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.