In lateral LMC neurons, ephrin-As are expressed at low levels and interact in trans with EphA4 receptors expressed in the limb mesenchyme leading to the attraction of lateral LMC into the dorsal limb nerve. In medial LMC neurons,

ephrin-As are expressed at much higher levels and attenuate coexpressed EphAs in cis, enabling medial LMC axons to grow into the ventral limb where ephrin-As are present. Mirroring these interactions, lateral LMC neurons express high levels of ephrin-Bs that attenuate endogenous EphB receptors in cis, enabling lateral LMC axons to grow into the dorsal limb where ephrin-Bs abound, while medial LMC axons express low levels of ephrin-Bs Afatinib that mediate attractive responses PD-1/PD-L1 activation to EphB receptors expressed in the ventral limb ( Kania and Jessell, 2003). Thus, in addition to restriction at the protein expression level, we propose that Eph receptor function is also regulated by ephrins in cis such that even though some Ephs are apparently expressed in all LMC neurons, they exert their function only in neurons with low levels of same-class ephrin. For example, our findings explain a recent observation where, although EphB2 and EphB3 are expressed in apparently all LMC neurons, EphB2−/−/EphB3−/− knockout mice display a phenotype

only in medial LMC neurons ( Luria et al., 2008), presumably because EphB function is normally blocked in lateral LMC neurons by high ephrin-B expression levels. Similarly, the ventral limb projection phenotype of lateral LMC neurons overexpressing ephrin-A is stronger than EphA4 loss of function ( Luria et al., 2008) probably because of increased global cis-attenuation of all EphAs that are normally present in lateral LMC neurons. Axon sorting through axon-axon interactions has been proposed as a key event in Olopatadine the establishment of neural maps (Brown et al., 2000, Feinstein and Mombaerts, 2004 and Imai et al., 2009), implying that Eph-ephrin interactions might direct

the selection of limb trajectory by modulating the fasciculation of LMC axons. For example, Ephs and ephrins function in the context of sensory and motor axon sorting in the periphery, which in turn influences the trajectory choices made by these axons (Gallarda et al., 2008). Our observation of differential expression of Ephs and ephrins in LMC divisions implied a possible involvement of fasciculation in modulating LMC axon limb trajectory choice. However, our in vitro results show that both ephrin:Eph forward signaling and ephrin-mediated cis-attenuation of Eph function are retained in low-density cultures with negligible axon-axon interactions; thus, the phenotype of LMC axon misrouting in ephrin loss of function is probably not secondary to changes in fasciculation properties of LMC axons.