Microvillous Olfactory Sensory Neurons Research Articles

Ancient and nonuniform loss of olfactory receptor expression renders the shark nose a de facto vomeronasal organ.

Cartilaginous fishes are renowned for a keen sense of smell, a reputation based on behavioral observations and supported by the presence of large and morphologically complex olfactory organs. At the molecular level, genes belonging to the four families coding for most olfactory chemosensory receptors in other vertebrates have been identified in a chimera and a shark, but it was unknown whether they actually code for olfactory receptors in these species. Here we describe the evolutionary dynamics of these gene families in cartilaginous fishes using genomes of a chimera, a skate, a sawfish and eight sharks. The number of putative OR, TAAR and V1R/ORA receptors is very low and stable whereas the number of putative V2R/OlfC receptors is higher and much more dynamic. In the catshark Scyliorhinus canicula, we show that many V2R/OlfC receptors are expressed in the olfactory epithelium in the sparsely distributed pattern characteristic for olfactory receptors. In contrast, the other three vertebrate olfactory receptor families are either not expressed (OR) or only represented with a single receptor (V1R/ORA and TAAR). The complete overlap of markers of microvillous olfactory sensory neurons with panneuronal marker HuC in the olfactory organ suggests the same cell type specificity of V2R/OlfC expression as for bony fishes, i.e. in microvillous neurons. The relatively low number of olfactory receptors in cartilaginous fishes compared to bony fishes could be the result of an ancient and constant selection in favor of a high olfactory sensitivity at the expense of a high discrimination capability.

Response of Olfactory Sensory Neurons to Mercury Ions in Zebrafish: An Immunohistochemical Study.

Olfactory sensory neurons (OSNs) of fish belong to three main types: ciliated olfactory sensory neurons (cOSNs), microvillous olfactory sensory neurons (mOSNs), and crypt cells. Mercury is a toxic metal harmful for olfaction. We exposed the olfactory epithelium of zebrafish to three sublethal Hg2+ concentrations. Molecular markers specific for the different types of OSNs were immunohistochemically detected. Image analysis of treated sections enabled counting of marked cells and measurement of staining optical density indicative of the response of OSNs to Hg2+ exposure. The three types of OSNs reacted to mercury in a different way. Image analysis revealed that mOSNs are more susceptible to Hg2+ exposure than cOSNs and crypt cell density decreases. Moreover, while the ratio between sensory/nonsensory epithelium areas is unchanged, epithelium thickness drops, and dividing cells increase in the basal layer of the olfactory epithelium. Cell death but also reduction of apical processes and marker expression could account for changes in OSN immunostaining. Also, the differential results between dorsal and ventral halves of the olfactory rosette could derive from different water flows inside the olfactory chamber or different subpopulations in OSNs.

Comparative analysis of the olfactory organs in selected species of marine sharks and freshwater batoids

We present comparative analysis of the micro- and macro-morphology of the olfactory organ of six neoselachians living in different habitats: two freshwater benthopelagic batoids, Potamotrygon motoro and Potamotrygon hystrix (Potamotrygonidae) as well as four species of sharks occupying the outer continental shelves and upper slopes Mustelus mustelus (Trikidae) and Scyliorhinus canicula (Scyliorhinidae), and deep-water Galeus melastomus (Scyliorhinidae) and Etmopterus spinax (Etmopteridae). Our research used light and electron (TEM, SEM) microscopes as well as macroscopic observations. The position of the nostrils in relation to the mouth varied in these species but all of them possessed elongate olfactory rosettes with primary lamellae arranged over two rows. The types of primary lamellae were distinguished based on their arrangement on the opposite sides of the median raphe as well as morphology of secondary lamellae and distribution of sensory and non-sensory regions of olfactory epithelium lining the lamellae; these were different between batoids and sharks. Analysis of olfactory epithelium revealed the presence of microvillous olfactory sensory neurons as the only type of receptor cells in all studied species whereas ionocytes were recognized only in sharks. All species possessed one type of goblet cells (Alcian Blue or Periodic Acid Schiff positives) except for P. motoro in which both types of goblet cells were present. Moreover, apical parts of non-sensory microvillous cells that lined the basal parts of the primary lamellae, possessed PAS-positive mucous granules; these were present in both species of batoids. This analogous region was abundant in goblet cells in the species of sharks studied here. Results indicated that differences between freshwater and marine species as well as between marine species occupying various habitat zones include mainly nostrils position, density of occurrence of ionocytes and goblet cells within olfactory epithelium as well as biochemical character of mucus.

