Thesis (M.S.)--Wake Forest University. Dept. of Biology, 2008. / Vita. Includes bibliographical references (leaves 14-17)

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / "July 1999." Typescript. Vita. Includes bibliographical references (leaves 128-129). Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, June 2003. / Includes bibliographical references. Also available on the Internet.

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Retinal degeneration is the progressive loss of neurons lining the posterior surface of the eye. Loss of a certain group of neurons called rod photoreceptors can occur as the result of genetic mutation. In humans, and in mammalian models of retinal degeneration, the death of these cells is permanent, and often followed by cone photoreceptor death, which leads to blindness. As a step towards understanding the implications of rod cell death in the retina, we generated transgenic X. laevis that expressed a novel form of caspase-9, with binding domains specific to the compound AP20187. We treated these transgenic animals with AP20187 and caused rod cell death by apoptosis in tadpoles and post metamorphic animals. Peak rod apoptosis occurred two days after drug exposure. We adapted an electroretinography apparatus, and protocols designed for mammals to measure functional changes in X. laevis rod and cone derived responses. We observed delayed secondary cone cell dysfunction after induced rod cell apoptosis, which was subsequently restored. These animals provide a simple and clinically relevant model of diseases like Retinitis pigmentosa, in which we will be able to probe in detail the mechanisms that govern cone cell dysfunction as a consequence of rod apoptosis. The unique ability of this species to recover from this insult will provide clues towards initiating similar recovery in humans. / Medicine, Faculty of / Graduate

At the outset of my PhD, my aim was to investigate the mechanisms responsible for the directed migration of primitive myeloid cells (PMCs) to wounds in Xenopus embryos. PMCs are the first blood cells to differentiate and become functional in Xenopus embryos, and have a notable migratory ability to be recruited by embryonic wounds before a functional vasculature is established. To find the mechanism underlying PMCs migration toward embryonic wounds, I first performed a screen to identify candidate cytoskeleton related genes, which might be responsible for facilitating the inflammatory response to injury in embryos. In situ hybridization and RT-PCR showed that coronin1a and l-plastin were specifically expressed in PMCs. I carried out loss-of-function experiments for coronin 1a and l-plastin in Xenopus embryos. Unfortunately neither knockdown affected the ability for PMCs to migrate during embryonic development or during the wound healing process. Loss-of-function experiments on coronin 1a and l-plastin also did not affect epidermal wound closure speed. Thus, although coronin 1a and l- plastin are expressed specifically in PMCs, they do not appear to be necessary for the migration of PMCs during development and during wound healing in Xenopuos embryos. Since my initial aim failed to provide insight into the mechanisms that mediate 9the inflammatory response to embryonic wounds, I decided to investigate the function of a previously identified monomeric actin protein during embryonic wound healing and appendage regeneration: namely Thymosin beta4 (Tβ4). In situ hybridization experiments showed that Tβ4 is expressed exclusively in the epidermis of developing frog embryos. Tβ4 knockdown embryos resulted in a significantly delay in the speed of wound closure during the early phase of wound healing. This delay correlated with a decrease in the actin contractile ring at the wound margin. Furthermore I found that the cell shapes of epidermal cells in the Tβ4 knockdown embryos were different from epidermal cells in control embryos. I hypothesize that this reduction caused the actin filaments changes in the epidermal cells, and were responsible for the failure of the cells to form an actin contractile ring, thus delaying the initial speed of wound closure. I tried to confirm that most of these defects specific to Tβ4, by performing rescue experiments with Tβ4 mRNA injections. Furthermore, I discovered that Tβ4 knockdown embryos displayed defects in tail development, including the absence of blood vessel branching within the fin of the tail. Finally, I found that the tails in Tβ4 knocked-down tadpoles failed to regenerate, while tails in control embryos regenerated completely following amputation. Both in situ hybridization and real-time PCR showed that Tβ4 was up regulated in the regenerated part of the tail in Xenopus tadpoles. Together with the tail amputation results, Tβ4 might be important for tail development and regeneration. These findings suggest that Tβ4 might play an important roles in the modulation of the actin cytoskeleton, which are essential for the proper behavior of epidermal cells during wound healing and appendage regeneration.

