by Pui-kuen Lee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 168-182). / ACKNOWLEDGMENT --- p.I / ABSTRACT --- p.II / TABLE OF CONTENTS --- p.IV / ABBREVIATIONS --- p.X / LIST OF FIGURES --- p.XII / LIST OF TABLES --- p.XIV / Chapter CHAPTER1 --- INTRODUCTION AND BACKGROUND --- p.1 / Chapter 1.1 --- ROLE OF CYTOKINES IN MOUSE EMBRYONIC DEVELOPMENT --- p.1 / Chapter 1.1.1 --- Why mouse model --- p.1 / Chapter 1.1.2 --- Embryonic development of mouse --- p.1 / Chapter 1.1.3 --- An overview of cytokines --- p.4 / Chapter a. --- Classes of cytokines --- p.5 / Chapter i) --- Growth factors --- p.5 / Chapter ii) --- Interleukins --- p.7 / Chapter iii) --- Colony-stimulating factors --- p.9 / Chapter iv) --- Interferons --- p.10 / Chapter v) --- Tumor necrosis factor --- p.11 / Chapter b. --- Cytokine networks --- p.12 / Chapter c. --- Role of cytokines in the whole organism --- p.13 / Chapter 1.1.4 --- Cytokine and receptor gene expression in mouse embryonic development --- p.15 / Chapter a. --- Murine embryonic stem cell model --- p.15 / Chapter b. --- Leukemia Inhibitory Factor (LIF) in mouse embryos --- p.16 / Chapter c. --- IL-6 in mouse embryo --- p.19 / Chapter d. --- Ciliary Neurotrophic Factor (CNTF) in mouse embryo --- p.19 / Chapter e. --- TNF-a and TNF-β in mouse embryos --- p.20 / Chapter f. --- TGF-a in mouse embryos --- p.20 / Chapter g. --- TGF-P in mouse embryos --- p.20 / Chapter h. --- Stem cell factor / c-kit --- p.21 / Chapter i. --- Other cytokines in mouse embryos --- p.22 / Chapter j. --- Cytokine receptors --- p.24 / Chapter 1.2 --- NEUROPOIETIC CYTOKINES --- p.28 / Chapter 1.2.1 --- Family members --- p.28 / Chapter 1.2.2 --- Shared signal transducer gpl30 --- p.29 / Chapter 1.2.3 --- "LIF, CNTF and OSM inhibit differentiation of embryonic stem cells" --- p.31 / Chapter 1.3 --- BIOLOGY OF ONCOSTATIN M (OSM) --- p.33 / Chapter 1.3.1 --- Physical properties of OSM --- p.33 / Chapter 1.3.2 --- Biological activities of OSM --- p.34 / Chapter 1.3.3 --- Molecular aspect of OSM --- p.35 / Chapter 1.4 --- AIMS OF THE STUDY --- p.38 / Chapter CHAPTER2 --- CYTOKINE GFNE EXPRESSION DURING MOUSE EMBRYONIC DEVELOPMENT --- p.40 / Chapter 2.1 --- INTRODUCTION --- p.40 / Chapter 2.1.1 --- Rationale --- p.40 / Chapter 2.1.2 --- Design of primers --- p.43 / Chapter 2.2 --- MATERIALS --- p.44 / Chapter 2.2.1 --- Chemicals and Reagents --- p.44 / Chapter 2.2.2 --- Enzymes --- p.45 / Chapter 2.2.3 --- Buffers --- p.45 / Chapter 2.2.4 --- Solutions --- p.47 / Chapter 2.2.5 --- Probe labeling and detection kits --- p.48 / Chapter 2.2.6 --- Primers and internal probes --- p.49 / Chapter 2.3 --- METHODS --- p.52 / Chapter 2.3.1 --- Preparation of total RNA from mouse embryos at different stages --- p.52 / Chapter a. --- Mice dissection for embryo --- p.52 / Chapter b. --- Guanidinium thiocyanate cell lysate --- p.52 / Chapter c. --- Isolation of RNA by centrifugation through CsCl gradient --- p.53 / Chapter d. --- Spectrophotometric determination of RNA amount --- p.54 / Chapter 2.3.2 --- Preparation of embryo sections --- p.54 / Chapter 2.3.3 --- Primers and internal probes --- p.55 / Chapter 2.3.4 --- Cytokine mRNA Phenotyping by Reverse transcription-Polymerase chain reaction --- p.56 / Chapter a. --- Reverse transcription (First strand cDNA synthesis) --- p.56 / Chapter b. --- Polymerase chain reaction (PCR) --- p.56 / Chapter 2.3.5 --- Analysis of PCR products with agarose gel electrophoresis --- p.57 / Chapter 2.3.6 --- Analysis of PCR products with Southern blotting --- p.58 / Chapter a. --- DNA transfer from gel to nylon membrane --- p.58 / Chapter b. --- Probe