Woolley, Katie Jane; (2001) Early development and wound healing in the Zebrafish embryo. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In this thesis I use the Zebrafish embryo as a model to study tissue movements of embryonic wound healing as a model of natural morphogenetic movements that shape the embryo during development. In particular I am interested in the signals that initiate embryonic tissue movements, the signals that stop them, and the cytoskeletal machinery that drives them. Re-epithelialisation of a wound is accomplished by a concerted set of cell shape changes and rearrangements which drive the epithelium over the wound area. I have used the Zebrafish embryo to study these cell shape changes both by Scanning Electron Microscopy (SEM) and in real time using time-lapse analysis of vitally-stained embryos, and made direct comparisons with the analogous natural morphogenetic tissue movement of epiboly. The cell shape changes of re-epithelialisation appear to be driven by contraction of an actin/myosin purse string which draws the epithelium forward. I have examined reorganisation of the actin cytoskeleton in leading edge cells during both wound closure and epiboly using phalloidin-staining to reveal filamentous actin and have used a specific inhibitor of a key effector of the small GTPase Rho, Rho-kinase, to test its function in directing these key actin polymerisation events. I have also analysed reorganisation of the microtubule network in wound edge cells and report that while microtubules reorientate following wounding and are essential for epiboly, they are not required for initiation or maintenance of epithelial movements. In order to correlate the cell shape changes and cell shufflings of re-epithelialisation with the signals driving these tissue movements I have examined various signalling pathways that might be regulating cell behaviours at the wound site, including calcium influx and the MAP kinase pathways. For each of these signals I present experiments that test their functional involvement in directing re-epithelialisation of a wound in the embryo. Finally, I have taken advantage of Zebrafish mutants available from various genetic screens that fail in epiboly or to test whether they also fail in wound repair, in order to address whether the same signalling and cytoskeletal machinery used during morphogenesis is being re-used when a wound repairs.

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