Ephrin-B3 is the midline barrier that prevents corticospinal tract axons from recrossing, allowing for unilateral motor control.

Growing axons follow highly stereotypical pathways, guided by a variety of attractive and repulsive cues, before establishing specific connections with distant targets. A particularly well-known example that illustrates the complexity of axonal migration pathways involves the axonal projections of motor neurons located in the motor cortex. These projections take a complex route during which they first cross the midline, then form the corticospinal tract, and ultimately connect with motor neurons in the contralateral side of the spinal cord. These obligatory contralateral connections account for why one side of the brain controls movement on the opposing side of the body. The netrins and slits provide well-known midline signals that regulate axonal crossings at the midline. Herein we report that a member of the ephrin family, ephrin-B3, also plays a key role at the midline to regulate axonal crossing. In particular, we show that ephrin-B3 acts as the midline barrier that prevents corticospinal tract projections from recrossing when they enter the spinal gray matter. We report that in ephrin-B3(-/-) mice, corticospinal tract projections freely recross in the spinal gray matter, such that the motor cortex on one side of the brain now provides bilateral input to the spinal cord. This neuroanatomical abnormality in ephrin-B3(-/-) mice correlates with loss of unilateral motor control, yielding mice that simultaneously move their right and left limbs and thus have a peculiar hopping gait quite unlike the alternate step gait displayed by normal mice. The corticospinal and walking defects in ephrin-B3(-/-) mice resemble those recently reported for mice lacking the EphA4 receptor, which binds ephrin-B3 as well as other ephrins, suggesting that the binding of EphA4-bearing axonal processes to ephrin-B3 at the midline provides the repulsive signal that prevents corticospinal tract projections from recrossing the midline in the developing spinal cord.