Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references.

by Sze, Lung Yam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 106-112). / Abstract --- p.i / Acknowledgements --- p.iv / Contents --- p.v / Chapter Chapter 1 --- General Introduction / Chapter 1.1. --- Morphology of the Mammalian Somite and Limb --- p.1 / Chapter 1.1.1. --- The Somite --- p.1 / Chapter 1.1.2. --- The Limb --- p.3 / Chapter 1.2. --- Somite-Limb Relationship --- p.5 / Chapter 1.2.1. --- Somite Contribution to the Appendicular Musculature --- p.5 / Chapter 1.2.2. --- Somite Contribution to Limb Morphogenesis --- p.6 / Chapter 1.3. --- Control of Directionality of Somitic Cell Migration in Appendicular Environment --- p.8 / Chapter 1.4. --- Reasons and Objective of the Present Study --- p.10 / Chapter Chapter 2 --- The Origin of the Mammalian Limb Skeletal Muscles / Chapter 2.1. --- Introduction --- p.16 / Chapter 2.2. --- Materials and Methods --- p.19 / Chapter 2.2.1. --- Embryo Collection --- p.19 / Chapter 2.2.2. --- Isolation of Somites --- p.20 / Chapter 2.2.3. --- DiI-labelling of Rat Donor Somites --- p.21 / Chapter 2.2.4. --- Somite Transplantation --- p.21 / Chapter 2.2.5. --- Embryo Culture --- p.22 / Chapter 2.2.6. --- Analysis of Cultured Embryos --- p.22 / Chapter 2.2.7. --- Cryosection --- p.23 / Chapter 2.2.8. --- Limb Explant Cultures --- p.23 / Chapter 2.2.9. --- Immunohistochemistry --- p.24 / Chapter 2.2.10. --- X-gal Staining --- p.25 / Chapter 2.2.11. --- Histology --- p.25 / Chapter 2.3. --- Results --- p.27 / Chapter 2.3.1. --- Gross Morphology of Cultured Embryos --- p.27 / Chapter 2.3.2. --- Distribution of DiI Labelled Somitic Cells in Rat Embryos --- p.27 / Chapter 2.3.3. --- Histogenetic Potential of Labelled Somitic Cells in the Limbs --- p.29 / Chapter 2.3.4. --- Chimeaic Limb Culture --- p.30 / Chapter 2.4. --- Discussion --- p.32 / Chapter 2.4.1. --- Relationship Between the Somites and the Limb Musculature in Rat Embryos --- p.33 / Chapter 2.4.2. --- Myogenic Potential of Somitic Cells in the Mouse Limb Bud --- p.36 / Chapter 2.4.3. --- The Regulatory Potentials of Mammalian Somites --- p.37 / Chapter Chapter 3 --- The Migration of Somitic Cells into the Mammalian Fore- limb Bud / Chapter 3.1. --- Introduction --- p.52 / Chapter 3.2. --- Materials and Methods --- p.57 / Chapter 3.2.1. --- Embryo Collection --- p.57 / Chapter 3.2.2. --- Embryo Culture and Analysis of Cultured Embryos --- p.58 / Chapter 3.2.3. --- Experimental Series I --- p.58 / Chapter A. --- Micro-injection of DiI --- p.58 / Chapter B. --- Explant Cultures of Rat Fore-limb Bud --- p.59 / Chapter C. --- Histology and Immunohistochemistry --- p.60 / Chapter 3.2.4. --- Experimental Series II --- p.61 / Chapter A. --- Preparation of Conditioned and Unconditioned Medium --- p.61 / Chapter B. --- Coating of Nucleopore Membrane with Fibronectin --- p.62 / Chapter C. --- Preparation of Somitic Cells --- p.62 / Chapter D. --- Analysis of Chemotatic Effect --- p.63 / Chapter 3.2.5. --- Experiment Series III --- p.64 / Chapter A. --- Micro-injection of Latex Beads --- p.64 / Chapter B. --- Isolation of Somatopleure and Transplantation --- p.64 / Chapter C. --- "Somite Isolation, Labelling, and Transplantation" --- p.65 / Chapter D. --- Histology --- p.65 / Chapter 3.3. --- Results --- p.66 / Chapter 3.3.1. --- Development of Embryos In vitro --- p.66 / Chapter 3.3.2. --- Experimental Series I --- p.66 / Chapter A. --- Distribution of Somitic Cells in DiI Injected Embryos --- p.66 / Chapter B. --- Histogenetic Potential of Limb Explants Cultured Under the Kidney Capsule --- p.68 / Chapter C. --- Histogenetic Potential of Limb Explants Cultured In vitro --- p.69 / Chapter 3.3.3. --- Experimental Series II --- p.70 / Chapter A. --- Chemotatic Behaviour of Somitic Cells --- p.70 / Chapter 3.3.4. --- Experimental Series III --- p.71 / Chapter A. --- Ability of Latex Beads to Invade the Limb Bud --- p.71 / Chapter B. --- Distribution Pattern of Somatopleural and Somitic Cells --- p.71 / Chapter 3.4. --- Discussion --- p.75 / Chapter 3.4.1. --- Experimental Series I --- p.75 / Chapter A. --- Distribution of Somitic Cells in DiI-Injected Rat Embryos --- p.75 / Chapter B. --- Histogenetic Potential of Rat Fore-limb Bud --- p.77 / Chapter 3.4.2. --- Experimental Series II --- p.80 / Chapter A. --- Chemotatic Behaviour of Somitic Cells --- p.80 / Chapter 3.4.3. --- Experimental Series III --- p.80 / Chapter A. --- Ability of Latex Beads to Invade the Limb Bud --- p.80 / Chapter B. --- Ability of Somatopleure and Somite to Invade Limb Bud --- p.83 / Chapter 3.4.4. --- Conclusion --- p.83 / References --- p.106 / Appendix --- p.113

