Our data so far indicate that motor axonal EphA3/4 act in a non-cell-autonomous manner to determine sensory axon projections in vitro and in vivo. This prompted us to ask whether EphA proteins would directly influence sensory axon extension in a simplified in vitro environment. To test SRT1720 research buy this, sensory axons were allowed to extend on control substrates or substrates containing recombinant EphA3 ectodomain (EphA3ECD) or paralogous EphA7ECD protein. Exposure to the EphAECD-containing substrates resulted

in markedly enhanced sensory axon extension compared to the control substrates (Figures 8A and 8B). The activity of the EphAECD proteins on sensory axon extension was observed irrespective of whether nerve growth factor (NGF) or neurotrophin-3 (NT-3) was used as neurotrophic supplements (Figures 8A and 8B). This was consistent with the requirements of EphA3/4 observed by us in vivo, which comprised both NGF-dependent cutaneous and NT3-dependent muscle sensory projections. We next asked whether EphAECD would act through ephrin-As to promote sensory axon extension. Sensory axons derived from Efna2/5null embryos displayed significantly

reduced extension in response to EphA3ECD compared to control sensory axons ( Figures 8C to 8E). Thus, EphAECDs are sufficient to promote sensory axon extension in vitro, at least in part by operating through Afatinib concentration sensory neuron-expressed ephrin-As. The present study reveals an absolute requirement of motor axon-derived signals for establishing normally patterned peripheral sensory projections and provides mechanistic insights into the axonal interactions that couple peripheral sensory and motor pathways. Below, we discuss these findings in light of previous data by us and others. In a previous study we have shown that EphA3/4

contribute to the anatomical and functional segregation of epaxial motor projections from sensory pathways and DRGs (Gallarda et al., 2008). In EphA3/4 null mutant embryos, epaxial motor axons misproject into Linifanib (ABT-869) proximal sensory pathways and DRGs, while electrophysiological recordings revealed that this results in the aberrant incorporation of motor input into sensory afferents. Sensory and/or motor neuron culture assays further showed that these phenotypes reflect a requirement for EphA3/4 repulsive signaling in motor growth cones, likely activated by their cognate ephrin-As on sensory axons (see Figures 9A–9A″). Herein, loss of EphA3/4 abolished motor growth cone repulsion induced by recombinant ephrin-A proteins or wild-type sensory axons in vitro ( Gallarda et al., 2008).