labeling --- p.61 / Chapter c. --- Prehybridization --- p.61 / Chapter d. --- Hybridization --- p.62 / Chapter e. --- Detection of DIG-labeled probe --- p.62 / Chapter 2.3.7 --- Cycle titration of PCR and dot blotting of regulatory cytokine mRNA --- p.63 / Chapter a. --- Cycle titration of PCR --- p.63 / Chapter b. --- Dot blotting --- p.63 / Chapter 2.4 --- RESULTS --- p.65 / Chapter 2.4.1 --- Sagittal sections of mouse embryos --- p.65 / Chapter 2.4.2 --- Preparation of total RNA --- p.69 / Chapter 2.4.3 --- Cytokine mRNA phenotyping --- p.71 / Chapter a. --- Southern hybridization for 'no expression' cytokines --- p.74 / Chapter b. --- Consistent' and 'regulatory ´ة cytokines in embryo and placenta --- p.79 / Chapter 2.5 --- DISCUSSION --- p.95 / Chapter 2.5.1 --- Isolation of embryo RNA by guanidinium thiocyanate/ cesium chloride centrifugation --- p.95 / Chapter 2.5.2 --- mRNA Quantitation --- p.96 / Semi-quantitative PCR --- p.98 / Chapter 2.5.3 --- Cytokine mRNA phenotyping by RT-PCR --- p.99 / Chapter a. --- Reverse Transcription --- p.99 / Chapter b. --- GAPDH as a control for normalization --- p.100 / Chapter c. --- PCR for cytokine transcripts --- p.101 / Chapter 2.5.4 --- Cytokines and receptors in embryonic development --- p.103 / Chapter 2.5.4.1 --- Cytokines in hematopoietic development of mouse fetus --- p.104 / Chapter 2.5.4.2 --- Other cytokines --- p.113 / Chapter 2.5.5 --- Expression Pattern in placenta: maternal and fetal communication --- p.116 / Chapter CHAPTER3 --- MOLECULAR ANALYSTS OF MOUSE ONCOSTATIN M --- p.117 / Chapter 3.1 --- INTRODUCTION --- p.117 / Chapter 3.2 --- MATERIALS --- p.121 / Chapter 3.2.1 --- Chemicals and Reagents --- p.121 / Chapter 3.2.2 --- Enzymes --- p.121 / Chapter 3.2.3 --- Buffers --- p.122 / Chapter 3.2.4 --- Solutions --- p.122 / Chapter 3.2.5 --- Culture media --- p.124 / Chapter 3.2.6 --- Competent cell --- p.125 / Chapter 3.2.7 --- DNA materials --- p.125 / Chapter 3.2.8 --- Primers --- p.126 / Chapter 3.3 --- METHODS --- p.127 / Chapter 3.3.1 --- Primers and internal probes --- p.127 / Chapter 3.3.2 --- Cloning of human Oncostatin M exon 2 and exon 3 by PCR --- p.127 / Chapter 3.3.3 --- Subcloning of human OSM exons 2 and 3 into pUC18 --- p.128 / Chapter a. --- Preparation of human OSM exons and plasmid --- p.128 / Chapter i) --- Purification of PCR products --- p.128 / Chapter ii) --- T4 DNA polymerase ´بblunt-end´ة reaction for PCR products --- p.129 / Chapter iii) --- Sma I digestion of pUC18 --- p.129 / Chapter b. --- Ligation --- p.129 / Chapter c. --- Preparation of competent cell --- p.130 / Chapter d. --- Transformation --- p.131 / Chapter e. --- Screening of recombinants by PCR --- p.131 / Chapter f. --- Screening of recombinants by restriction enzyme digestion --- p.132 / Chapter i) --- Preparation of plasmids --- p.132 / Chapter ii) --- Double restriction enzymes digestion of pUC18 --- p.133 / Chapter 3.3.4 --- Verification of the clones of human OSM exons 2 and 3 by cycle sequencing --- p.135 / Chapter 3.3.5 --- Purification of human OSM exons from plasmid for making probe --- p.136 / Chapter 3.3.6 --- Southern blotting --- p.136 / Chapter a. --- Probe making and labeling --- p.136 / Chapter b. --- Preparation of mouse genomic DNAs --- p.137 / Chapter c. --- DNA transfer --- p.138 / Chapter i) --- Digestion of genomic DNA with restriction endonucleases --- p.138 / Chapter ii) --- Gel electrophoresis and DNA blotting --- p.139 / Chapter d. --- Hybridization --- p.139 / Chapter 3.4 --- RESULTS --- p.142 / Chapter 3.4.1 --- Cloning of human OSM exon 2 and exon 3 by PCR --- p.142 / Chapter 3.4.2 --- Subcloning