Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Axon Guidance of Olfactory Sensory Neurons in Zebrafish
Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute, Wako-shi, Saitama 351-0198, Japan
Correspondence to be sent to: Yoshihiro Yoshihara, e-mail: yoshihara{at}brain.riken.jp
Key words: axon guidance, olfactory sensory neurons, zebrafish
The guidance of growing axons to their appropriate targets is a crucial process
for the establishment of complex but highly ordered neural circuits and the proper
functioning of the nervous system. The axonal convergence of olfactory sensory neurons
(OSNs) expressing a given odorant receptor (OR) onto spatially invariant glomeruli
(Ressler et al., 1994
;
Vassar et al., 1994;
Mombaerts et al., 1996) is
one of the most exciting examples of precise axonal targeting. Genetic deletions or
substitutions of specific OR genes in mice suggested that the ORs themselves play an
instructive role in the guidance of OSN axons, but guidance molecules other than ORs are
also required for precise glomerular targeting (Mombaerts et al., 1996;
Wang et al., 1998). Indeed,
a large number of axon guidance and cell recognition molecules are expressed in the
developing olfactory system, with dynamic spatiotemporal patterns (Yoshihara et al., 1997;
Pasterkamp et al., 1999;
Astic et al., 2002;
St John et al., 2002).
However, functional evidence for their involvement in the precise glomerular targeting is
limited. To learn about molecular basis of specific connectivity of the olfactory neural
circuitry, we are studying molecular and cellular mechanisms underlying axon guidance of
OSNs in zebrafish, an excellent vertebrate model system.
The zebrafish offers many advantages over other model organisms. It is well suited to
forward genetics because of large clutch size and relatively short generation time.
Externally fertilized eggs can be easily manipulated. Its nervous system has reduced
complexity and high similarity to that of higher vertebrates: zebrafish olfactory bulb
(OB) contains only 
80 glomeruli, as compared with 18Ä00 in rodents
(Baier and Korsching, 1994); optical
imaging techniques revealed that individual odorants can elicit activity in specific
subsets of glomeruli in the OB as in rodents (Friedrich and Korsching, 1997, 1998). A great advantage for
studying axon guidance mechanisms is the optical transparency of zebrafish during early
development. By combining transgenesis with the use of fluorescent proteins such as green
fluorescent protein (GFP), it is possible to visualize dynamic axon behavior in living
embryos (Dynes and Ngai, 1998;
Higashijima et al., 2000).
Thus, we are taking a two-step strategy to identify genes that control precise axonal
targeting of OSNs: (i) establishment of transgenic zebrafish lines in which GFP is
expressed in specific subpopulations of OSNs and (ii) analysis of existing mutants by
crossing with the GFP-expressing transgenic lines or application of reverse genetic
approach using antisense morpholino oligonucleotides to the transgenic lines.
In many terrestrial vertebrates including rodents, there are two distinct olfactory
systems: main and accessory. In the main olfactory system, ciliated sensory neurons lying
in the olfactory epithelium (OE) sense volatile odorant molecules and transmit their
information to the main OB. In the accessory olfactory system, on the other hand,
microvillous sensory neurons populating the vomeronasal organ sense pheromone molecules
and send their information to the accessory OB. In contrast, fish including zebrafish
have a single OE and a single OB. Ciliated and microvillous OSNs occupy the same OE, and
the somata of each type of OSNs are situated at different positions along the
apical-basal axis (Yamamoto, 1982;
Morita and Finger, 1998). We have
established two transgenic zebrafish lines in which GFP is expressed in either ciliated
or microvillous OSNs (unpublished data). Immunohistochemical analysis of the OB from each
transgenic line revealed that ciliated and microvillous OSNs projected their axons to
mutually exclusive sets of glomeruli in the adult OB. Furthermore, we were able to obtain
high-resolution images of the axonal trajectories of each type of OSNs in living embryos
throughout development. Our ongoing analysis of existing mutants under the transgenic
background has identified an interesting one in which the navigation of OSN axons to the
OB is perturbed. Furthermore, these GFP-expressing transgenic lines can be used as
starting strains for mutagenesis to find new mutations that affect OSN axon pathfinding.
We expect that these transgenic lines will allow us to discover many genes critical for
the formation and maintenance of precise neural connectivity patterns in the zebrafish
olfactory system.
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