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Leung, Chun Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 155-169). / Abstracts in English and Chinese. / Title Page --- p.i / Acknowledgements --- p.ii / Table of Content --- p.iii / List of Figures --- p.viii / List of Graphs --- p.x / List of Tables x --- p.iv / Abbreviations --- p.xvii / Abstract --- p.xviii / Abstract (Chinese) --- p.xx / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Introduction to retinoids --- p.2 / Chapter 1.2 --- Role of endogenous retinoic acid in embryonic development --- p.3 / Chapter 1.3 --- Regulation of retinoic acid in embryonic development --- p.5 / Chapter 1.3.1 --- Retinoic acid synthesis and degradation --- p.5 / Chapter 1.3.2 --- Retinoic acid signaling --- p.8 / Chapter 1.4 --- Effect of excess vitamin AJ RA on embryogenesis --- p.8 / Chapter 1.4.1 --- Examples of human animal studies --- p.9 / Chapter 1.4.2 --- Mechanisms of retinoid teratogenesis --- p.11 / Chapter 1.4.2.1 --- Apoptosis --- p.11 / Chapter 1.4.2.2 --- Altered proliferation --- p.12 / Chapter 1.4.2.3 --- Altered cell migration --- p.12 / Chapter 1.4.2.4 --- Altered differentiation --- p.13 / Chapter 1.4.3 --- Critical period of RA administration caused specific Malformations --- p.14 / Chapter 1.5 --- Effect of vitamin A/ RA deficiency on embryogenesis --- p.15 / Chapter 1.6 --- Excess and deficiency of RA cause similar types of malformations --- p.17 / Chapter 1.6.1 --- Retinoic acid-induced renal malformations mouse model --- p.18 / Chapter 1.7 --- Strategy of thesis --- p.19 / Chapter Chapter 2: --- General Materials and Methods / Chapter 2.1 --- Mouse maintenance and mating methods --- p.23 / Chapter 2.2 --- All-trans retinoic acid preparation and injection --- p.23 / Chapter 2.3 --- Whole mount in situ hybridization --- p.24 / Chapter 2.3.1 --- Riboprobe synthesis --- p.24 / Chapter 2.3.1.1 --- Bacterial culture --- p.24 / Chapter 2.3.1.2 --- DNA plasmids extraction --- p.24 / Chapter 2.3.1.3 --- Linearization of plasmid --- p.25 / Chapter 2.3.1.4 --- Purification of linearized plasmid --- p.26 / Chapter 2.3.1.5 --- In vitro transcription and labeling --- p.26 / Chapter 2.3.2 --- Sample collection --- p.27 / Chapter 2.3.3 --- Hybridization --- p.28 / Chapter 2.3.4 --- Post hybridization wash and antibody development --- p.29 / Chapter 2.3.4.1 --- Embryo powder preparation --- p.30 / Chapter 2.3.4.2 --- Pre-absorption of antibody --- p.30 / Chapter 2.3.5 --- Post-antibody and staining --- p.31 / Chapter 2.4 --- Real-time quantitative reverse transcription -polymerase chain reaction (RT-PCR) --- p.32 / Chapter 2.4.1 --- Sample collection --- p.32 / Chapter 2.4.2 --- RNA extraction --- p.32 / Chapter 2.4.3 --- Reverse transcription into cDNA --- p.33 / Chapter 2.4.4 --- Quantitative real-time PCR --- p.33 / Chapter 2.4.5 --- Preparation of cDNA standards --- p.34 / Chapter 2.5 --- High pressure liquid chromatography (HPLC) --- p.35 / Chapter 2.5.1 --- Chromatographic system --- p.35 / Chapter 2.5.2 --- Standards preparation --- p.35 / Chapter 2.5.3 --- Embryo sample collection and preparation --- p.36 / Chapter 2.5.4 --- HPLC conditions --- p.36 / Chapter 2.5.5 --- Sample recovery --- p.37 / Chapter 2.5.6 --- Bradford assay --- p.38 / Chapter 2.6 --- RA-responsive cell line --- p.38 / Chapter 2.6.1 --- Cell culture --- p.39 / Chapter 2.6.2 --- Seeding and loading sample to 96-well plate --- p.40 / Chapter 2.6.3 --- X-gal staining --- p.41 / Chapter Chapter 3: --- Time and Dose Responses to RA / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.1.1 --- Time response to RA --- p.43 / Chapter 3.1.2 --- Dose response to RA --- p.45 / Chapter 3.1.3 --- Other factors affecting susceptibilities to RA --- p.46 / Chapter 3.2 --- Experimental design --- p.48 / Chapter 3.3 --- Materials and methods --- p.50 / Chapter 3.3.1 --- Time response to RA --- p.50 / Chapter 3.3.2 --- Dose response to RA --- p.50 / Chapter 3.3.3 --- Examination of fetuses --- p.51 / Chapter 3.3.4 --- Statistical analysis --- p.51 / Chapter 3.4 --- Results --- p.53 / Chapter 3.4.1 --- Time response --- p.53 / Chapter 3.4.1.1 --- Time response to RA-induced resorption --- p.53 / Chapter 3.4.1.2 --- Time response to RA-induced renal malformations --- p.54 / Chapter 3.4.1.3 --- Time response to RA-induced changes in growth parameters --- p.57 / Chapter 3.4.1.4 --- Time response to RA-induced non-renal malformations --- p.60 / Chapter 3.4.2 --- Dose response --- p.64 / Chapter 3.4.2.1 --- Dose response to RA-induced resorption --- p.64 / Chapter 3.4.2.2 --- Dose response to RA-induced renal malformations --- p.65 / Chapter 3.4.2.3 --- Dose response to RA-induced changes in growth parameters --- p.68 / Chapter 3.4.2.4 --- Dose response to RA-induced non-renal malformations --- p.71 / Chapter 3.5 --- Discussion --- p.74 / Chapter Chapter 4: --- Effect of Teratogenic Dose of RA on RA Synthesis and Endogenous RA Levels in the Embryo / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.1.1 --- RA synthesis in embryo --- p.79 / Chapter 4.1.2 --- Detection of endogenous RA in embryo --- p.81 / Chapter 4.2 --- Experimental design --- p.83 / Chapter 4.3 --- Materials and methods --- p.84 / Chapter 4.3.1 --- Localization of mRNA transcripts in whole embryo by in situ hybridization --- p.84 / Chapter 4.3.2 --- Vibratome sectioning --- p.85 / Chapter 4.3.2.1 --- Preparation of Gloop --- p.85 / Chapter 4.3.2.2 --- Sample preparation and sectioning --- p.85 / Chapter 4.3.3 --- Quantification of mRNA expression levels in whole embryo and in metanephros by real-time RT-PCR --- p.86 / Chapter 4.3.4 --- Detection of RA levels in whole embryo by HPLC --- p.87 / Chapter 4.3.5 --- Detection of RA levels in metanephros by RA-responsive cell line --- p.87 / Chapter 4.3.6 --- Statistical analysis --- p.88 / Chapter 4.4 --- Results --- p.89 / Chapter 4.4.1 --- Comparison of mRNA expression levels of different iso forms of RA synthesizing enzymes Raldh and RA catabolizing enzymes Cyp26 between embryos of RA-treated and vehicle-treated control mice at various time points after treatment --- p.89 / Chapter 4.4.2 --- Comparison of mRNA expression levels of different iso forms of RA synthesizing enzymes Raldh and RA catabolizing enzymes Cyp26 between metanephroi of embryos of RA-treated and vehicle-treated control mice at various time points after treatment --- p.93 / Chapter 4.4.3 --- Comparison of the in situ hybridization pattern of different iso forms of Raldh between embryos of RA-treated and vehicle-treated control mice at different time points after treatment --- p.95 / Chapter 4.4.3.1 --- In situ hybridization pattern of Raldh 1 --- p.96 / Chapter 4.4.3.2 --- In situ hybridization pattern of Raldh2 --- p.97 / Chapter 4.4.3.3 --- In situ hybridization pattern of Raldh3 --- p.100 / Chapter 4.4.4 --- Comparison of the in situ hybridization pattern of Cyp26al and Cyp26bl between embryos of RA-treated and vehicletreated control mice at different time points after treatment --- p.101 / Chapter 4.4.4.1 --- In situ hybridization pattern of Cyp26al --- p.101 / Chapter 4.4.4.2 --- In situ hybridization pattern of Cyp26bl --- p.102 / Chapter 4.4.5 --- Comparison of RA levels between embryos of RA-treated and vehicle-treated control mice at different time points after treatment --- p.103 / Chapter 4.4.6 --- Comparison of RA levels between metanephroi of embryos of RA-treated and vehicle-treated control mice at different time points after treatment --- p.105 / Chapter 4.5 --- Discussion --- p.106 / Chapter Chapter 5: --- Effect of Supplementation with Low Doses of RA on RA Teratogenesis / Chapter 5.1 --- Introduction --- p.111 / Chapter 5.1.1 --- RA supplementation --- p.111 / Chapter 5.1.2 --- Wilms' tumor suppressor gene Wtl --- p.112 / Chapter 5.1.3 --- Apoptosis --- p.113 / Chapter 5.2 --- Experimental design --- p.115 / Chapter 5.3 --- Materials and methods --- p.117 / Chapter 5.3.1 --- Oral gavage of low dose of RA --- p.117 / Chapter 5.3.2 --- Determination of Wtl expression level by real-time quantitative RT-PCR --- p.117 / Chapter 5.3.3 --- Preparation of paraffin sections and TUNEL staining --- p.118 / Chapter 5.3.3.1 --- Sample collection --- p.118 / Chapter 5.3.3.2 --- "Dehydration, embedding and sectioning" --- p.118 / Chapter 5.3.3.3 --- TUNEL staining --- p.119 / Chapter 5.3.4 --- Statistical analysis --- p.121 / Chapter 5.4 --- Results --- p.122 / Chapter 5.4.1 --- Time response to RA supplementation in rescuing kidney development --- p.122 / Chapter 5.4.2 --- Dose response to RA supplementation in rescuing kidney development --- p.127 / Chapter 5.4.3 --- RA supplementation restored various growth parameters --- p.132 / Chapter 5.4.4 --- RA supplementation rescued non-renal malformations --- p.134 / Chapter 5.4.5 --- Wtl expression in the metanephros after RA supplementation --- p.142 / Chapter 5.4.6 --- Apoptotic cell death in the metanephros after RA supplementation --- p.143 / Chapter 5.5 --- Discussion --- p.145 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.150 / References --- p.155 / Figures / Graphs

In two separate experiments, weight adjusted doses of nocodazole and acrylamide were injected intraperitoneally at various time intervals into twelve week old female mice. Within the nocodazole experiment, the doses were injected at varying time intervals before and after mating. On day seventeen of gestation, the female mice were sacrificed and their uterine contents examined. Nocodazole induced a significant increase in reproductive pathology per total implants when administered one hour after mating to the (SECxC57BL)F, stock: 5.00% total deads, 70.23% moles, and 3.41% abnormal fetuses. Acrylamide treatment produced a significant reduction in live births when administered six hours after mating: 50.86% moles and 46.46% living fetuses per total implants.

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Previous research has shown a relationship between prenatal teratogenic exposure and impaired cognitive functioning. However, data regarding the long-term outcome of prenatal teratogenic exposure are minimal. The present study investigated the long-term neuropsychological functioning (specifically attention and memory) of adults prenatally exposed to alcohol or phenylalanine, and examined whether there was evidence to suggest that there are effects specific to individual teratogens. Using a battery of attention and memory measures the performance of 17 adults diagnosed with Fetal Alcohol Syndrome (FAS) and 13 adults with Maternal Phenylketonuria Syndrome (MPKUS) was assessed. In order to identify the pattern of deficits associated with prenatal teratogenic exposure, an age and CA and IQ matched control group was assessed. Attention was broadly assessed using Mirsky et al.'s (1991) neuropsychological model of attention. The memory and learning tests administered included a number of well standardized measures of verbal learning, verbal and visual recall, delayed recall, and recognition. Paired comparisons between the FAS group and age and CA and IQ matched controls indicated a unique pattern of attention and memory deficits consistent with previous research with children and adolescents. Specifically, adult individuals with FAS appear to have deficits in acquisition of new material, delayed recall of verbal material and in response inhibition. Paired comparisons between the MPKUS group and CA and IQ matched controls indicated that the pattern of attention and memory deficits seen in adults with MPKUS is difficult to distinguish when the effect of IQ is removed. A randomized block design using IQ as the blocking variable and group (FAS, MPKUS, or Controls) as the treatment variable was utilized to examine the question of whether the two prenatal teratogen groups differ from one another and from Controls in terms of attention and memory ability. Ten blocks of three participants (FAS, MPKUS and Control) matched on IQ were formed. The randomized block analyses revealed few differences between the groups and failed to reveal a number of the differences found in the paired comparisons between the prenatal teratogen groups and the CA and IQ matched Control group. Possible reasons for these differences are discussed.

