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 galliforms. The morphology and wiring at brachial levels of the chicken embryonic spinal cord resemble those of ephrin-B3 null mice. 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.

Peripheral and intraspinal feedback is required to shape and update the output of spinal networks that execute motor behavior. We report that lumbar dI2 spinal interneurons in chicks receive synaptic input from afferents and premotor neurons. These interneurons innervate contralateral premotor networks in the lumbar and brachial spinal cord, and their ascending projections innervate the cerebellum. These findings suggest that dI2 neurons function as interneurons in local lumbar circuits, are involved in lumbo-brachial coupling, and that part of them deliver peripheral and intraspinal feedback to the cerebellum. Silencing of dI2 neurons leads to destabilized stepping in P8 hatchlings, with occasional collapses, variable step profiles and a wide-base walking gait, suggesting that dI2 neurons may contribute to the stabilization of the bipedal gait.

Peripheral and intraspinal feedback is delivered to the cerebellum through the spinocerebellar tracts in order to shape and update the output of spinal networks that execute motor behavior. However, genetic inaccessibility of the tract neurons prevented further elucidation of the ascending circuits. We report that genetically targeted lumbar dI2 interneurons, form a part of the avian ventral spinocerebellar tract. Lumbar dI2s receive synaptic input from afferents and pre-motoneurons. They innervate contralateral dI2s and pre-motoneurons at lumbar cord and their ascending axons give off collaterals to innervate contralateral brachial dI2s and premotoneurons, enroute to the cerebellum. Collectively these findings suggest that dI2s deliver peripheral and intraspinal feedback to the cerebellum, that they also function as interneurons in local lumbar circuits and involved in lumbo-brachial coupling. Targeted silencing of dI2s leads to destabilized stepping in P8 hatchlings, suggesting that the activated dI2s may contribute to stabilization of the bipedal gait.

No abstract

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.TeaserWalking vs flying: Deciphering the organization and evolution of the neuronal network that controls wing flapping in birds.