Developing neuronal growth cones respond to a number of post‐transcriptionally modified guidance cues to establish functional neural networks. The Semaphorin family has well‐established roles as both secreted and transmembrane guidance cues. Here, we describe the first evidence that a transmembrane Semaphorin, Semaphorin 5B (Sema5B), is proteolytically processed from its transmembrane form and can function as a soluble growth cone collapsing guidance cue. Over‐expression of A Disintegrin and Metalloprotease (ADAM)‐17, results in an enhanced release of the Sema5B ectodomain, while removal of a predicted ADAM‐17 cleavage site prevents its release. In contrast, knockdown of ADAM‐17 does not significantly reduce Sema5B release, indicating there are additional unknown compensating proteases. This modulation of the transmembrane Sema5B to a diffusible cue represents a sophisticated method to regulate neuronal guidance in vivo.

During vertebrate development, centrally projecting sensory axons of the dorsal root ganglia neurons first reach the embryonic spinal cord at the dorsolateral margin. Instead of immediately projecting into the grey matter, they bifurcate and extend rostrally and caudally to establish the longitudinal dorsal funiculus during a stereotyped waiting period of approximately 48 h. Collateral fibres then extend concurrently across multiple spinal segments and project to their appropriate targets within the grey matter. This rostrocaudal extension of sensory afferents is crucial for the intersegmental processing of information throughout the spinal cord. However, the precise cues that prevent premature entry during the waiting period remain to be identified. Here, we show that semaphorin 5B (Sema5B), a member of the semaphorin family of guidance molecules, is expressed in the chick spinal cord during this waiting period and dorsal funiculus formation. Sema5B expression is dynamic, with a reduction of expression apparent in the spinal cord concomitant with collateral extension. We show that Sema5B inhibits the growth of NGF-dependent sensory axons and that this effect is mediated in part through the cell adhesion molecule TAG-1. Knockdown of Sema5B in the spinal cord using RNA interference leads to the premature extension of cutaneous nociceptive axons into the dorsal horn grey matter. These premature projections predominantly occur at the site of dorsal root entry. Our results suggest that Sema5B contributes to a repulsive barrier for centrally projecting primary sensory axons, forcing them to turn and establish the dorsal funiculus.

The semaphorins are a large family of secreted and membrane associated proteins that play numerous key roles in the development and function of the nervous system and other tissues. They have been primarily associated with their function as guidance cues in the developing nervous system. In general, semaphorins have been shown to function as inhibitory guidance cues; however there are also numerous examples where they can function as attractive or permissive cues. Thus it is important to employ a variety of assays to test for semaphorin function. While numerous assays have been established for secreted semaphorins, testing the function of transmembrane semaphorins has been challenging. In this chapter we outline two assays that we have used extensively to test their function. In one assay we examine the effect of a constant source of a transmembrane semaphorin on neurite outgrowth and in a second assay we examine whether neurons will actively avoid growing across islands of cells expressing a transmembrane semaphorin. We have found both assays to be relatively easy to perform and useful to test semaphorin function and signaling.