Robo2 is required for establishment of a precise glomerular map in the zebrafish olfactory system

Abstract

Olfactory sensory neurons (OSNs) expressing a given odorant receptor project their axons to specific glomeruli, creating a topographic odor map in the olfactory bulb (OB). The mechanisms underlying axonal pathfinding of OSNs to their precise targets are not fully understood. Here, we demonstrate that Robo2/Slit signaling functions to guide nascent olfactory axons to the OB primordium in zebrafish. robo2 is transiently expressed in the olfactory placode during the initial phase of olfactory axon pathfinding. In the robo2 mutant, astray (ast), early growing olfactory axons misroute ventromedially or posteriorly, and often penetrate into the diencephalon without reaching the OB primordium. Four zebrafish Slit homologs are expressed in regions adjacent to the olfactory axon trajectory,consistent with their role as repulsive ligands for Robo2. Masking of endogenous Slit gradients by ubiquitous misexpression of Slit2 in transgenic fish causes posterior pathfinding errors that resemble the astphenotype. We also found that the spatial arrangement of glomeruli in OB is perturbed in ast adults, suggesting an essential role for the initial olfactory axon scaffold in determining a topographic glomerular map. These data provide functional evidence for Robo2/Slit signaling in the establishment of olfactory neural circuitry in zebrafish.

Requirement for Slit-1 and Robo-2 in Zonal Segregation of Olfactory Sensory Neuron Axons in the Main Olfactory Bulb

“…Members of the Robo (roundabout) family of axon guidance cue receptors have been implicated in the targeting of OSN axons in Drosophila and zebrafish (Jhaveri et al, 2004;Miyasaka et al, 2005). Moreover, we demonstrated previously that the Slit and Robo families of axon guidance cues can promote zonal segregation of vomeronasal axons in the accessory olfactory system in mice (Cloutier et al, 2004).…”

Mutually Exclusive Glomerular Innervation by Two Distinct Types of Olfactory Sensory Neurons Revealed in Transgenic Zebrafish

“…This feature will enable us to efficiently screen and analyze mutant zebrafish that show impairment in the projection of OSN axons. For instance, we have identified Robo2 as a crucial axon guidance molecule for the establishment of a precise glomerular map in the OB by crossing the OMP:YFP transgenic fish with the robo2 mutant fish astray (Miyasaka et al, 2005).…”

“…The 6 and 2 kb fragments upstream of the zOMP translation initiation site were used to drive the expression of EYFP-Mem (BD Biosciences Clontech, Palo Alto, CA), a fusion protein consisting of the N-terminal 20 aa of GAP-43 (termed gap) and enhanced yellow fluorescent protein (EYFP), as described previously (Miyasaka et al, 2005). pOMP 2k :gap-CFP-S.…”

“…The generation of transgenic zebrafish with pOMP 6k :gap-YFP and pOMP 2k :gap-YFP were described previously (Miyasaka et al, 2005), and one of the established lines, Tg(pOMP 2k :gap-YFP) rw032a , was used in this study and abbreviated as OMP:YFP. Plasmid constructs for transgenesis with I-SceI were purified using the QIAfilter Plasmid Maxi kit (Qiagen, Hilden, Germany) and prepared at 10 ng/l in 0.5ϫ commercial I-SceI buffer containing 1 U/l I-SceI (New England Biolabs, Beverly, MA) and 0.05% Phenol Red.…”

Robo1 and Robo2 Control the Development of the Lateral Olfactory Tract

“…Robo receptors play a major role in guiding forebrain axons in vertebrates (Miyasaka et al, 2005;Andrews et al, 2006).…”