3.2 --- Analysis of DNA fragmentation of tail bud cells --- p.66Chapter 4.3.3 --- TUNEL --- p.67Chapter 4.3.4 --- Wnt-5A gene expression --- p.68Chapter 4.4 --- Discussion --- p.71Chapter Chapter 5: --- Retinoic Acid Receptors (RARs) --- p.75Chapter 5.1 --- Introduction --- p.76Chapter 5.1.1 --- RARs and their isoforms --- p.76Chapter 5.1.2 --- Expression pattern of RARs during embryogenesis --- p.77Chapter 5.1.3 --- RAR mutants and functional redundancy of RARs --- p.78Chapter 5.1.4 --- RARs and SA --- p.79Chapter 5.2 --- Materials and methods --- p.80Chapter 5.2.1 --- In situ hybridization of RAR-α --- p.80Chapter 5.2.1.1 --- Sample collection --- p.80Chapter 5.2.1.2 --- Probe preparation --- p.80Chapter 5.2.2 --- In situ hybridization of RAR-β --- p.81Chapter 5.2.2.1 --- Sample collection --- p.81Chapter 5.2.2.2 --- Probe preparation --- p.81Chapter 5.2.3 --- In situ hybridization of RAR-γ --- p.82Chapter 5.2.3.1 --- Sample collection --- p.82Chapter 5.2.3.2 --- Probe preparation --- p.82Chapter 5.3 --- Results --- p.83Chapter 5.3.1 --- RAR-α gene expression --- p.83Chapter 5.3.2 --- RAR-β gene expression --- p.84Chapter 5.3.3 --- RAR-γ gene expression --- p.86Chapter 5.4 --- Discussion --- p.88Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.92Chapter 6.1 --- Conclusion and future perspectives --- p.93References --- p.97Appendices --- p.114Figures --- p.11