the role of thymosin beta4 during embryonic wound healing and tail regeneration in Xenopus

UoM administered thesis: Phd

  • Authors:
  • Yanan Zhao

Abstract

Submitted by Yanan Zhao for the Degree of Doctor of Philosophy in The University of Manchester and entitled 'The Role of Thymosin β4 during embryonic wound healing and tail regeneration in Xenopus' in September, 2012.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 knock-down 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 the 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). This part of my PhD project was more fruitful. 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 membranes of epidermal cells in the Tβ4 knockdown embryos were less stiff and had more ruffles than epidermal cells in control embryos. These results indicated that knocking-down Tβ4 in embryos might reduce high-tension actin filament formation in the epidermis. I hypothesize that this reduction caused the membrane ruffles 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 was able 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, I suggest that Tβ4 is important for tail development and regeneration. These findings suggest that Tβ4 plays 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.

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Original languageEnglish
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Award date31 Dec 2013