Natural loss of function of ephrin-B3 shapes spinal flight circuitry in birds

Flight in birds evolved through patterning of the wings from forelimbs and transition from alternating gait to synchronous flapping. In mammals, the spinal midline guidance molecule ephrin-B3 instructs the wiring that enables limb alternation, and its deletion leads to synchronous hopping gait. Here we show that the ephrin-B3 protein in birds lacks several motifs present in other vertebrates, diminishing its affinity for the EphA4 receptor. The avian ephrin-B3 gene lacks an enhancer that drives midline expression, and is missing in Galliformes. The morphology and wiring at brachial levels of the chick spinal cord resemble those of ephrin-B3 null mice. Importantly, dorsal midline decussation, evident in the mutant mouse, is apparent at the chick brachial level, and is prevented by expression of exogenous ephrin-B3 at the roof plate. Our findings support a role for loss of ephrin-B3 function in shaping the avian brachial spinal cord circuitry and facilitating synchronous wing flapping.