The primary aim of my PhD thesis was to identify and characterise novel modulators of intracellular signalling during early vertebrate development. The first phase of my thesis was to design and execute a large-scale gain of function screen in order to identify novel modulators of various important signal transduction pathways during early Xenopus development. From this screen I identified twenty novel of growth factor signalling. In the second phase of my PhD study, I concentrated on the characterization and mode of action of one of the genes I identified in the screen; namely fezf2. I showed that Fezf2 regulates neurogenesis in the diencephalon by locally promoting Wnt signalling through repression of lhx2 and lhx9. Notably, this investigation on the function of fezf2 not only revealed the previously undiscovered role of fezf2-mediated Wnt regulatory mechanism during diencephalon development, but also confirmed our in vivo screening approach in identifying potential regulators of signalling pathways. To the end, my PhD project has provided me with a fruitful journey of discovery, which started with the design and execution of a large-scale screen, ending with the detailed characterization of a factor involved in the modulation of signalling and forebrain development. This study is has broadened our understanding of how intracellular and extracellular signals are integrated during embryonic development process, which forms an interactive network ultimately resulting in appropriate cell differentiation, organ formation, and regional patterning.

For years, it has been appreciated that embryos have remarkable abilities to heal wounds efficiently and perfectly, without scar formation. However, the molecular mechanisms underlying embryonic wound healing, especially how they coordinate and function in an efficient way, remains poorly understood. The primary aim of my PhD thesis was to use Xenopus as a model system to investigate the molecular and cellular mechanisms which are responsible for the regulation and coordination of embryonic wound healing. More specifically, my thesis includes the study of three signalling pathways during embryonic wound healing; namely the Erk MAPK pathway, PI3K pathway and inositol phosphate pathways. Erk and PI3K signalling are sequentially activated post injury, during separate phases of wound closure. The initial activation of Erk signalling governs the initial stage of wound closure, by mediating myosin-2 phosphorylation and actomyosin contraction through Rho activity. PI3K signalling increases in the late stage of wound closure, promotes leading edge migration and zippering via Rac and Cdc42 activity (Manuscript #1). From the findings of this study, I proposed a novel model, which suggests a cooperation of these two signalling pathways in orchestrating distinct cytoskeletal events during in tissue morphogenesis. In the second part of my thesis, I studied the role of inositol phosphate signalling during wound healing. In particular, I studied the role of the enzyme Itpkb and its product InsP4, in promoting rapid wound healing (Manuscript #2). Itpkb colocalizes with F-actin cable and promotes its formation at the wound edge in both single cell and multicellular wounds, enhancing the activity of three Rho GTPases Rac, Cdc42 and Rho at the same time. In addition, itpkb is required for calcium propagation from the wound edge to distant cells, suggesting a role in transmitting the wound signal across the tissue, resulting in the coordination of healing in multicellular wounds. Together, these PhD work provided more insights into the in vivo regulation of intracellular and intercellular signals in coordinating cell behavior in tissue movement during embryonic wound healing.

Xenopus Laevis has provided, during the last ten years, the basis of a growing body of physiological enquiries initiated by Jolly's research on reflex action. Its viability in the laboratory and amenability to operative procedure in particular, make it a peculiarly suitable object for investigation. It is regrettable therefore, that existing literature on the anatomy of Xenopus has been directed to elucidating those characteristics which are of especial interest to the Systematists and Morphologists rather than detailed information of a type which is essential to operative procedure. There is in particular no extant account of the endocrine system of Xenopus, although it is evident from superficial inspection that the suprarenal complex differs from that of the more familar Anura. The present enquiry concerns the Thyroid Gland, Pituitary and Epiphyseal Complexes with some observations concerning the possible occurrence of chromophil cells in the kidney of Xenopus laevis. The data have been placed on record specifically and constitution of these organs in physiological operations.