001 for both), as previously shown ( Marquardt et al., 2005). In medial LMC neuron growth cones labeled by e[Isl1]:GFP electroporation, however, we observed that the majority of patches contained both ephrin-A5 and EphA3 protein ( Figures 7D–7F; p = 0.124 and 0.236). These observations thus suggest a vastly different distribution EphAs and ephrin-As in medial LMC versus lateral LMC growth Apoptosis inhibitor cones. To examine the effect of ephrin expression on the distribution pattern of Ephs and ephrins, we knocked down ephrin-A5 expression in medial LMC neurons. Similar to the control

medial LMC growth cones, those treated with scrambled [eA5]siRNA via in ovo electroporation, showed obvious copatching of ephrin-A5 and EphA3 ( Figures 7G–7I; p = 0.538 and 0.169). In contrast, in medial LMC neuron growth cones electroporated with ephrin-A5 siRNA, ephrin-A5-containing patches were occasionally observed, but they no longer contained any obvious EphA3 protein ( Figures 7J–7L; p < 0.001 for

both), a configuration similar to that found in lateral LMC neurons. These findings suggest that ephrin expression levels control the subcellular distribution pattern of Ephs and ephrins, and their consequence is a shift between cis-attenuation and trans-signaling modes, PCI-32765 order increasing the precision of axon trajectory selection. Concurrent trans-reverse and trans-forward signaling versus cis-attenuation have been proposed as two divergent modes of Eph and ephrin interaction. To understand their relative contribution to axon guidance in vivo, we studied them in the context of the choice of LMC motor axon trajectory

in the limb and showed that (1) limb trajectory selection by LMC axons is specified by ephrins in LMC neurons, (2) in addition to their signaling in trans, ephrins expressed in LMC neurons contribute to guidance of LMC axons by cis-attenuation of Eph receptor signaling, and (3) the balance between cis- and trans-interaction appears to be determined by ephrin protein levels. Here, we discuss the role of the molecular symmetry of ephrin-A and ephrin-B cis-attenuation function in the fidelity of LMC axon guidance, secondly the possible mechanisms and modes of Eph cis-attenuation by ephrins, and in-cis receptor-ligand interactions as a common strategy for axon guidance signaling refinement. Based on our gain and loss of ephrin function experiments, we propose that a molecular symmetry of ephrin cis- and trans-signaling in LMC neurons controls LMC axon guidance ( Figure 7M). Our model builds on the previous in vitro observation that ephrin-A5 in LMC axons can elicit attractive EphA:ephrin-A reverse signaling ( Marquardt et al., 2005) in parallel to forward ephrin:Eph signaling ( Eberhart et al., 2002, Helmbacher et al., 2000, Kania and Jessell, 2003 and Luria et al., 2008). This model is based on our observation that increasing or decreasing ephrin levels in LMC neurons leads to, correspondingly, attenuated or augmented sensitivity to ephrins provided in trans.