of human OSM exons 2 and 3 into pUC18 --- p.142 / Chapter a. --- Screening of recombinants by PCR --- p.142 / Chapter b. --- Screening of recombinants by restriction enzymes digestion --- p.143 / Chapter 3.4.3 --- Sequence of subcloned exons 2 and3 --- p.147 / Chapter 3.4.4 --- Southern hybridization --- p.149 / Chapter a. --- Genomic DNA preparation --- p.149 / Chapter b. --- Digestion of genomic DNAs --- p.151 / Chapter c. --- Hybridization signal --- p.154 / Chapter 3.5 --- DISCUSSION --- p.158 / Chapter 3.5.1 --- Cross-species hybridization --- p.158 / Chapter 3.5.2 --- Hybridization of human OSM exon fragments against mouse genome --- p.158 / Chapter a. --- hOSM exon 2 as probe --- p.158 / Chapter b. --- hOSM exon 3 as probe --- p.160 / Chapter c. --- Feasibility of using hOSM as probe for fishing out the mOSM gene --- p.160 / Chapter d. --- The cloning of mouse OSM by Yoshimura's group --- p.161 / Chapter CHAPTER4 --- CONCLUSION --- p.162 / Chapter 4.1 --- SUMMARY OF CYTOKINE AND CYTOKINE RECEPTOR GENES EXPRESSION DURING EMBRYONIC DEVELOPMENT --- p.162 / Chapter 4.2 --- FURTHER STUDIES OF THE CYTOKINE ACTIONS ON EMBRYOGENESIS --- p.165 / Chapter 4.3 --- MOLECULAR ANALYSIS OF MOUSE OSM GENE --- p.167 / REFERENCES --- p.168

No description available.

Cited2 is a member of the Cited gene family, which has no homology to any other genes. It encodes a transcriptional co-factor that is expressed during early heart formation (cardiogenesis). Embryos lacking Cited2 display a range of cardiac defects including bilaterally identical atria, aortic arch abnormalities, rotation of the aorta and pulmonary artery, and malseptation of the cardiac chambers. The latter results in communication between the aorta and pulmonary artery, the aorta and both ventricles, and the atria and ventricles (with themselves and each other). Cardiogenesis is complex, and requires many different cell types and processes to occur correctly. Some of these cells and processes are external to the primary heart. For example, once the initial muscle cells of the heart form a tube, cells from other regions such as the secondary heart field (adjacent mesoderm) and cardiac neural crest (ectoderm) migrate into this tube, and are required for the formation of additional muscle cells and septa. Furthermore, cardiogenesis also requires correct left-right patterning of the embryo to be established prior to heart formation. To understand the developmental origins of the cardiac defects observed in Cited2-null embryos, the expression pattern of Cited2 and the anatomy of Cited2-null embryo hearts were studied. Subsequently, the expression of genes required for left-right patterning were studied in both Cited2-null and Cited2 conditionally-deleted embryos. This demonstrated that Cited2 may be required in many, possibly all, of the processes required for cardiogenesis. Next this study focused on the role of Cited2 in patterning the left-right axis of the embryo. Firstly, Cited2 was found to regulate the expression of the master regulator of left-right patterning (Nodal). Secondly, Cited2 was shown to regulate the expression of the left-specific transcription factor Pitx2 independently of Nodal. Thirdly, gene expression and conditional deletions of Cited2 suggested that Cited2 might regulate left-right patterning in the paraxial mesoderm, a tissue which has not previously been shown to regulate the left-right axis in the mouse. Lastly, an argument is made suggesting the possibility that all the cardiac defects found in Cited2-null embryos may directly or indirectly stem from a failure of correct left-right patterning.