Although there have been two-dimensional electrophoretic studies on fetal brain tissue (for instance, Yoshida and Takahashi, 1980), the emphasis in most of this work has been on developmental changes in protein expression, and not on the effects that drugs have on fetal brain protein complement. Klose and co-workers (1977) did an early study using two-dimensional gel electrophoresis to determine the effects of various teratogens on whole embryos. No protein changes were found and that line of research was not continued. In this study two-dimensional gel electrophoresis is extensively used, in the belief that the usefulness of this technique to experimental teratology has not been fully evaluated. It is reasonable to suppose that a central nervous system teratogen administered during critical periods of susceptibility will led to perturbations of orderly brain development, and that these perturbations will be reflected as changes to the protein complement. The total brain protein complement of mice that have been exposed to drugs in utero will therefore be analysed, in the hope that any inductions or deletions of proteins as a result of drug exposure may provide a clue to the molecular events underlying drug injury to the fetus.

Indiana University-Purdue University Indianapolis (IUPUI) / Fanconi anemia (FA) is a heterogenous genetic syndrome characterized by progressive bone marrow failure, aneuploidy, and cancer predisposition. It is incompletely understood why FA-deficient cells develop gross aneuploidy leading to cancer. Since the mitotic spindle assembly checkpoint (SAC) prevents aneuploidy by ensuring proper chromosome segregation during mitosis, we hypothesized that the FA signaling network regulates the mitotic SAC. A genome-wide RNAi screen and studies in primary cells were performed to systematically evaluate SAC activity in FA-deficient cells. In these experiments, taxol was used to activate the mitotic SAC. Following taxol challenge, negative control siRNA-transfected cells appropriately arrested at the SAC. However, knockdown of fourteen FA gene products resulted in a weakened SAC, evidenced by increased formation of multinucleated, aneuploid cells. The screen was independently validated utilizing primary fibroblasts from patients with characterized mutations in twelve different FA genes. When treated with taxol, fibroblasts from healthy controls arrested at the mitotic SAC, while all FA patient fibroblasts tested exhibited weakened SAC activity, evidenced by increased multinucleated cells. Rescue of the SAC was achieved in FANCA patient fibroblasts by genetic correction. Importantly, SAC activity of FANCA was confirmed in primary CD34+ hematopoietic cells. Furthermore, analysis of untreated primary fibroblasts from FA patients revealed micronuclei and multinuclei, reflecting abnormal chromosome segregation. Next, microscopy-based studies revealed that many FA proteins localize to the mitotic spindle and centrosomes, and that disruption of the FA pathway results in supernumerary centrosomes, establishing a role for the FA signaling network in centrosome maintenance. A mass spectrometry-based screen quantifying the proteome and phospho-proteome was performed to identify candidates which may functionally interact with FANCA in the regulation of mitosis. Finally, video microscopy-based experiments were performed to further characterize the mitotic defects in FANCA-deficient cells, confirming weakened SAC activity in FANCA-deficient cells and revealing accelerated mitosis and abnormal spindle orientation in the absence of FANCA. These findings conclusively demonstrate that the FA signaling network regulates the mitotic SAC, providing a mechanistic explanation for the development of aneuploidy and cancer in FA patients. Thus, our study establishes a novel role for the FA signaling network as a guardian of genomic integrity.