We reasoned that if ephrins expressed in LMC neurons interact with Eph receptors in cis and attenuate their sensitivity to ephrins in the limb, loss of ephrin function in LMC neurons MAPK inhibitor should affect the fidelity of axon trajectory choice in the limb. To test this idea, we knocked down ephrin-A5 or ephrin-B2 expression by introducing inhibitory siRNAs against ephrin-A5 mRNA ([eA5]siRNA) or ephrin-B2 mRNA ([eB2]siRNA) into LMC neurons. To do this, we coelectroporated either siRNA with a GFP expression plasmid into the chick lumbar neural tube before LMC neuron specification and axon entry into the limb

(HH st. 18/19), and examined GFP+ motor axons in dorsal and ventral divisional nerve branches exiting the crural plexus at HH st. 28/29 ( Kania and Jessell, 2003). Electroporation of [eA5]siRNA or [eB2]siRNA and GFP plasmid significantly reduced ephrin-A5 or ephrin-B2 protein expression compared with embryos electroporated with a control GFP plasmid or scrambled siRNAs, and did not cause any change in their differentiation or Eph receptor expression ( Figure S2; Figure S3). Quantification of GFP+ LMC neurons indicated nearly equal proportions of electroporated

cells in both LMC divisions ( Figure S2; Figure S3). Cyclopamine mw To determine whether a knockdown of ephrin-A5 or ephrin-B2 affected the choice of limb trajectory by LMC axons, we quantified the proportions of GFP+ axons in the dorsal (d) and ventral (v) divisional limb nerve branches by integrating fluorescence intensities of a series of hindlimb section images in multiple embryos MYO10 for each experimental condition (Kao et al., 2009 and Luria et al., 2008). In embryos coelectroporated

with [eA5]siRNA and GFP, significantly higher proportions of GFP+ axons were observed in the dorsal nerve branches when compared with both GFP and scrambled [eA5]siRNA controls ( Figures 2A–2C; p < 0.001 versus controls). This axonal misrouting defect was rescued by mouse ephrin-A5 expression ( Figure S2). To determine whether ephrin-A5 knockdown results in redirection of medial LMC axons into the dorsal limb mesenchyme, we coelectroporated [eA5]siRNA with the e[Isl1]::GFP plasmid, which preferentially labels medial LMC motor neurons and their axons ( Kao et al., 2009), and as controls, the e[Isl1]::GFP plasmid only. In embryos coelectroporated with [eA5]siRNA and e[Isl1]::GFP, a significantly higher proportion of GFP+ axons was observed in the dorsal limb nerve when compared with e[Isl1]::GFP controls ( Figures 2D and 2E; p < 0.001). These findings indicate that ephrin-A5 is required for the fidelity of limb trajectory selection by medial LMC axons ( Figure 2N). We next asked whether LMC-expressed ephrin-B2 is required for the choice of limb trajectory.