Alpha2-chimaerin as a molecular switch which regulates motor axon growth and guidance. (360G-Wellcome-091637_Z_10_Z)

We will first establish that Semaphorin3A/C and CXCL12 signal in oculomotor development, by assessing the responses of oculomotor neurons in vitro to Semaphorin3A/C and CXCL12 ligands, singly and in combination. We will then evaluate the role of the cognate receptors and of alpha2-chimaerin signalling, by determining whether these responses are abolished/modified when neurons express appropriate dominant-negative receptors (Plexins/Neuropilins/CXCR4), or alpha2-chimaerin gain- or loss-of-functi on mutant forms. Secondly, we will analyse the molecular basis of changes in alpha2-chimaerin dynamics and trafficking in response to Semaphorins/CXCL12. We will use fluorescent live cell imaging to visualise and quantify the interaction of aplha2-chimaerin with Plexin and CXCR4 receptors in response to their physiological ligands. Using photoconvertible fluorescent tags and subcellular markers we will analyse how alpha2-chimaerin signalling is controlled and how the mutated proteins escape these regulatory mechanisms. Finally we will determine the in vivo role of CXCL12/Sema3A/C signalling via alpha2-chimaerin by electroporation of appropriate gain- or loss-of-function constructs into the oculomotor/abducens nerves of the chick embryo in ovo, singly or in combination. We will assess phenotypes of axon guidance defects and nerve-muscle patterning and look for evidence of complementation or rescue of defects by manipulation of molecules which lie within the same pathway.