The proteins and molecular machinery mediating the release of cortical granule (CG) contents from fertilized embryos is not completely understood. The process of vesicle fusion involves linking chaperones prior to vesicle to membrane contact. Rab3A, a member of a low-molecular weight GTP-binding protein superfamily has been detected in mouse embryos from the unfertilized meiotic II stage to the 2-cell. It is believed to positively regulate the final step of CG exocytosis by binding to Rabphillin, calcium ions (Ca2+), and phospholipids. SNAP23 a member of soluble NSF [N-ethylmaleimidesensitive factor] attachment protein receptors (SNAREs) binds together with parts of the Rab3A-rabphilin3A complex and is believed to be involved in the Ca2+-dependent exocytosis of non-neuronal systems. In this study we observed the mRNA expression for SNAP23 and Rab3A in pre-Meiotic I, post-Meiotic I unfertilized eggs (pre-MI UFE and post-MI UFE), and fertilized eggs (FE) utilizing RT-PCR. The products were analyzed in 2% agarose gel stained with ethidium bromide. Density analysis using a globin external standard showed that the levels of mRNA transcripts declined from the UFE to the FE in both genes, SNAP23 and Rab3A. Immunofluorescence was used for the detection and localization of Rab3A protein within the pre-MI and post-MI UFE and FE mouse egg. Eggs were stained with anti-Rab3A primary antibody and lens culinaris agglutinin (LCA) conjugated to FITC. Rab3A showed punctate staining in pre- and post-MI UFEs on small vesicles assumed to be CGs and in FEs on vesicles of a larger size. Uniform cytoplasmic expression was also seen, throughout the cells cortical and subcortical regions in each stage (pre- and post-MI UFEs and FEs), but with decreasing intensity as the eggs matured. This cytoplasmic stain may represent inactive Rab3A in the cytosol. The LCA stain showed punctate expression of cortical granules with localization within the cortical region and the plasma membrane. The addition of information on SNAP23 and Rab3A will aid in the process of studying CG exocytosis as well as in understanding the temporal and spatial development pathways involved in stimulating the cortical reaction. / Department of Biology

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Dilantin (DPH) is a common anticonvulsant drug used to prevent seizures. It is known to be a human teratogen causing fetal hydantoin syndrome (FHS). FHS is characterized by multiple developmental and growth related abnormalities and mental retardation. Previous studies demonstrated that DPH slowed growth and division in preimplantation mouse embryos in vivo and in vitro. DHP exposure in utero decreased the crown to rump length and weight of 25-35% of embryos and reduced the rate of endochondral bone conversion from cartilage. In vitro preimplantation mouse embryos treated with DPH at 5, 10 and 20 μg/ml showed a reduction of 25-35% in their development, and block at 2-cell or 3-4-cell stages. These embryos also showed a prolonged DNA synthesis (S) phase during the second cell cycle. Nuclear localization and concentration levels of cyclin A , the S phase cyclin, were also altered in vivo in 2-cell DPH treated embryos compared with NaOH control embryos during G1, S phase and G2 of the first, second and third cell cycles. DPH altered patterns of expression of cyclin A were associated with cell cycle disregulation during preimplantation development. The purpose of the current study was to determine whether DPH also affects the concentration of DNA pol δ catalytic subunit in 2-cell preimplantation mouse embryos at G1 and S phases, thus delaying DNA synthesis and contributing to FHS. Immunofluorescence and confocal microscopy were used as tools to determine relative levels and distribution of DNA pol δ (for consistency with text) in the cytoplasm and the nuclei of DPH and NaOH treated 2-cell embryos at G1 and S phase of the second cell cycle. DPH decreased DNA pol δdelta total embryo and nuclear levels by 43% and 36%, respectively, in G1 compared with NaOH controls. Similarly, nuclear levels of DNA pol δ in DPH embryos in S phase near the G2 transition of the second cell cycle increased to 144% of NaOH control levels; there was not a statistically significant difference between total embryonic levels of late S phase DNA pol δ in DPH and NaOH treated control embryos. The results indicated that DPH affects the levels of DNA pol δduring G1 and S phase near the G2 transition of the second cell cycle in preimplantation mouse embryos. The significant alteration in the levels of DNA pol δ during S phase and its probable consequent altered polymerase activity could contribute to an explanation for the extension of S phase in preimplantation embryos observed by Blosser and Chatot. Even more, the alteration in the levels of DNA pol δ and potentially in its exonuclease activity could lead to an increase in the rate of mismatches and mutations suggesting a likely explanation for some features of FHS. / Department of Biology

