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|Title:||SRC FAMILY KINASES FUNCTION IN SPINAL MOTOR AXON GUIDANCE|
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|Authors/Affiliations:||1 Tzu-Jen Kao*; 2 Artur Kania; |
1 Institut de recherches cliniques de Montréal, QC, Canada
2 Institut de recherches cliniques de Montréal, Department of Anatomy & Cell Biology, Div Experimental Medicine, McGill University & Faculty of Medicine, QC, Canada
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|Content:||Objectives: The relay of axon guidance signals to the actin cytoskeleton at the growth cone remains poorly understood. A simple system well-suited to address this issue are the axon projections of spinal motor neurons of the lateral motor column (LMC). Lateral and medial LMC axons arrive at the base of the limb and make an invariant decision to project to, respectively, dorsal and ventral limb mesenchyme. Src family kinases (SFKs) have been implied to mediate growth cone neurite extension/retraction with ephrin:Eph signaling being one of the axon guidance effector pathways linked to SFK function. EphA tyrosine kinase receptors are predominantly expressed in lateral LMC neurons while their ephrin-A ligands are present in the ventral limb. In parallel, EphB tyrosine kinase receptors are predominantly expressed in medial LMC neurons while their ephrin-B ligands are present in the dorsal limb. Previous studies provide compelling evidence for ephrin:Eph signaling functions in the control of fidelity of LMC axon projections, and suggest that this occurs, in part, through repulsion away from ephrin ligands expressed in the limb mesenchyme. However, the intracellular cascade relaying ephrin:Eph signals and leading to growth cone cytoskeleton rearrangement underlying axonal trajectory choices remains unclear. Because of the role of SFKs in Eph signaling, we began to examine whether SFKs are involved in Eph-mediated LMC motor neuron axon guidance in vivo.|
Materials and Methods: In situ mRNA hybridization was performed to characterise SFK expression in chick and mouse embryonic spinal cords. In ovo electroporation of expression plasmid DNA was performed using standard methods.
Results: Src and fyn mRNAs are expressed in LMC motor neurons of chick and mouse embryos at the time of limb trajectory selection. The inhibition of SFKs by over-expression of C-terminal Src kinase (Csk), an SFK inhibitor, in chick LMC neurons leads to the selection of inappropriate LMC axon trajectories, with medial LMC projection being severely misrouted. To determine the extent to which SFKs contribute to Eph signaling, Csk was co-electroporated with EphA4::GFP or EphB2::GFP fusion protein in chick LMC neurons. We found that Csk attenuated the retargeting of LMC axons caused by EphA4 or EphB2 expression. To determine whether Src and Fyn are essential for normal LMC axonal pathfinding we are currently examining Src and fyn mutant mice, and to further assess the role of SFKs in ephrin:Eph signaling we will overexpress EphA4 and EphB2 receptors with mutations at SFK binding sites.
Conclusion: The misrouting of LMC axons induced by SFK inhibition suggests that SFKs are required for the selection of appropriate LMC motor neuron axon trajectory. In addition, SFKs appear to be at least partially involved in Eph signaling mediating LMC motor axon guidance.
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