Thesis (M.S.)--University of Missouri-Columbia, 2008. / "May 2008" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Includes bibliographical references.

Successful mammalian development to term requires that embryonic and extra-embryonic tissues communicate and grow in coordination, to form the body. After implanting into the uterus, the mouse embryo is comprised of three cell lineages: first, the embryonic epiblast (EPI) that forms the embryo proper, second, the extra-embryonic ectoderm (ExE) which contributes to the foetal portion of the placenta, and third, the visceral endoderm (VE) that contributes to the yolk sac. These three tissues form a characteristic ‘egg-cylinder’ structure, which allows signals to be exchanged between them and sets the stage for body axis establishment and subsequent tissue patterning. The mechanisms underlying this process are difficult to study in vivo because a different genetically manipulated mouse line must be generated to investigate each factor involved. This difficulty has prompted efforts to model mammalian embryogenesis in vitro, using cell lines, which are more amenable to genetic manipulation. The pluripotent state of the EPI can be captured in vitro as mammalian embryonic stem cells (ESCs). Although mouse ESCs have been shown to contribute to all adult tissues in chimeric embryos, they cannot undertake embryogenesis when allowed to differentiate in culture. Previous studies have shown that ESCs formed into three-dimensional (3D) aggregates, called embryoid bodies, can become patterned and express genes associated with early tissue differentiation. However, embryoid bodies cannot recapitulate embryonic architecture and therefore may not accurately reflect what happens in the embryo. In this study, a new technique was developed to model early mouse development which is more faithful to the embryo. ESCs were co-cultured with stem cells derived from the ExE, termed trophoblast stem cells (TSCs), embedded within extracellular matrix (ECM). These culture conditions lead to the self-assembly of embryo-like structures with similar architecture to the mouse egg cylinder. They were comprised of an embryonic compartment derived from ESCs abutting an extra-embryonic compartment derived from TSCs, and hence were named ‘ETS-embryos’. These structures developed a continuous cavity at their centre, which formed via a similar sequence of events to those that lead to pro-amniotic cavity formation in the mouse embryo, and required active Nodal/Activin signalling. After cavitation, ‘ETS-embryos’ developed regionalised mesodermal tissue and primordial germ cell-like cells originating at the boundary between embryonic and extra-embryonic compartments. Inhibitor studies revealed that this occurred in response to endogenous Wnt and BMP signalling, pathways which also govern these tissue specification events in the early mouse embryo. To demonstrate that ‘ETS-embryos’ were comparable to mouse embryos at the global transcriptional level, RNA-sequencing was then performed on different tissue regions of ‘ETS-embryos’ and the resulting transcriptomes were compared to datasets from mouse embryos. These data showed that ‘ETS-embryos’ were highly similar to mouse embryos at post-implantation stages in their overall gene expression patterns. Taken together, these results indicate that ‘ETS-embryos’ are an accurate in vitro model of mammalian embryogenesis, which can be used to complement studies undertaken in vivo to investigate early development.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 13, 2009). Includes bibliographical references.

