During development, growing motor axons are excluded from the ventral midline of the neural tube by diffusible chemorepellents emanating from this region. Molecular candidates for this chemorepellent activity include semaphorin D and netrin-1; the latter is known to repel trochlear motor axons. Qualitatively or quantitatively different responses to these molecules might underlie the initial deflection from the midline and subsequent segregation of motor axon trajectories. To test this idea, we have cocultured cell aggregates secreting netrin-1 or semaphorin D at a distance from tissue explants containing different motor neuron subpopulations, in collagen gels. Cranial motor axons that project dorsally in vivo such as those of the trigeminal, facial, and glossopharyngeal nuclei were repelled by both netrin-1 and semaphorin D. By contrast, ventrally projecting spinal motor axons and abducens axons were not affected by netrin-1. Spinal and abducens motor neurons also responded to semaphorin D. The ventrally projecting axons of oculomotor neurons were not repelled by netrin-1 or semaphorin D. Differential responsiveness to netrin-1 and semaphorin D could thus contribute to the generation of dorsal and ventral motor axon pathways during development.

Within the developing chick hindbrain, motor neurons differentiate in columns on either side of the ventral midline floor plate. Along the rostrocaudal axis, populations of motor neurons are organized segmentally with the trigeminal (V) and facial (VII) nuclei occupying successive pairs of rhombomeres. To reach their targets, motor axons follow stereotyped pathways. Branchiomotor and visceral motor axons of the Vth and VIIth nerves first project in a dorsal (lateral) direction away from the floor plate and towards the nerve exit point located in the alar plate of the even-numbered rhombomere of the pair. Having exited the hindbrain, axons grow in association with the cranial sensory ganglia before branchiomotor axons enter the branchial arches. We have investigated some of the factors that might guide cranial motor axons using a three-dimensional collagen gel culture system. When explants of hindbrain basal plate containing trigeminal or facial motor neurons were co-cultured with floor plate explants, axon outgrowth from the side facing the floor plate was inhibited in a manner consistent with chemorepulsion. When basal plate explants that contained an exit point were cultured alone, motor axons grew to the exit point and then stopped. When basal plate explants were co-cultured with trigeminal ganglia, motor outgrowth was increased in comparison with that in control cultures, suggesting a trophic influence. The findings presented here indicate that motor pathways are elaborated due to a progression of signals to which the growth cones respond in sequence.