Forward signaling mediated by ephrin-B3 prevents contralateral corticospinal axons from recrossing the spinal cord midline

To investigate Eph-ephrin bidirectional signaling, a series of mutations we re generated in the ephrin-B3 locus. The absence of both forward and revers e signaling resulted in mice with mirror movements as typified by a hopping locomotion. The corticospinal tract was defective as axons failed to respe ct the midline boundary of the spinal cord and bilaterally innervated both contralateral and ipsilateral motor neuron populations. A second mutation t hat expresses a truncated ephrin-B3 protein lacking its cytoplasmic domain did not lead to hopping, indicating that reverse signaling is not required for corticospinal innervation. Ephrin-B3 is concentrated at the spinal cord midline, while one of its receptors, EphA4, is expressed in postnatal cort icospinal neurons as their fibers pathfind down the contralateral spinal co rd. Our data indicate ephrin-B3 functions as a midline-anchored repellent t o stimulate forward signaling in EphA4-expressing axons.