Lbx1-expressing cells lacking the repellent EphA4 receptor are involved in axonal midline crossing in the spinal cord and evoke a minor gait defect

Most limbed animals, including mice and human beings, show alternating hindlimb movement mediated by neuronal circuits in the spinal cord. However, when the repulsive EphA4 receptor expressed by subsets of spinal neurons is mutated, hindlimbs lose their typical left-right alternating pattern and as a consequence, mice exhibit a hopping gait. EphA4 tyrosine kinase receptor binds to its ligand ephrinB3 at the midline of the spinal cord resulting in axonal growth cone collapse. Therefore, in wild type mice, EphA4-expressing axons are prevented from crossing the midline towards the contralateral side. In full EphA4-/- mutants, it has been suggested that the hopping gait is caused by an increased number of axons derived from excitatory neurons crossing the spinal midline (Kullander 2003, Restrepo 2011). However, it remained unclear, which subpopulations of spinal interneurons are misguided towards the contralateral side and are involved in the observed hopping gait phenotype. Hence, we aim to determine the cellular origin contributing to axon misguidance and hopping gait in EphA4-/- mutant mice, by influencing the balance between excitation and inhibition across the spinal midline. Among 11 main neuron populations in the spinal cord, the interneurons derived from the progenitor domain territory dorsal dI4-6 and marked by the transcription factor Lbx1, were targeted in this study. Here, we investigated the premotor interneuron distribution of motor neurons targeting specific hindlimb muscles in EphA4 mutant mice by means of monosynaptic rabies tracing technique. We also assessed the gait behavior on a treadmill in the conditional EphA4 mutant mice, whose EphA4 receptor was deleted in Lbx1-expressing neurons. We found that a deletion of EphA4 in Lbx1-positive neurons resulted in aberrant axon guidance of dorsal neurons across the spinal midline and minor gait defects such as a hopping gait at low velocities on the treadmill and a reduced swing time during alternating gait. Moreover, 3-week old conditional EphA4 mutants perfomed a slight aberrant hopping gait at higher velocities compared to adults. Therefore, Lbx1-expressing interneurons appear to be partially responsible for the phenotypes observed in full EphA4 mutant mice. In conclusion, we show that the EphA4 receptor plays an important role in preventing axons of Lbx1-expressing interneurons from crossing the spinal midline. Further, EphA4-expression in Lbx1-positive neurons is essential to conserve a complete alternating gait. Lbx1-expressing neurons might be one component of several cell types contributing to the locomotor CPG. Moreover, we also found that deletion of the EphA4 receptor in all inhibitory neurons of conditional EphA4flox/-vGATCre/+ mutant mice caused a partial hopping gait demonstrating that proper axon guidance of inhibitory neurons beside excitatory neurons is important to maintain alternating gait. Finally, although alpha2 chimaerin was shown to be an EphA4 downstream effector and full alpha2 chimaerin mutant mice exhibited a hopping gait (Beg 2007; Wegmeyer 2007), we found no anatomical and gait behavioral defects in the conditional alpha2 chimaerin mutant mice, lacking alpha2 chimaerin in Lbx1-positive cells. In addition, full alpha2 chimaerin-/- mutants displayed significantly decreased synchronous hindlimb movement compared to the full EphA4-/- mutant. These findings suggests that deletion of a single EphA4 effector has less effect on the anatomical and gait behavioral phenotypes than it was observed for the EphA4 receptor itself

Context-Dependent Gait Choice Elicited by EphA4 Mutation in Lbx1 Spinal Interneurons

The most commonly used locomotor strategy in rodents is left-right limb alternation. Mutation of the axon guidance molecule EphA4 profoundly alters this basic locomotor pattern to synchrony. Here we report that conditional mutation of EphA4 in spinal interneurons expressing the transcription factor Lbx1 degrades the robustness in the expression of left-right alternating gait during development. Lbx1 EphA4 conditional mice exhibit alternating gait when walking on ground, but synchronous gait in environments with decreased weight-load, like swimming and airstepping. Using cell-type-specific, transient pharmacogenetic silencing approaches, we attribute this behavioral gait switch to neuronal activity of dorsal Lbx1 spinal interneurons. We also found that in Lbx1 EphA4 conditional mice these dorsal interneurons form aberrant bilateral connections to motor neurons, thereby indirectly transmitting received unilateral proprioceptive sensory information to both spinal sides. Together, our findings reveal the behavioral and circuit-level impact of conditional EphA4 mutation in a transcriptionally defined spinal interneuron subpopulation