| Proper pathfinding of axons to their correct targets is fundamental to nervous system development. This pathfinding process is orchestrated by axon guidance molecules that can act either as attractants and support axon growth, or as inhibitory signals steering axons away from incorrect destinations. In the visual system, the axons of retinal ganglion cells are directed by a series of guidance molecules along the developing visual pathway. The earliest retinal axon guidance decisions include exiting the retina through the optic disc and traversing the length of the developing optic nerve. These tasks are known to involve the guidance cues Netrin-1, L1, and laminin. Netrin-1 is expressed by neuroepithelial cells surrounding the optic disc and along the optic nerve, and is necessary for retinal axon, exit through the optic disc. Laminin and L1 are growth substrates available to retinal axons throughout the early pathfinding process, and both have been reported to modulate axon responses to other- guidance molecules. Here we identified, through a mRNA expression screen, the presence of transmembrane Sema5A at the optic disc, and along the optic nerve, at times when retinal axons were passing through these regions. Given that semaphorins are a large, and generally inhibitory, family of axon guidance molecules, we tested how Sema5A influenced retinal axons, and whether this response was modulated by other guidance signals encountered along the retinal pathway. In both collapse assays and neurite outgrowth assays, our results showed that Sema5A invariantly inhibited retinal axons in the context of Netrin-1, L1, or laminin signaling. This suggested that Sema5A inhibited these same axons in vivo as they traveled through the optic disc and along the optic nerve. A polyclonal antibody confirmed that Sema5A was distributed in a ring-like pattern surrounding axons in the retinal pathway. In addition, antibody perturbation of Sema5A function led to optic nerve defasciculation. Thus, these studies show that Sema5A acts invariantly on retinal axons as they progress through pathfinding tasks directed by other guidance molecules, and that the use of inhibitory sheaths is a mechanism for maintaining nerve integrity during central nervous system development. |