Differential nickel-induced responses of olfactory sensory neuron populations in zebrafish

The olfactory epithelium of fish includes three main types of olfactory sensory neurons (OSNs). Whereas ciliated (cOSNs) and microvillous olfactory sensory neurons (mOSNs) are common to all vertebrates, a third, smaller group, the crypt cells, is exclusive for fish. Dissolved pollutants reach OSNs, thus resulting in impairment of the olfactory function with possible neurobehavioral damages, and nickel represents a diffuse olfactory toxicant. We studied the effects of three sublethal Ni2+ concentrations on the different OSN populations of zebrafish that is a widely used biological model. We applied image analysis with cell count and quantification of histochemically-detected markers of the different types of OSNs. The present study shows clear evidence of a differential responses of OSN populations to treatments. Densitometric values for Gα olf, a marker of cOSNs, decreased compared to control and showed a concentration-dependent effect in the ventral half of the olfactory rosette. The densitometric analysis of TRPC2, a marker of mOSNs, revealed a statistically significant reduction compared to control, smaller than the decrease for Gα olf and without concentration-dependent effects. After exposure, olfactory epithelium stained with anti-calretinin, a marker of c- and mOSNs, revealed a decrease in thickness while the sensory area appeared unchanged. The thickness reduction together with increased densitometric values for HuC/D, a marker of mature and immature neurons, suggests that the decrements in Gα olf and TRPC2 immunostaining may depend on cell death. However, reductions in the number of apical processes and of antigen expression could be a further explanation. We hypothesize that cOSNs are more sensitive than mOSNs to Ni2+ exposure. Difference between subpopulations of OSNs or differences in water flux throughout the olfactory cavity could account for the greater susceptibility of the OSNs located in the ventral half of the olfactory rosette. Cell count of anti-TrkA immunopositive cells reveals that Ni2+ exposure does not affect crypt cells. The results of this immunohistochemical study are not in line with those obtained by electro-olfactogram.

Differential copper-induced death and regeneration of olfactory sensory neuron populations and neurobehavioral function in larval zebrafish

Fish rely heavily on their sense of smell to maintain behaviors essential for survival, such as predator detection and avoidance, prey selection, social behavior, imprinting, and homing to natal streams and spawning sites. Due to its direct contact with the outside environment, the peripheral olfactory system of fish is particularly susceptible to dissolved contaminants. In particular, environmental exposures to copper (Cu) can cause a rapid loss of olfactory function. In this study, confocal imaging of double-transgenic zebrafish larvae with differentially labeled ciliated and microvillous olfactory sensory neurons (OSNs) were used to examine cell death and regeneration following Cu exposure. Changes in cell morphologies were observed at varying degrees within both ciliated and microvillous OSNs, including the presence of round dense cell bodies, cell loss and fragmentation, retraction or loss of axons, disorganized cell arrangements, and loss of cells and fluorescence signal intensity, which are all indicators of cell death after Cu exposure. A marked loss of ciliated OSNs relative to microvillous OSNs occurred after exposure to low Cu concentrations for 3 h, with some regeneration observed after 72 h. At higher Cu concentrations and 24-h exposures, ciliated and microvillous OSNs were damaged with increased severity of injury with longer Cu exposures. Interestingly, microvillous, but not ciliated OSNs, regenerated rapidly within the 72-h time period of recovery after death from Cu exposure, suggesting that microvillous OSNs may be replaced in lieu of ciliated OSNs. An increase in bromodeoxyuridine labeling was observed 24 h after Cu-induced OSN death, suggesting that increased proliferation of the olfactory stem cells replaced the damaged OSNs. Olfactory behavioral analyses supported our imaging studies and revealed both initial loss and restoration of olfactory function after Cu exposures. In summary, our studies indicate that following zebrafish OSN damage by Cu, regeneration of microvillous OSNs may occur exceeding ciliated OSNs, likely via increased proliferation of the cellular reservoir of neuronal OSC precursors. Transgenic zebrafish are a valuable tool to study metal olfactory injury and recovery and to characterize sensitive olfactory neuron populations in fish exposed to environmental pollutants.

