Polarity during migration and asymmetric cell division in fate determination of thymocytes

The thymus is the site that generates many of the T cell subsets for our adaptive immunity. The T cells that mediate immune responses are generated in the thymus from thymocytes. Elucidating the mechanisms for thymocyte fate determination has the potential for profound impact upon autoimmunity, vaccination, recovery from immunosuppression during chemotherapy and leukemogenesis. In solid tissues, fate determination is dictated by asymmetric cell division (ACD). ACD involves the induction and maintenance of polarity during division, resulting in two daughter cells with different molecular composition and fates. Recently, studies have shown that the fate of hematopoietic stem cells and T cells are influenced by ACD, and that the mechanisms of polarity are conserved. It is unknown whether ACD occurs in thymocytes to determine fate outcome. In this thesis I investigated whether ACD occurs in DN3 thymocytes. Using an in vitro system of T cell development, I established protocols for time lapse imaging of thymocytes, and co-developed a software platform to quantify polarisation in migrating and dividing DN3 thymocytes. A number of proteins previously implicated in ACD polarise in DN3 thymocytes, and excitingly, two of these, Numb and Ap2a2, polarise in DN3 thymocytes during division. These experiments provide the first quantified evidence that DN3 thymocytes undergo ACD. To investigate the molecular mechanisms and consequences of DN3 ACD, I used several in vitro approaches and a mouse model of T cell leukaemia driven by overexpression of Lmo2. Abrogation of Numb phosphorylation by the polarity protein aPKC prevents Numb polarisation during DN3 division, but not during migration. Disruption of chemokine signalling reduces Ap2a2 polarisation, providing a mechanism through which ACD may be controlled. Using Ap2a2 polarisation as a marker for ACD, I demonstrate that the altered fate of leukaemic DN3 thymocytes is associated with a reduction in ACD. Combinatorial effects of Lmo2 overexpression, inhibition of chemokine signalling and Ap2a2 overexpression correlate with effects on polarity and differentiation of DN3 thymocytes, suggesting that ACD of DN3 thymocytes impacts upon DN3 fate decisions. These results indicate for the first time that thymocytes undergo ACD, and suggest a novel molecular mechanism for control of fate in thymocytes. Furthermore, they suggest that disruption of ACD is associated with development of leukemia. My preliminary evidence, together with the technological platform that I developed provide a foundation for future experiments to investigate the impact of ACD with thymocyte fate outcome in normal development and leukaemogenesis.