Singleton Escofet, R; (2013) Role of CXCR4 Dynamics and Activity in Neural Crest Migration. Doctoral thesis (PhD), UCL (University College London).

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Abstract

During early embryonic development the neural crest migrates from its site of induction to various locations within the embryo; a peculiarity of this movement is that cells move collectively. Contact Inhibition of Locomotion (CIL) is required for directional migration of the neural crest cells. In addition, negative and positive signals present in neighbouring tissues help guide the group. Previous experiments have shown that group of cells are able to respond to these signals more efficiently than single cells, but how clustering neural crest cells contribute to a better response to chemoattractants is poorly understood. The major neural crest chemoattractant is SDF1 that activates the receptor CXCR4 expressed in the neural crest cells and is required to guide neural crest migration. In this thesis we analysed the dynamic of CXCR4 during neural crest chemotaxis. We found that the CXCR4 is constantly internalised and trafficked within the cell, even in absence of SDF1. However, treatment of neural crest cells with SDF1 promotes a change in the endocytic pathways of the receptor. Importantly, we found that endocytosis of CXCR4 is polarized in neural crest cells, with higher rates of endocytosis at the free edge compared to the cells contact. The overall effect is that the free edge membrane is primed respond efficiently to SDF1. We show that this endocytic polarity is Rac1 and RhoA dependent, two GTPases whose activity is controlled by cell-cell contact. In order to study the role of CXCR4 endocytosis in neural crest migration several mutant forms of the receptor were expressed and the response to SDF1 was analysed in vivo and in vitro. When a mutated CXCR4 that cannot be internalized is expressed, a dramatic effect on directional cell migration was observed. Cells are still able to respond to SDF1 but they exhibit a diminished capacity to adapt to changes in the position of the SDF1 source. When a constitutively active CXCR4 receptor is expressed in neural crest cells, their migration is also affected. Cell migration becomes independent on SDF1 and they disperse colonizing tissues that never contain neural crest cells. In conclusion, our results show that cell-cell contact controls CXCR4 endocytosis, which is likely to explain why groups of cells respond better than single cell to the ligand SDF1. In addition, we show that the endocytosis and activity of CXCR4 need to be tightly regulated to allow a correct neural crest migration in vivo and in vitro.

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