Comparative effects of cadmium, zinc, arsenic and chromium on olfactory-mediated neurobehavior and gene expression in larval zebrafish (Danio rerio)

Studies have shown that olfactory-mediated behaviors that are critical to survival can be disrupted by exposure to certain metals. Polluted waterways often contain elevated levels of metals, yet only a subset have been characterized for their potential to cause olfactory toxicity. A larval zebrafish behavioral assay was developed to characterize concentration-response curves for zinc (Zn), hexavalent chromium (Cr), and arsenate (As) olfaction inhibition. Cadmium (Cd), an established olfactory toxicant, was used as a positive control. As expected, following a 24-hour exposure to Cd, we observed a reduced response to taurocholic acid (TCA), a substrate for ciliated olfactory sensory neurons (OSNs), thus validating the behavioral assay. Zn exposure similarly decreased the olfactory response toward TCA, (IC50: 36 μg/L and 76 μg/L, for Cd and Zn, respectively). The response towards a secondary odorant L-cysteine (Cys), a substrate for ciliated and microvillous OSNs, was significantly altered by both Cd and Zn exposure, although the response to Cys was not completely removed in Zn treated larvae, suggesting preferential toxicity towards ciliated OSNs. No significant changes in olfactory responses were observed following Cr and As exposures. Exposures to binary mixtures of Cd and Zn indicated that Zn had a protective effect against Cd toxicity at low Zn concentrations. QuantiGene (QDP) RNA analysis revealed Cd to be a potent inducer of metallothionein 2 (mt2) mRNA in zebrafish larvae, and Zn to be a weak mt2 inducer, suggesting a protective role of mt2 in Cd and Zn olfactory injury. By contrast, QDP analysis of eight other genes important in mitigating the effects of oxidative stress suggested an antioxidant response to Cd, but not Zn, As, and Cr suggesting that oxidative stress was not a primary mechanism of Zn-induced olfactory dysfunction. In summary, our study indicates that Zn inhibits zebrafish olfaction at environmental concentrations and may potentially mitigate Cd induced olfactory dysfunction when present in mixtures. The zebrafish behavioral trough assay incorporating the odorants L-cysteine and TCA is an effective assay to assess the effects of metals on olfactory function.

Organization of glomerular territories in the olfactory bulb of post-embryonic wild chinook salmon Oncorhynchus tshawytscha.

The post-embryonic odor imprinting paradigm suggests Chinook salmon (Oncorhynchus tshawytscha) acquire memory to stream-specific amino acid olfactory odors prior to emergence as fry. Because effects of olfactory experience on development can be examined by mapping olfactory sensory neurons extending into distinct territories of glomerular neuropil in the olfactory bulb, glomerular patterning from early yolk-sac larva to fry was documented in wild salmonids, a temporal scale not yet thoroughly explored. Labeling olfactory sensory neurons with anti-keyhole limpet hemocyanin (anti-KLH) revealed seven spatially conserved glomerular territories visible at hatch and well established by the late yolk-sac larva developmental stage. Because of the responsiveness of microvillous olfactory sensory neurons to amino acids, corresponding glomeruli in the lateral bulbar region were mapped using anti-calretinin. The dorsolateral territory, distinct glomeruli of the lateral glomerular territory and the ventromedial glomeruli were immunoreactive to both KLH and calretinin. This study offers a morphological description of glomerular patterning in post-embryonic stages in wild Chinook salmon, a temporal window previously shown to be significant for olfactory imprinting. J. Morphol. 278:464-474, 2017. © 2017 Wiley Periodicals, Inc.

Differential response of olfactory sensory neuron populations to copper ion exposure in zebrafish

The peripheral olfactory system of fish is in direct contact with the external aqueous environment, so dissolved contaminants can easily impair sensory functions and cause neurobehavioral injuries. The olfactory epithelium of fish is arranged in lamellae forming a rosette in the olfactory cavity and contains three main types of olfactory sensory neurons (OSNs): ciliated (cOSNs) and microvillous olfactory sensory neurons (mOSNs), common to all vertebrates, and a third minor group of olfactory neurons, crypt cells, absent in tetrapods. Since copper is a ubiquitously diffusing olfactory toxicant and a spreading contaminant in urban runoff, we investigated the effect of low copper concentration on the three different OSNs in the olfactory epithelium of zebrafish, a model system widely used in biological research. Image analysis was applied for morphometry and quantification of immunohistochemically detected OSNs. Copper exposure resulted in an evident decrease in olfactory epithelium thickness. Moreover, after exposure, the lamellae of the dorsal and ventral halves of the olfactory rosettes showed a different increase in their sensory areas, suggesting a lateral migration of new cells into non-sensory regions. The results of the present study provide clear evidence of a differential response of the three neural cell populations of zebrafish olfactory mucosa after 96h of exposure to copper ions at the sublethal concentration of 30μgL−1. Densitometric values of cONS, immunostained with anti-G αolf, decreased of about 60% compared to the control. When the fish were transferred to water without copper addition and examined after 3, 10 and 30days, we observed a partial restoration of anti-G αolf staining intensity to normal condition. The recovery of cOSNs appeared sustained by neuronal proliferation, quantified with anti-PCNA immunostaining, in particular in the early days after exposure. The densitometric analysis applied to mOSNs, immunostained with anti-TRPC2, revealed a statistically significant decrease of about 30% compared to the control. For cOSNs and mOSNs, the decrement in staining intensity may be indicative of cell death, but reduction in antigen expression may not be excluded. In the post-exposure period of 1 month we did not find recovery of mOSNs. We hypothesize that cOSNs are more sensitive than mOSNs to copper treatment, but also more prompted to tissue repair. Anti-TrkA-immunopositive crypt cells appeared not to be affected by copper exposure since statistical analysis excluded any significant difference between the control and treated fish. Comparative studies on OSNs would greatly enhance our understanding of the mechanisms of olfaction.

