An important problem in developmental biology is to understand how precise patterns of cell types are maintained during development. Eph receptor tyrosine kinases and ephrins have key roles in stabilising these patterns of cell organisation and segregation during development and can restrict the movement of cells by promoting cell repulsion. Previous work by Alexei Poliakov in the Wilkinson lab has shown that Eph-ephrin signalling leads to directional persistence of migration, and modelling suggests that this can contribute to cell segregation. In order to test experimentally the contribution of directional persistence in cell segregation, I have used and developed in vitro assays to dissect the roles of EphB2-ephrinB1 signalling in cell segregation, boundary sharpening and directional persistence. In these assays, stable HEK293 cell lines expressing EphB2 or ephrinB1 are mixed in cell culture and this leads to segregation of the two cell populations. Plating these cells either side of a removable barrier and allowing migration of cells towards each other leads to the formation of a sharp boundary on interaction. Analysis of cell behaviour shows EphB2 cells to move more persistently after interaction with ephrinB1 cells. To analyse how EphB2-ephrinB1 interactions lead to directional persistence of migration, my studies have focussed on the role of components potentially involved in directional persistence that act downstream of EphB2-ephrinB1 signalling, including the planar cell polarity (PCP) pathway (Dishevelled and Daam1) and core polarity components such as the PAR proteins (PAR-3 and PAR-6B). The PCP and PAR components were all found to have roles in cell segregation, as siRNA-mediated knockdown of each of these components disrupted EphB2-ephrinB1 mediated cell segregation and boundary sharpening. However, cell behaviour studies showed that only Dishevelled and PAR-6B have roles in EphB2-ephrinB1 mediated directional persistence, whilst Daam1 knockdown has no effect on the migratory response of cells. PAR-3 knockdown affects the basal ability of cells to migrate, potentially due to its role in establishing front-rear polarity. Taken together, these findings can be explained by a model in which Dishevelled and PAR-6B have a role in EphB2-ephrinB1 mediated directional persistence required for cell segregation and boundary sharpening. I propose that Daam1 may function in the contact inhibition of locomotion between cells also required for segregation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:565353 |
| Date | January 2011 |
| Creators | Gregory, L. G. L. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1318081/ |
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