Cyclin B1 is a cell cycle protein typically associated with the regulation of cellular division (mitosis). Previous studies in this laboratory involving preimplantation mouse embryos found that cyclin B1, or a cyclin B 1-related protein, were present at both G1 and G2/M phase of the cell cycle. Not only was cyclin Bi detected during G1 phase in this study, it was found to be present in higher concentrations at G1 phase through the first three cell cycles. These findings were unexpected, because most of the literature suggests that cyclin B1 is normally degraded during G1 phase. Using immunoprecipitation and immunoblot techniques, a more detailed study of cyclin B1 expression was inititated. Using two different primary antibodies direct against cyclin B1, a 48.97 kDa protein band, which is believed to be cyclin B1, was detected at both G1 and G2/M phases in 1-cell mouse embryos. Using another antibody directed against Cdk1, the kinase that forms a complex with cyclin B1 in order to direct the G2/M transition, a 37 kDa protein band was also detected at both G1 and G2/M phases in 1-cell mouse embryos. In order to determine whether cyclin B1 was present as a complex with Cdk1, immunoblotting with the anti-Cdk1 antibody. Again, a 37kDa protein band was detected at both G1 and G2/M phases. Finally, in order to determine whether the cyclin B1/Cdk1 complex exists in its active form, histone kinase assays were performed using anti-cyclin B1 immunoprecipitates. Kinase activity was detected in immunoprecipitates collected from G2/M phase 1-cell embryos, but no kinase activity was detected from immunoprecipitates collected from G1 phase 1-cell embryos. These data indicate that cyclin B1 and Cdk1 are present and exist as a complex in both G1 and G2/M phases of 1-cell mouse embryos, although the complex only appears to be active at the G2/M phase. / Department of Biology

Signaling molecules play essential roles in communication between cells. Wnt signaling molecules are critical for embryonic development of several organisms. I examined the involvement of Wnt signaling during two major developmental processes, namely embryo implantation and formation of the embryonic body axes. Using RT-PCR analysis, I showed that multiple Wnt genes are expressed in the blastocyst at the time of implantation. Moreover, expression of Wnt 11 requires both estrogen produced by the mother and the uterine environment. Using a transgenic approach, I showed that beta-catenin-regulated transcriptional activity, which is a major transducer of Wnt signaling, is activated in the uterus specifically at the site of implantation in an embryo-dependent manner. These results introduce Wnts as candidate signaling factors that may mediate the communication between the embryo and uterus that initiates implantation. / Wnt/beta-catenin signaling triggers axis formation in Xenopus and zebrafish embryos. I showed that, during embryonic development, beta-catenin-regulated transcriptional activity is first detected in the prospective primitive streak region prior to gastrulation. This demarcates the posterior region of the embryo. This activity then becomes restricted to the elongating primitive streak and to the node. In Xenopus embryos, beta-catenin participates in the formation of the organizer through the activation of the homeodomain transcription factors Siamois and Twin. I obtained evidence that a Siamois/Twin-like binding activity exists in mouse embryos and is localized in the node. These results strongly suggest that, as the case in Xenopus and zebrafish, the Wnt/beta-catenin pathway is involved in establishing embryonic body axes. / Furthermore, using the transgenic mouse line that I generated for these studies, I mapped the transcriptional activity of beta-catenin during mouse embryonic development. These results revealed when and where this activity, and presumably Wnt signaling, is active during the development of several organs and embryonic structures.