Exposure to Zinc Sulfate Results in Differential Effects on Olfactory Sensory Neuron Subtypes in Adult Zebrafish.

Zinc sulfate is a known olfactory toxicant, although its specific effects on the olfactory epithelium of zebrafish are unknown. Olfactory organs of adult zebrafish were exposed to zinc sulfate and, after 2, 3, 5, 7, 10 or 14 days, fish were processed for histological, immunohistochemical, ultrastructural, and behavioral analyses. Severe morphological disruption of the olfactory organ was observed two days following zinc sulfate exposure, including fusion of lamellae, epithelial inflammation, and significant loss of anti-calretinin labeling. Scanning electron microscopy revealed the apical surface of the sensory region was absent of ciliated structures, but microvilli were still present. Behavioral analysis showed significant loss of the ability to perceive bile salts and some fish also had no response to amino acids. Over the next several days, olfactory organ morphology, epithelial structure, and anti-calretinin labeling returned to control-like conditions, although the ability to perceive bile salts remained lost until day 14. Thus, exposure to zinc sulfate results in rapid degeneration of the olfactory organ, followed by restoration of morphology and function within two weeks. Zinc sulfate appears to have a greater effect on ciliated olfactory sensory neurons than on microvillous olfactory sensory neurons, suggesting differential effects on sensory neuron subtypes.

Scanning electron microscopy of development of the olfactory organ in ontogeny of grass carp Ctenopharyngodon idella

Cytoand morphogenesis of the olfactory organ of grass carp Ctenopharyngodon idella are followed using the method of electron scanning microscopy. Dates of formation of olfactory placode and olfactory pit, olfactory sensory neurons, ciliated nonsensory cells, and other types of cells of olfactory mucosa are determined. In embryos, ciliated olfactory sensory neurons appear, then microvillous olfactory sensory neurons; the ratio of their numbers (approximately 2: 1) is retained throughout the entire fish ontogeny. In embryos, rod-bearing olfactory sensory neurons were found, whose numbers increase and then successively decrease, and they almost completely disappear at the beginning of the fry period. Towards this age, olfactory sensory epithelium is transformed from continuous into discontinuous at the expense of appearance of a large number of sites of ciliated nonsensory epithelium. With formation of olfactory rosette, sensory epithelium covers only lateral surfaces of olfactory lamellae. Numerous ciliated nonsensory cells appear in larvae on the head around the olfactory opening and rapidly disappear in fry with formation of the nasal bridge and entry and exit olfactory nares. The rate of growth of cilia and microvilli of olfactory sensory neurons and kinocilia of ciliated nonsensory cells was determined. In fish of all age groups, the density of distribution of olfactory sensory neurons in the olfactory epithelium is similar and comprises approximately 51000 cells/mm2. The amount of olfactory sensory neurons in the olfactory organ in fish of different age was assessed; in mature fish their number is approximately 40 millions. The relationship between olfactory sensory equipping and development of other structures of the olfactory organ and olfactory sensitivity of fish to odors was traced.

Combinatorial analysis of calcium-binding proteins in larval and adult zebrafish primary olfactory system identifies differential olfactory bulb glomerular projection fields.

