Tang, Walfred. / "November 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 151-165). / Abstracts in English and Chinese. / Title Page --- p.i / Thesis/Assessment Committee (English) --- p.ii / Acknowledgements --- p.iii / Table of Content --- p.iv / List of Figures --- p.ix / List of Graphs --- p.xi / List of Tables --- p.xiv / Abbreviations --- p.xviii / Abstract (English) --- p.xx / Abstract (Chinese) --- p.xxii / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Renal Development --- p.2 / Chapter 1.1.1 --- The three embryonic excretory systems --- p.2 / Chapter 1.1.1.1 --- Pronephros and mesonephros --- p.2 / Chapter 1.1.1.2 --- Metanephros --- p.4 / Chapter 1.1.2 --- Renal malformations --- p.6 / Chapter 1.1.2.1 --- Causes of renal malformations --- p.8 / Chapter 1.1.2.1.1 --- Physical obstruction --- p.9 / Chapter 1.1.2.1.2 --- Mutation --- p.9 / Chapter 1.1.2.1.3 --- Environmental insults --- p.10 / Chapter 1.2 --- Retinoic Acid --- p.11 / Chapter 1.2.1 --- "Retinoic acid synthesis, signaling and degradation in the Embryo" --- p.12 / Chapter 1.2.2 --- Retinoic acid and embryonic development --- p.14 / Chapter 1.2.2.1 --- Retinoic acid and renal development --- p.15 / Chapter 1.2.3 --- Retinoic acid teratogenicity --- p.17 / Chapter 1.2.3.1 --- Retinoic acid teratogenic mechanism --- p.18 / Chapter 1.2.3.2 --- Retinoic acid-induced renal agenesis mouse model --- p.21 / Chapter 1.2.4 --- Induction of RA deficiency --- p.24 / Chapter 1.3 --- Strategy of the Thesis --- p.28 / Chapter Chapter 2: --- General Materials and Methods / Chapter 2.1 --- Mouse Maintenance and Mating Method --- p.31 / Chapter 2.2 --- Bisdiamine Preparation --- p.32 / Chapter 2.3 --- All-trans Retinoic Acid Preparation --- p.32 / Chapter 2.4 --- Embryo Dissection --- p.32 / Chapter 2.5 --- Real-time Quantitative Reverse Transcription- Polymerase Chain Reaction (RT-PCR) --- p.33 / Chapter 2.5.1 --- Sample collection --- p.33 / Chapter 2.5.2 --- Total RNA extraction --- p.33 / Chapter 2.5.3 --- Reverse transcription --- p.34 / Chapter 2.5.4 --- Polymerase chain reaction --- p.35 / Chapter 2.5.5 --- Preparation of DNA standards --- p.36 / Chapter 2.6 --- Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-end Labeling (TUNEL) Staining --- p.38 / Chapter 2.6.1 --- "Fixation, dehydration and embedding" --- p.38 / Chapter 2.6.2 --- Microtome sectioning --- p.39 / Chapter 2.6.3 --- TUNEL staining --- p.39 / Chapter Chapter 3: --- Induction of Renal Malformations by Bisdiamine via RA Deficien --- p.cy / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.1.1 --- Time and dose responses to bisdiamine-induced renal malformations --- p.43 / Chapter 3.1.2 --- Methods to detect endogenous RA in embryonic tissues --- p.44 / Chapter 3.2 --- Experimental Design --- p.46 / Chapter 3.3 --- Materials and Methods --- p.48 / Chapter 3.3.1 --- Time and dose responses to bisdiamine administration --- p.48 / Chapter 3.3.2 --- Quantification of RA and retinol content in whole embryo by high pressure liquid chromatography (HPLC) --- p.49 / Chapter 3.3.2.1 --- Bisdiamine injection and sample collection --- p.49 / Chapter 3.3.2.2 --- Chromatographic system --- p.50 / Chapter 3.3.2.3 --- Preparation of standards --- p.50 / Chapter 3.3.2.4 --- Extraction of embryo samples --- p.51 / Chapter 3.3.2.5 --- Conditions of HPLC --- p.52 / Chapter 3.3.2.6 --- Recovery of sample --- p.53 / Chapter 3.3.2.7 --- Bradford protein assay --- p.53 / Chapter 3.3.3 --- X-gal staining of RARE-hsp-lacZ embryos --- p.54 / Chapter 3.3.4 --- Quantification of RA content in metanephroi by the RA-responsive cell line --- p.55 / Chapter 3.3.4.1 --- Bisdiamine injection and sample collection --- p.55 / Chapter 3.3.4.2 --- Maintenance of the RA-responsive cell line --- p.56 / Chapter 3.3.4.3 --- Seeding of cells and addition of samples --- p.57 / Chapter 3.3.4.4 --- X-gal staining --- p.58 / Chapter 3.3.5 --- TUNEL staining --- p.59 / Chapter 3.3.6 --- Real-time quantitative RT-PCR --- p.60 / Chapter 3.3.7 --- Statistical analysis --- p.61 / Chapter 3.4 --- Results --- p.63 / Chapter 3.4.1 --- Time response to bisdiamine treatment --- p.63 / Chapter 3.4.1.1 --- Bisdiamine administration increased resorption and affected various growth parameters of the fetuses --- p.64 / Chapter 3.4.1.2 --- Bisdiamine administration resulted in renal malformations --- p.68 / Chapter 3.4.1.3 --- Bisdiamine administration resulted in non-renal malformations --- p.71 / Chapter 3.4.2 --- Dose response to bisdiamine treatment --- p.76 / Chapter 3.4.2.1 --- Dose response of resorption and various growth parameters --- p.77 / Chapter 3.4.2.2 --- Dose response to bisdiamine in inducing renal malformations --- p.80 / Chapter 3.4.2.3 --- Dose response to non-renal malformations --- p.83 / Chapter 3.4.3 --- RA deficiency induced by bisdiamine --- p.88 / Chapter 3.4.3.1 --- Comparison of endogenous RA and retinol levels in control and bisdiamine-treated whole embryos at different time points after treatment --- p.88 / Chapter 3.4.3.2 --- Comparison of RA signaling patterns in control and bisdiamine-treated embryos at different time points after treatment --- p.90 / Chapter 3.4.3.3 --- Comparison of endogenous RA levels in control and bisdiamine-treated metanephroi at different time points after treatment --- p.93 / Chapter 3.4.4 --- Increase in the number of apoptotic nuclei in the metanephros after bisdiamine treatment --- p.95 / Chapter 3.4.5 --- Alteration of genes expression in the metanephros after bisdiamine treatment --- p.96 / Chapter 3.5 --- Discussion --- p.99 / Figures / Graphs / Chapter Chapter 4: --- Rescuing Bisdiamine-treated Metanephroi by In Vitro Supplementation with Low Concentrations of RA / Chapter 4.1 --- Introduction --- p.107 / Chapter 4.1.1 --- Embryonic kidney culture --- p.107 / Chapter 4.1.2 --- In vitro culture of the RA-treated metanephros --- p.108 / Chapter 4.1.3 --- Effect of exogenous retinoic acid on in vitro development of metanephros --- p.109 / Chapter 4.2 --- Experimental Design --- p.111 / Chapter 4.3 --- Materials and Methods --- p.113 / Chapter 4.3.1 --- Supplementation of low concentrations of RA to metanephric explant culture --- p.113 / Chapter 4.3.1.1 --- Preparation of culture medium supplemented with low concentrations of RA --- p.113 / Chapter 4.3.1.2 --- Metanephric explant culture --- p.114 / Chapter 4.3.2 --- Whole-mount immunohistochemical staining of ureteric epithelium and nephric tubules in metanephric explants --- p.115 / Chapter 4.3.3 --- TUNEL staining of metanephric explants --- p.116 / Chapter 4.3.4 --- Real-time quantitative RT-PCR --- p.117 / Chapter 4.3.5 --- Statistical analysis --- p.117 / Chapter 4.4 --- Results --- p.119 / Chapter 4.4.1 --- Rescue of bisdiamine-treated metanephric explants by in vitro culture in medium supplemented with low concentrations of RA --- p.119 / Chapter 4.4.1.1 --- Assessment of metanephric development under various concentrations of RA by morphological grading of UB tips at different day of culture --- p.119 / Chapter 4.4.1.2 --- Effect of various concentrations of RA on the number of UB tips and nephric tubules in metanephric explants at day 6 of culture --- p.126 / Chapter 4.4.2 --- Effect of RA supplementation on apoptosis in bisdiamine-treated metanephric explants --- p.131 / Chapter 4.4.3 --- Effect of RA supplementation on genes expression in bisdiamine-treated metanephric explants --- p.133 / Chapter 4.5 --- Discussion --- p.136 / Figures / Graphs / Chapter Chapter 5: --- Conclusion and Future Perspectives --- p.141 / References --- p.150