The role of class 3 semaphorins in guiding olfactory sensory axons to their protoglomerular targets in the zebrafish olfactory bulb

The convergence of olfactory sensory axons onto topographically fixed, odorant receptor specific glomeruli in the olfactory bulb (OB) is a complex biological problem. Early in development, axons from olfactory sensory neurons (OSNs) extend into the OB where they innervate distinct neuropilar structures called protoglomeruli. Protoglomeruli subsequently segregate into mature glomeruli. The zebrafish is an excellent system in which to study early protoglomerular targeting. At 72 hours post-fertilization, the zebrafish OB contains 12 easily identifiable protoglomeruli. The objective of this thesis work was to identify axon guidance cues and receptors that direct olfactory sensory axons to their protoglomerular targets. Here, I describe a novel role for Sema3D/Nrp1a signaling in protoglomerular targeting. An OR111-7:IRES:Gal4 transgene labels a subset of olfactory axons that target the ventromedially located central zone (CZ) protoglomerulus. Sema3D is expressed in the anterior OB. OR111-7 transgene expressing axons terminate posterior to sema3D expression. In sema3D mutants, a subset of OR111-7 transgene expressing axons misproject to the dorsal zone (DZ) protoglomerulus which is dorsal and anterior to the CZ. Four zebrafish neuropilins (nrps), essential receptor components for class 3 semaphorins, are differentially expressed in OSNs. OR111-7 transgene expressing neurons express nrp1a, nrp1b, and/or nrp2b. Loss of either nrp1a or nrp2b phenocopies sema3D mutants. OR111-7 transgene expressing axons are misguided in nrp1a-/+;sema3D-/+ embryos, but not in nrp2b-/+;sema3D-/+ embryos. The phenotype is not accentuated in sema3D;nrp1a double mutants as compared to sema3D or nrp1a mutants. Re-expression of nrp1a in OR111-7 transgene expressing OSNs corrects the DZ targeting error observed in nrp1a mutants. These data support a model wherein sema3D expressed in the anterior OB repels nrp1a and OR111-7 transgene expressing axons towards the CZ. Additional class 3 semaphorins, not previously implicated in olfactory development, are expressed in the olfactory system during development. Mutations in nrp1a, nrp1b and nrp2b result in sema3D independent targeting errors. These findings suggest that many class 3 semaphorins cooperate in protoglomerular map formation. These studies highlight the utility of the zebrafish as a model for the study of early olfactory axon guidance, and provide the foundation for the future study of Semaphorin/Neuropilin signaling in the developing olfactory system