In the zebrafish (Danio rerio) olfactory epithelium, the calcium-binding proteins (CBPs) calretinin and S100/S100-like protein are mainly expressed in ciliated or crypt olfactory sensory neurons (OSNs), respectively. In contrast parvalbumin and calbindin1 have not been investigated. We present a combinatorial immunohistological analysis of all four CBPs, including their expression in OSNs and their axonal projections to the olfactory bulb in larval and adult zebrafish. A major expression of calretinin and S100 in ciliated and crypt cells, respectively, with some expression of S100 in microvillous cells is confirmed. Parvalbumin and calbindin1 are strongly expressed in ciliated and microvillous cells, but not in crypt cells. Moreover, detailed combinatorial double-label experiments indicate that there are eight subpopulations of zebrafish OSNs: S100-positive crypt cells (negative for all other three CBPs), parvalbumin only, S100 and parvalbumin, parvalbumin and calbindin1, and parvalbumin and calbindin1 and calretinin-positive microvillous OSNs, as well as a major parvalbumin and calbindin1 and calretinin, and minor parvalbumin and calbindin1 and calretinin-only-positive ciliated OSN populations. CBP-positive projections to olfactory bulb are consistent with previous reports of ciliated OSNs projecting to dorsal and ventromedial glomerular fields and microvillous OSNs to ventrolateral glomerular fields. We newly describe parvalbumin-positive fibers to the mediodorsal field which is calretinin free, with its anterior part showing additionally calbindin1-positive fibers, but absence thereof in the posterior part, indicating an origin from microvillous OSNs in both parts. One singular glomerulus (mdG2) exhibits S100 and parvalbumin-positive fibers, apparently originating from all crypt cells plus some microvillous OSNs. Arguments for various olfactory labeled lines are discussed.

Olfactory Sensory Neuron Morphotypes in the Featherback Fish, Notopterus notopterus (Osteoglossiformes: Notopteridae)

As in other vertebrates, olfactory sensory neurons (OSNs) in fishes are the main components of sensory part of olfactory epithelium that relay olfactory information (smell and taste) to the brain. Objective of the present study was to analyze if any polymorphism occurs in the OSNs in a featherback fish, Notopterus notopterus as far as the teleost lineage is concerned. With the help of neuronal staining technique, polymorphism of OSNs in N. notopterus was studied. Three polymorphic forms of OSNs were identified which are ciliated OSNs, microvillus OSNs and crypt OSNs. These morphotypes were identified on the basis of location of their somata within the depth of olfactory epithelium and resulting length of their dendrites. The ciliated OSNs have basally situated somata and long, thin dendrites with a few apically arranged cilia while microvillous OSNs have somata located midway in the epithelium and thick moderate-length dendrites with microvilli. Third cell type is crypt OSNs which are spherical or pear-shaped, located apically just close to the epithelial surface having cilia and microvilli in an invagination and devoid of any dendrite. N. notopterus belongs to order Osteoglossiformes which is a representative of an early evolutionary lineage of teleost fishes. OSN polymorphism reported in the present work indicates that it is a fairly conserved trait throughout the evolution of teleosts. To our knowledge, we are the first ones to report OSN polymorphism in a member of the order Osteoglossiformes.

Light and transmission electron microscopy study of the peripheral olfactory organ of the guppy, Poecilia reticulata (Teleostei, Poecilidae)

A study of the peripheral olfactory organ, with special attention to the olfactory epithelium, has been carried out in the guppy (Poecilia reticulata). Guppy is well known to have a vision-based sexual behavior. The olfactory chamber caudally opens directly in an accessory nasal sac, which is bent medially and gives rise to two recesses that can be considered secondary accessory nasal sacs, antero-medial and postero-medial, respectively. The sensory epithelium, which lines only the medial wall of the nasal cavity, is basically flat rising in a very low lamella only in the posterior part. The olfactory receptors are not evenly distributed in the olfactory mucosa, but aggregate in shallow folds separated by epithelial cells with evident microridges. Ciliated olfactory sensory neurons and microvillous olfactory sensory neurons are clearly identified by transmission electron microscopy (TEM). Scarce crypt olfactory neurons are found throughout the sensory folds. The nasal sacs indicates the capacity to regulate the flow of odorant molecules over the sensory epithelium, possibly through a pump-like mechanism associated with gill ventilation. The organization of the olfactory organ in guppy is simple and reminds what is found in early posthatching stages of fish which at the adult state have a well developed olfactory organ. This simple organization supports the idea that the guppy rely on olfaction less than other fish species provided with more extended olfactory receptorial surface.

Axon Guidance of Olfactory Sensory Neurons in 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 ∼18 00 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.