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S.F. Oster, M. Bodeker, F. He, D.W. Sretavan; Inhibitory Function of Transmembrane Semaphorin5A During Optic Nerve Development . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1659.
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Purpose: Previous studies have shown that the attractive axon guidance molecules L1, laminin, and Netrin-1 govern retinal ganglion cell (RGC) axon pathfinding in the developing retina and optic nerve. In a screen for additional molecules contributing to this process, we found that Sema5A, a transmembrane semaphorin, was expressed at the embryonic mouse optic disc and nerve during the period of axon pathfinding through these regions. Given that semaphorins are generally inhibitory molecules, and that growth cone responses to a given guidance cue can be altered by concomitant exposure to a second cue, we studied how RGC axon responses to Sema5A were affected by co-exposure to L1, laminin, and Netrin-1. Methods: Recombinant protein consisting of Sema5A extracellular domain was fused to human Fc. Embryonic mouse retinal axons from explants were used in growth cone collapse, substrate choice, and neurite outgrowth assays. A Sema5A specific rabbit polyclonal antibody was made and used in immunostaining and function blocking studies. Results: Sema5A inhibited RGC growth cones in the context of L1, laminin, or Netrin-1 dependent axon outgrowth in vitro, suggesting that Sema5A invariantly inhibited retinal axons throughout their course in the optic disc and nerve. Immunostaining using a Sema5A specific antibody showed Sema5A protein distribution in a ring-like pattern surrounding RGC axons exiting the retina, consistent with an inhibitory ensheathing function. Antibody blockade of Sema5A function in living embryo preparations resulted in RGC axons defasiculation from the optic nerve bundle, demonstrating an involvement of Sema5A in normal optic nerve development. Conclusions: In addition to growth promoting axon guidance molecules, the visual system also uses inhibitory molecules such as Sema5A to serve an ensheathing function, to ensure proper development of the embryonic optic nerve.
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