Poster abstracts

Poster number 50 submitted by Jona Hilario

Semaphorin 5A is a bifunctional axon guidance cue for axial motoneurons in vivo.

Jona D. Hilario (Molecular, Cellular and Developmental Biology Program, The Ohio State University), Louise Rodino-Klapac (Nationwide Childrens Hospital), Chunping Wang (Center for Molecular Neurobiology, The Ohio State University), Christine Beattie (Department of Neuroscience, Center for Molecular Neurobiology, The Ohio State University)

Abstract:
Although numerous axon guidance cues have been identified, few cues have been characterized that function in vertebrate motor axon guidance. Class 5 Semaphorins (Sema) are transmembrane protein ligands that are unique in that they contain a Sema domain and multiple thrombospondin repeats in their extracellular domain. Semaphorin 5A (Sema5A), a class 5 semaphorin has been shown to have a bifunctional role in guiding axons both in culture and in the rat diencephalon. Here, we show that Sema5A is expressed in the ventral myotome of developing zebrafish during the period of motor axon outgrowth leading us to question whether this molecule functions in motor axon guidance in zebrafish. To test this, we knocked down Sema5A using anti-sense morpholino. Knocking down Sema5A results in 20% of CaP axons observed being delayed in extending to the ventral muscle and 27% of CaP axons observed displaying an aberrant branching phenotype. Both phenotypes were rescued by injection of full-length rat sema5A mRNA. Adding back the rat sema5A sema domain mRNA alone into sema5A morphants also rescued both phenotypes though the rescue of the delay phenotype was relatively moderate. Conversely, adding back the rat sema5A thrombospondin repeat (TSR) domain mRNA alone into sema5A morphants rescued CaP axon extension but not CaP axon branching. To elucidate the Sema5A receptor involved in these functions, we looked at interactions of Sema5A with known Plexins by doing subthreshold co-injections of morpholinos against Sema5A and specific Plexins. Plexin B1-A, Plexin B1-B, and Plexin A3 all did not synergistically interact with Sema5A to promote CaP axon branching and CaP axon extension into the ventral myotome. Sema5A and PlexinA3 subthreshold MO co-injections did, however, triple the number of axons observed per embryo exiting from ectopic points along the spinal cord; a phenotype previously observed in PlexinA3 morphants and mutants. These data suggest that Sema5a and PlexinA3 may be interacting to define specific exit points along the spinal cord. Our data suggest that Sema5a is a bifunctional axon guidance cue in CaP axon pathfinding in the developing zebrafish embryo through the different functions of its thrombospondinrepeat and sema domains and that it could potentially be a ligand for the semaphorin receptor PlexinA3.

Keywords: zebrafish, axon guidance, semaphorins