In the present study, we have tackled this issue by the extensive use of targeted cell lineage and conditional gene manipulation in mouse, combined with in vitro live axon imaging. First, genetic manipulations that completely blocked motor projections

triggered randomized formation of either epaxial or hypaxial sensory nerves. Second, conditional or systemic removal of motor axonal EphA3/4 triggered selective loss of epaxial sensory projections, while preserving epaxial motor projections. Third, subsequent gene replacement experiments in mice revealed that, intriguingly, the requirement of EphA3/4 for determining epaxial sensory projections operates independently from the EphA3/4 repulsive forward signaling involved in sensory-motor axon segregation. LY294002 concentration Palbociclib in vivo Herein, reconstituting EphA4 extracellular domain expression on epaxial motor axons in EphA3/4-deficient mice effectively rescued epaxial sensory projections, but not the misrouting of motor axons into DRGs triggered by the loss of EphA3/4 repulsive forward signaling. Fourth, in vivo genetic interaction data and in vitro experiments indicated that motor axonal EphAs act by reverse signaling through cognate ephrin-A binding partners on sensory growth cones. Fifth, live axon imaging revealed that motor axons pre-extending in vitro induced sensory growth cones to track along their

trajectories. Sixth, these sensory growth cone tracking behaviors required EphA3/4 ectodomain expression on motor axons or ephrin-A2/5 expression on sensory axons, but did not require EphA3/4 signaling in motor axons proper. Seventh, recombinant EphA ectodomains were sufficient to induce sensory axon extension in vitro, which involved ephrin-A2/5

expressed by sensory axons. EphA3/4 therefore fulfills two diametrically opposed functions during peripheral nerve assembly. Initially, EphA3/4 repulsive forward signaling assures Fossariinae the segregation of epaxial motor axons from proximal sensory pathways (Figures 9A–9A″) (Gallarda et al., 2008). Subsequently, EphA3/4 operate through the reverse activation of ephrin-As on sensory growth cones to couple sensory projections to epaxial motor pathways (Figures 9B–9B″) (this study). What determines whether kinase-dependent EphA3/4 forward signaling or kinase-independent EphA3/4 reverse signaling are elicited between epaxial motor and sensory axons? A key factor is likely the developmental status of epaxial motor axon extension relative to sensory projections, because it dictates the specific growth cone-axon encounters possible between epaxial motor and sensory axons (Figures S8A and S8B). Herein, the initial extension of epaxial motor axons is predicted to favor interactions of epaxial motor growth cones with sensory growth cones and axons extending from DRGs within the same spinal segment (Figure S8A).