The cellular and molecular mechanisms of olfactory ensheathing cell and axon migration.

The regenerative capacity of the olfactory system is partly attributed to the presence of olfactory ensheathing cells (OECs) as they are intimately associated with primary olfactory sensory neuron (OSN) axons from the olfactory epithelium until the final targeting of axons within the olfactory bulb. However, fundamental aspects of OEC and OSN axon biology remain unclear and little is understood about how OECs and OSN axons interact and are capable of detecting and responding to each other. This thesis addresses the cellular and molecular processes and mechanisms that mediate OEC-OEC and OEC-axon interactions and migration in vitro. To start to understand how OECs interact in vitro and in vivo, we generated transgenic mice expressing a variant of the Discosoma species red fluorescent protein (DsRed) under the control of 9.6 kb of the human S100s gene regulatory sequence. Primary cultures derived from the neuroepithelium of these mice allowed for the first time clear identification and visualisation of OECs in vitro and in vivo. Using high resolution time-lapse microscopy we report here that novel lamellipodial protrusions, termed lamellipodial waves, are integral to peripherally derived OEC migration. Time-lapse imaging of migrating OECs revealed that these highly dynamic waves progress along the shaft of the cells and are crucial for mediating cell-cell adhesion. Without these waves, cell-cell adhesion does not occur and migrational rates decline. The activity of waves is modulated by both GDNF and inhibitors of the JNK and SRC kinases. Furthermore, the activity of lamellipodial waves can be modulated by Mek1, independently of leading edge activity. OECs are not a uniform population of cells and express distinct markers and are thought to play different roles depending on their anatomical position in the olfactory nerve pathway. As OECs arise from a common progenitor and migrate to populate the primary olfactory nerve, the different subpopulations must intermingle and sort out with considerable cell-cell interactions occurring. However, little is understood about how OECs interact and how the different subpopulations of OECs are capable of detecting and responding to each other. We have micro-dissected anatomically distinct subpopulations of OECs from the olfactory bulb and from the peripheral nerve and performed cell behaviour assays to reveal that the behaviour of OECs dramatically alters depending on their anatomical location and developmental age. In particular, centrally-derived OECs are a heterogeneous population of cells that respond to cell-cell contact with a mix of adhesion, retraction and indifference. In contrast, OECs derived from the peripheral olfactory nerve are clearly a homogeneous population. We have further determined that lamellipodial waves along the shaft of centrally-derived OEC processes are imperative for initiating and mediating behaviour during cell-cell contact. Inhibition of lamellipodial waves via Mek-1 resulted in OECs losing their ability to distinguish between different subpopulations. These results demonstrate that centrally-derived OECs are a heterogeneous population of cells and that cell-cell recognition and responses are regulated through lamellipodial waves. To start to understand how olfactory axons interact in vitro and in vivo, we generated transgenic mice expressing a coral ZsGreen fluorescent protein under the control of the full length (5.5kb) olfactory marker protein (OMP) promoter. Primary cultures derived from the neuroepithelium of these mice allowed clear identification and visualisation of olfactory axons in vitro. Using high resolution time-lapse microscopy, we reveal here that OSN pioneer axons play a passive role in axon guidance; the motility of the underlying OECs is primarily responsible for pathfinding and migration of OSN axons. This intimate association is in part initiated and mediated by lamellipodial waves on the shaft of OEC processes. Moreover, pioneer OSN axon migration can be indirectly modulated via GDNF and inhibitors of the JNK and SRC kinases. We also reveal that inhibition of NCAM results in the disruption of OEC cell-cell recognition. Loss of the OEC scaffold by selective ablation of OECs resulted in reduced axon survival. These results suggest that OEC growth and guidance are crucial to primary olfactory axon navigation in the olfactory system. This thesis demonstrates that OECs utilize novel cellular mechanisms to facilitate migration and cellular interactions. These findings have important implications for our understanding of OECs in the primary olfactory system. Moreover, the ability to selectively regulate cell migration via lamellipodial waves has implications for manipulating the migratory behaviour of OECs during neural repair.