Thesis (Ph.D.). / Written for the Dept. of Experimental Medicine. Title from title page of PDF (viewed 2009/06/09). Includes bibliographical references.

Hydroxyurea was used as a model teratogen to investigate the role of oxidative stress and stress-response pathways in mediating developmental toxicity. When administered to pregnant mice during early organogenesis, hydroxyurea induced fetal death and growth retardation, as well as external and skeletal malformations. The malformed fetuses displayed hindlimb, vertebral column, and tail defects. Hydroxyurea treatment enhanced the production of 4-hydroxynonenal, a lipid peroxidation end product, in malformation sensitive regions of the embryo. Depletion of glutathione, a major cellular antioxidant, specifically enhanced hydroxyurea-induced malformations and elevated the region-specific production of 4--hydroxynonenal protein adducts in the embryo, without affecting the incidence or extent of hydroxyurea-induced fetal death or growth retardation. The major proteins modified by 4-hydroxynonenal were involved in energy metabolism. Thus, oxidative stress is important in the induction of malformations by hydroxyurea. / Exposure to hydroxyurea stimulated the DNA binding activity of activator protein 1 (AP-1), an early response redox-sensitive transcription factor. Activated AP-1 was composed mainly of c-Fos heterodimers. Glutathione depletion did not change the effects of hydroxyurea on AP-1/c-Fos DNA binding activities despite an augmentation of the incidence of embryo malformations. Mitogen-activated protein kinases (MAPKs) activate AP-1 in response to stress by post-transcriptional phosphorylation of AP-1 proteins. Hydroxyurea treatment dramatically enhanced the activation of stress-responsive p38 MAPKs and JNKs (c-Jun N-terminal protein kinases). Selectively blocking p38 MAPKs enhanced the incidence of fetal death, whereas selective inhibition of JNKs specifically elevated the limb defects induced by hydroxyurea. Thus, activation of stress-response pathways impacts on the response of the embryo to a teratogenic insult.

No description available.

5-Bromo-2'-deoxyuridine (BrdU), a thymidine analogue, is genotoxic and teratogenic. The exposure of mouse embryos to BrdU at doses that cause malformations induces oxidative stress and an embryonic stress response characterized by an increase in c-Fos dependent AP-1 DNA binding. The goal of this thesis was to test the hypothesis that development is disturbed at sites where BrdU is incorporated into DNA, triggering oxidative stress and c-Fos induction. Gestation day 9 CD-1 mice were treated with BrdU and embryos were obtained for immunolocalization of BrdU, 8-oxoguanine, a biomarker for oxidative stress, and c-Fos. BrdU incorporation into DNA was dispersed throughout the embryo. In contrast, the staining for 8-oxoguanine and c-Fos were highest in the neuroepithelium. BrdU incorporation was not affected by the pre-administration of N-acetyl-cysteine (NAC), an anti-oxidant, although both 8-oxoguanine and c-Fos staining were decreased. Thus, the response of the embryo to insult is tissue specific.

Cell proliferation and differentiation are tightly regulated processes required for the proper development of multi-cellular organisms. To understand the effects of cell proliferation on embryo patterning in mice, we inactivated Aurora A, a gene essential for completion of the cell cycle. We discovered that inhibiting cell proliferation leads to different outcomes depending on the tissue affected. If the epiblast, the embryonic component, is compromised, it leads to gastrulation failure. However, when Aurora A is inactivated in extra-embryonic tissues, mutant embryos fail to properly establish the anteroposterior axis. Ablation of Aurora A in the epiblast eventually leads to abnormal embryos composed solely of extra-embryonic tissues. We took advantage of this phenomenon to generate embryonic stem (ES) cell-derived mice. We successfully generated newborn pups using this epiblast ablation chimera strategy. Our results highlight the importance of coordinated cell proliferation events in embryo patterning. In addition, epiblast ablation chimeras provide a novel in vivo assay for pluripotency that is simpler and more amenable to use by stem cell researchers.