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Title: Genetic control of corneal epithelial cell migration by Pax6
Author: Kucerova, Romana
ISNI:       0000 0001 3602 7297
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2008
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Coordinated cell migration is essential for tissue regeneration and· development. During migration cells sense various directional cues from the surrounding environment, and intracellular mechanisms process this extracellular information resulting in appropriate vectorial cell movement. During corneal epithelial maintenance, cells migrate centripetally from the limbal region towards the centre of the cornea. This centripetal movement is genetically controlled by Pax6 (Collinson et aI., 2004), one of the master genes in eye development (Kozmik, 2005). The guidance cues and cellular mechanisms controlling centripetal migration of corneal epithelial cells remain to be elucidated. Clarifying the mechanisms guiding cell migration in an experimentally accessible model of corneal epithelial cells would be relevant to mechanisms controlling vectorial cell migration in other cell types. In this project the roles of endogenous electrical and chemical cues in directing corneal epithelial cell migration were tested using in-vitro and ex-vivo culture systems in which migration was induce by wounding. It was ~hown that Pax6+1 - cells suffer from a 2 h wound healing delay, but at later time points, the migration is not significantly different from pax~/+ cells (Leiper et aI., 2006). Additionally Pax6+1 - cells showed defective glycoprotein trafficking which is als~ affecting tJ-le molecules not directly controlled by Pax6 at the transcriptional level and potentially increasing the number of molecular defects contributing to the Pax6+1 - phenotype.. An ex-vivo whole eye system was developed which subsequently demonstrated growth factor dependent woiInd healing defect of pax~/- cells, in contrast to Pax6+1+ epithelia which healed and migrated normally regardless externally supplied growth factors. Exogenous application ofEGF was found to restore wound healing in pax~/cells to wild-type levels. The extensive part of the work was focused on elucidating the role of electrical guidance in wound healing and migration. Recent publications have established roles for endogenous electric current in guiding epithelial ceIl migration (Zhao et aI., 2006)but until this project, there was no genetic model 'of defective 'wound-induced currents. paxrt'l- eyes were found to have compromised or reversed wound mediated currents and were therefore used as model to investigate the importance of electrical cues for normal wound healing. The study represented the most rigorous challenge yet to the hypothesised roles of endogenous electric fields, and surprisingly the analysis showed no significant correlation between electrical cues and directional migration, based on the observation that Pax6+1 - with defective signals healed at rates not significantly different from control wild type epithelia. Further investigation showed no significant correlation ofelectric current and wound healing even in Pax6+1+eyes. In searching for molecular mechanis1J1s controlling directional migration in applied EFs, Pax6+1 - cells were used as model because the cells previously showed compromised or reverse response to in-vitro applied EFs. Src was identified as a candidate signalling molecule playing an important role in mediating directional migration and additionally failure of pSrc to polarise in paxrt'l- cells in applied electric fields correlated with their impaired migration response. Finally wound healing acceleration by addition of Shh peptide was shown to be Pax6 dosage dependent. Investigation of Hh pathway components in Pax6+1+ and paxrt'l- corneal epithelial cells suggested important differences at the level of Gli transcription factors which may underlie the failure of paxrt'l- cell~ to show a migratory respOIise to exogenous Shh.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available