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Motor neurons of the spinal cord are responsible for the assembly of neuromuscular connections indispensable for basic locomotion and skilled movements. A precise spatial relationship exists between the position of motor neuron cell bodies in the spinal cord and the course of their axonal projections to peripheral muscle targets. Motor neuron innervation of the vertebrate limb is a prime example of this topographic organization and by virtue of its accessibility and predictability has provided access to fundamental principles of motor system development and neuronal guidance. The seemingly basic binary map established by genetically defined motor neuron subtypes that target muscles in the limb is directed by a surprisingly large number of directional cues. Rather than being simply redundant, these converging signaling pathways are hierarchically linked and cooperate to increase the fidelity of axon pathfinding decisions. A current priority is to determine how multiple guidance signals are integrated by individual growth cones and how they synergize to delineate class-specific axonal trajectories., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

When the axons that carry signals to muscles are growing, they rely on help from Frizzled3-a protein that is known to perform a number of other important functions in cells-to reach their final destination.

Disruption of the Frizzled3 (Fz3) gene leads to defects in axonal growth in the VII(th) and XII(th) cranial motor nerves, the phrenic nerve, and the dorsal motor nerve in fore- and hindlimbs. In Fz3(-/-) limbs, dorsal axons stall at a precise location in the nerve plexus, and, in contrast to the phenotypes of several other axon path-finding mutants, Fz3(-/-) dorsal axons do not reroute to other trajectories. Affected motor neurons undergo cell death 2 days prior to the normal wave of developmental cell death that coincides with innervation of muscle targets, providing in vivo evidence for the idea that developing neurons with long-range axons are programmed to die unless their axons arrive at intermediate targets on schedule. These experiments implicate planar cell polarity (PCP) signaling in motor axon growth and they highlight the question of how PCP proteins, which form cell-cell complexes in epithelia, function in the dynamic context of axonal growth. DOI: http://dx.doi.org/10.7554/eLife.01482.001.

Abstract: Our previous surgical manipulations have shown that the target ectoderm is necessary for the initial formation of one of the major cutaneous nerves in the embryonic chick limb (Honig, M.G., Camilli, S.J., Xue, Q.S., 2004. Ectoderm removal prevents cutaneous nerve formation and perturbs sensory axon growth in the chick hindlimb. Dev. Biol. 266, 27–42.). Moreover, the target ectoderm is required during a critical time period, at ∼St. 24, when those axons are about to diverge from the hindlimb plexus. To elucidate the underlying mechanisms, here we examined the effects of removing the ectoderm at St. 24 on a variety of molecules expressed within the limb. We find that, while ectoderm removal is accompanied by changes in the expression of Lmx1, fibronectin, EphA7, cDermo-1, and in the complement of muscle cells, these changes do not account for the cutaneous nerve deficit. In contrast, an upregulation of PNA-binding sites and a downregulation of Bmp4 appear to be associated with this nerve deficit. Exogenous BMP4 reversed the effect of ectoderm removal on cutaneous nerve formation, but did not act as a chemoattractant. Our results suggest that BMP4, together with permissive and repulsive molecules that growing cutaneous axons encounter in the local environment and with signaling molecules, originating from and/or dependent on the ectoderm, work in concert to ensure proper cutaneous nerve formation. [Copyright &y& Elsevier]

Target tissues are thought to provide important cues for growing axons, yet there is little direct evidence that they are essential for axonal pathfinding. Here we examined whether target ectoderm is necessary for the formation of cutaneous nerves, and for the normal growth and guidance of cutaneous axons as they first enter the limb plexus. To do this, we removed a patch of ectoderm from the chick hindlimb at various times during early axon outgrowth. We find there is a critical period when cutaneous nerve formation requires target ectoderm. When the ectoderm is absent during this time, axons progress into the limb more slowly and, although a few sensory axons occasionally diverge a short distance from the plexus, they do not form a discrete nerve that travels to the skin. A few days later, when the nerve pattern is mature, axons normally destined for the ‘deprived’ cutaneous nerve are not segregated appropriately within the plexus. Some cutaneous axons are instead misdirected along an inappropriate cutaneous nerve, while others have seemingly failed to reach their correct target, or a suitable alternative, and died. These results demonstrate that the target ectoderm is necessary for normal sensory axon growth and guidance in the hindlimb. [Copyright &y& Elsevier]

In the developing chick hindlimb, sensory axons, which grow together in bundles as they extend distally, and the motoneuron axons they encounter express the cell adhesion molecule L1. Following injection of function-blocking anti-L1 antibodies into the limb at stage 25, some sensory axons choose inappropriate peripheral nerves even though motoneuron pathfinding is unaffected. Here, to further elucidate L1''s role, we assessed the effects of this perturbation using pathway tracing, immune labeling, confocal microscopy, and electron microscopy. After L1 blockade, sensory axons were still bundled and closely apposed. However, clear signs of decreased adhesion were detectable ultrastructurally. Further, sensory axons grew into the limb more slowly than normal, wandering more widely, branching more frequently, and sometimes extending along inappropriate peripheral nerves. Sensory axons that ultimately projected along different cutaneous nerves showed increased intermixing in the spinal nerves, due to errors in pathfinding and also to a decreased ability to segregate into nerve-specific fascicles. These results suggest that, in the highly complex in vivo environment, as in tissue culture, L1 stimulates axon growth and enhances fasciculation, and that these processes contribute to the orderly, timely, and specific growth of sensory axons into the limb. [Copyright &y& Elsevier]