Cited2 is a member of the Cited gene family, which has no homology to any other genes. It encodes a transcriptional co-factor that is expressed during early heart formation (cardiogenesis). Embryos lacking Cited2 display a range of cardiac defects including bilaterally identical atria, aortic arch abnormalities, rotation of the aorta and pulmonary artery, and malseptation of the cardiac chambers. The latter results in communication between the aorta and pulmonary artery, the aorta and both ventricles, and the atria and ventricles (with themselves and each other). Cardiogenesis is complex, and requires many different cell types and processes to occur correctly. Some of these cells and processes are external to the primary heart. For example, once the initial muscle cells of the heart form a tube, cells from other regions such as the secondary heart field (adjacent mesoderm) and cardiac neural crest (ectoderm) migrate into this tube, and are required for the formation of additional muscle cells and septa. Furthermore, cardiogenesis also requires correct left-right patterning of the embryo to be established prior to heart formation. To understand the developmental origins of the cardiac defects observed in Cited2-null embryos, the expression pattern of Cited2 and the anatomy of Cited2-null embryo hearts were studied. Subsequently, the expression of genes required for left-right patterning were studied in both Cited2-null and Cited2 conditionally-deleted embryos. This demonstrated that Cited2 may be required in many, possibly all, of the processes required for cardiogenesis. Next this study focused on the role of Cited2 in patterning the left-right axis of the embryo. Firstly, Cited2 was found to regulate the expression of the master regulator of left-right patterning (Nodal). Secondly, Cited2 was shown to regulate the expression of the left-specific transcription factor Pitx2 independently of Nodal. Thirdly, gene expression and conditional deletions of Cited2 suggested that Cited2 might regulate left-right patterning in the paraxial mesoderm, a tissue which has not previously been shown to regulate the left-right axis in the mouse. Lastly, an argument is made suggesting the possibility that all the cardiac defects found in Cited2-null embryos may directly or indirectly stem from a failure of correct left-right patterning.

Dong Ming. / Thesis submitted in: December 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 118-152). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgement --- p.iv / Table of Contents --- p.v / Abbreviation list --- p.xi / Chapter Chapter 1 --- General introduction / Chapter 1.1 --- Preamble --- p.1 / Chapter 1.2 --- Neural Crest Cells (NCCs) --- p.2 / Chapter 1.3 --- Enteric Nervous System (ENS) and Vagal Neural Crest Cells (Vagal NCCs) --- p.4 / Chapter 1.4 --- Sacral Neural Crest Cells (Sacral NCCs) --- p.7 / Chapter 1.5 --- Signalling Mechanisms of Sacral Neural Crest Cells --- p.17 / Chapter 1.6 --- Hirschsprung's Disease (HSCR) --- p.20 / Chapter 1.7 --- Objective of the Study and Contents of the Following Chapters --- p.21 / Chapter Chapter 2 --- Migration from the dorsal neural tube to the pelvic mesenchyme / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and Methods --- p.32 / Chapter 2.2.1 --- Animal --- p.32 / Chapter 2.2.2 --- Isolation of embryos from pregnant mice at E9.5 to --- p.32 / Chapter 2.2.3 --- Histological preparation of the caudal segments --- p.33 / Chapter 2.2.4 --- p75 immunohistochemical staining --- p.33 / Chapter 2.2.5 --- Preparation of rat serum --- p.34 / Chapter 2.2.6 --- Preparation of the culture medium --- p.34 / Chapter 2.2.7 --- Preparation of wheat germ agglutinin-gold conjugates (WGA-Au) --- p.35 / Chapter 2.2.8 --- Preparation of CMFDA --- p.35 / Chapter 2.2.9 --- Preparation of DiI --- p.36 / Chapter 2.2.10 --- "Microinjection of WGA-Au, DiI and CMFDA" --- p.36 / Chapter 2.2.11 --- Whole embryo culture --- p.37 / Chapter 2.2.12 --- Examination of cultured embryos --- p.37 / Chapter 2.2.13 --- Histological preparation of WGA-Au labelled embryos --- p.38 / Chapter 2.2.14 --- Silver enhancement staining of the sections of WGA-Au labelled embryos --- p.39 / Chapter 2.2.15 --- Cryosectioning of the embryos labelled with DiI --- p.39 / Chapter 2.2.16 --- p75 immunohistochemical staining of DiI-labelled cells --- p.40 / Chapter 2.3 --- Results --- p.41 / Chapter 2.3.1 --- Observations on embryos developed in vivo --- p.41 / Chapter 2.3.2 --- Closed yolk sac culture vs open yolk sac culture --- p.42 / Chapter 2.3.3 --- Neural crest cell labelling in the caudal part of embryos --- p.43 / Chapter 2.3.4 --- Neural crest cell labelling with DiI in the caudal part of the neural tube followed by in vitro culture from E9.5 to E11.0 --- p.45 / Chapter 2.3.5 --- Neural crest labelling with DiI in the caudal part of the neural tube followed by in vitro culture from E10.5 to E11.5 --- p.46 / Chapter 2.3.6 --- Focal labelling at the levels of the 26th and 29th somites followed by in vitro culture --- p.48 / Chapter 2.3.7 --- p75 immunohistochemical staining on the caudal part of the embryo at E10.5 --- p.49 / Chapter 2.3.8 --- p75 immunohistochemical staining on embryos labelled with DiI --- p.50 / Chapter 2.4 --- Discussion --- p.51 / Chapter 2.4.1 --- Embryos at E9.5 cultured with an intact yolk sac membrane grew better than those with the yolk sac membrane cut open --- p.52 / Chapter 2.4.2 --- Migration at the levels of the 24th to 28th somite --- p.53 / Chapter 2.4.3 --- Migration at the levels of the 29th to 33th somite --- p.58 / Chapter 2.4.4 --- Sacral NCCs migrate along a straight dorsolateral pathway --- p.60 / Chapter 2.4.5 --- "Most of the DiI positive cells are p75 positive, but not all of the p75 positive cells are DiI positive" --- p.62 / Chapter Chapter 3 --- Migration from the pelvic mesenchyme to the hindgut / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.2 --- Materials and Methods --- p.73 / Chapter 3.2.1 --- Isolation of hindguts with or without adjacent tissues from embryos at E10.5 to E13.5 --- p.73 / Chapter 3.2.2 --- Microinjection of DiI into the pelvic mesenchymal tissue of the h indguts --- p.74 / Chapter 3.2.3 --- Preparation of the culture medium --- p.74 / Chapter 3.2.4 --- Preparation of the culture dish --- p.74 / Chapter 3.2.5 --- Gut culture --- p.75 / Chapter 3.2.6 --- Cryosections of the hindguts after in vitro culture --- p.75 / Chapter 3.3 --- Results --- p.76 / Chapter 3.3.1 --- "Hindguts isolated from embryos at E10.5, E11.5, E12.5 and E14.5" --- p.76 / Chapter 3.3.2 --- p75 immunohistochemical staining of the serial sections through the caudal part of the embryos --- p.78 / Chapter 3.3.3 --- Observations on hindgut without pelvic plexus cultured from E11.5 to E14.5 --- p.81 / Chapter 3.3.4 --- "Culture of hindguts with pelvic mesenchyme cultured from E11.5 to E14.25, E14.5 and E15.5" --- p.82 / Chapter 3.3.5 --- Culture of the whole length of the gut tube without pelvic mesenchyme from E11.5 to E15.5 --- p.84 / Chapter 3.3.6 --- Culture of the whole length of the gut tube with pelvic mesenchyme from E11.5 to E15.5 --- p.84 / Chapter 3.3.7 --- "Culture of hindguts with Dil labelling in the pelvic mesenchyme from E11.5 to E14.0, E14.2 5, E14.5 and" --- p.85 / Chapter 3.3.8 --- Culture of the whole length of the gut tube with DiI labelling in the pelvic mesenchyme from E11.5 to E14.5 --- p.86 / Chapter 3.4 --- Discussion --- p.88 / Chapter 3.4.1 --- Development of the hindgut and the urogenital system from E10.5 to E14.5 --- p.88 / Chapter 3.4.2 --- No p75 positive cells were found in the hindgut before E13.5 --- p.89 / Chapter 3.4.3 --- The sacral neural crest cells migrate into the hindgut at around E14.5 --- p.91 / Chapter 3.4.4 --- "The sacral neural crest cells migrated in the serosa, and entered the myenteric plexus prior to populating the submucosal plexus" --- p.95 / Chapter 3.4.5 --- Most of DiI labelled sacral neural crest cells in the hindgut also expressed p75 --- p.98 / Chapter Chapter 4 --- Migration from the neural tube to the hindgut / Chapter 4.1 --- Introduction --- p.101 / Chapter 4.2 --- Materials and Methods --- p.104 / Chapter 4.3 --- Results --- p.106 / Chapter 4.3.1 --- Morphology observations on the hindguts isolated from DiI-labelled embryos --- p.106 / Chapter 4.3.2 --- Distribution of the DiI-labelled cells and p75 positive cells before the culture of the hindgut explant --- p.106 / Chapter 4.3.3 --- Culture of hindgut explanted from Dil-labelled embyos for 3.5 days from E11.0 to E14.5 --- p.107 / Chapter 4.4 --- Discussion --- p.109 / Chapter Chapter 5 --- General discussion and conclusions --- p.113 / References --- p.118 / Figures and Legends --- p.153 / Tables and Graphs --- p.203 / Appendix --- p.209