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Abstract Background Among the EF-Hand calcium-binding proteins the subgroup of S100 proteins constitute a large family with numerous and diverse functions in calcium-mediated signaling. The evolutionary origin of this family is still uncertain and most studies have examined mammalian family members. Results We have performed an extensive search in several teleost genomes to establish the s100 gene family in fish. We report that the teleost S100 repertoire comprises fourteen different subfamilies which show remarkable similarity across six divergent teleost species. Individual species feature distinctive subsets of thirteen to fourteen genes that result from local gene duplications and gene losses. Eight of the fourteen S100 subfamilies are unique for teleosts, while six are shared with mammalian species and three of those even with cartilaginous fish. Several S100 family members are found in jawless fish already, but none of them are clear orthologs of cartilaginous or bony fish s100 genes. All teleost s100 genes show the expected structural features and are subject to strong negative selection. Many aspects of the genomic arrangement and location of mammalian s100 genes are retained in the teleost s100 gene family, including a completely conserved intron/exon border between the two EF hands. Zebrafish s100 genes exhibit highly specific and characteristic expression patterns, showing both redundancy and divergence in their cellular expression. In larval tissue expression is often restricted to specific cell types like keratinocytes, hair cells, ionocytes and olfactory receptor neurons as demonstrated by in situ hybridization. Conclusion The origin of the S100 family predates at least the segregation of jawed from jawless fish and some extant family members predate the divergence of bony from cartilaginous fish. Despite a complex pattern of gene gains and losses the total repertoire size is remarkably constant between species. On the expression level the teleost S100 proteins can serve as precise markers for several different cell types. At least some of their functions may be related to those of their counterparts in mammals. Accordingly, our findings provide an excellent basis for future studies of the functions and interaction partners of s100 genes and finally their role in diseases, using the zebrafish as a model organism.

The parthenogenetic life cycle of the stick insect Medauroidea extradentata offers unique advantages for the generation of genome-edited strains, as an isogenic and stable mutant line can in principle be achieved already in the first generation (G0). However, genetic tools for the manipulation of their genes had not been developed until now. Here, we successfully implement CRISPR/Cas9 as a technique to modify the genome of the stick insect M. extradentata. As proof-of-concept we targeted two genes involved in the ommochrome pathway of eye pigmentation (cinnabar and white, second and first exon, respectively), to generate knockout (KO) mutants. Microinjections were performed within 24 h after oviposition, to focus on the mononuclear (and haploid) stage of development. The KOs generated resulted in distinct eye and cuticle colour phenotypes for cinnabar and white. Homozygous cinnabar mutants showed pale pigmentation of eyes and cuticle. They develop into adults capable of producing viable eggs. Homozygous white KO resulted in a completely unpigmented phenotype in developing embryos that were unable to hatch. In conclusion, we show that CRISPR/Cas9 can be successfully applied to the genome of M. extradentata by creating phenotypically different and viable insects. This powerful gene editing technique can now be employed to create stable genetically modified lines using a parthenogenetic non-model organism., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Cartilaginous fishes (chondrichthyans: chimaeras and elasmobranchs -sharks, skates and rays) hold a key phylogenetic position to explore the origin and diversifications of jawed vertebrates. Here, we report and integrate reference genomic, transcriptomic and morphological data in the small-spotted catshark Scyliorhinus canicula to shed light on the evolution of sensory organs. We first characterise general aspects of the catshark genome, confirming the high conservation of genome organisation across cartilaginous fishes, and investigate population genomic signatures. Taking advantage of a dense sampling of transcriptomic data, we also identify gene signatures for all major organs, including chondrichthyan specializations, and evaluate expression diversifications between paralogs within major gene families involved in sensory functions. Finally, we combine these data with 3D synchrotron imaging and in situ gene expression analyses to explore chondrichthyan-specific traits and more general evolutionary trends of sensory systems. This approach brings to light, among others, novel markers of the ampullae of Lorenzini electro-sensory cells, a duplication hotspot for crystallin genes conserved in jawed vertebrates, and a new metazoan clade of the transient-receptor potential (TRP) family. These resources and results, obtained in an experimentally tractable chondrichthyan model, open new avenues to integrate multiomics analyses for the study of elasmobranchs and jawed vertebrates., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Trimeric G proteins transduce signals from a superfamily of receptors and each G protein controls a wide range of cellular and systemic functions. Their highly conserved alpha subunits fall in five classes, four of which have been well investigated (Gs, Gi, G12, Gq). In contrast, the function of the fifth class, Gv is completely unknown, despite its broad occurrence and evolutionary ancient origin (older than metazoans). Here we show a dynamic presence of Gv mRNA in several organs during early development of zebrafish, including the hatching gland, the pronephros and several cartilage anlagen, employing in situ hybridisation. Next, we generated a Gv frameshift mutation in zebrafish and observed distinct phenotypes such as reduced oviposition, premature hatching and craniofacial abnormalities in bone and cartilage of larval zebrafish. These phenotypes could suggest a disturbance in ionic homeostasis as a common denominator. Indeed, we find reduced levels of calcium, magnesium and potassium in the larvae and changes in expression levels of the sodium potassium pump atp1a1a.5 and the sodium/calcium exchanger ncx1b in larvae and in the adult kidney, a major osmoregulatory organ. Additionally, expression of sodium chloride cotransporter slc12a3 and the anion exchanger slc26a4 is altered in complementary ways in adult kidney. It appears that Gv may modulate ionic homeostasis in zebrafish during development and in adults. Our results constitute the first insight into the function of the fifth class of G alpha proteins., (© 2024. The Author(s).)

The odor space of aquatic organisms is by necessity quite different from that of air-breathing animals. The recognized odor classes in teleost fish include amino acids, bile acids, reproductive hormones, nucleotides, and a limited number of polyamines. Conversely, a significant portion of the fish olfactory receptor repertoire is composed of trace amine-associated receptors, generally assumed to be responsible for detecting amines. Zebrafish possess over one hundred of these receptors, but the responses of olfactory sensory neurons to amines have not been known so far. Here we examined odor responses of zebrafish olfactory epithelial explants at the cellular level, employing calcium imaging. We report that amines elicit strong responses in olfactory sensory neurons, with a time course characteristically different from that of ATP-responsive (basal) cells. A quantitative analysis of the laminar height distribution shows amine-responsive cells undistinguishable from ciliated neurons positive for olfactory marker protein. This distribution is significantly different from those measured for microvillous neurons positive for transient receptor potential channel 2 and basal cells positive for proliferating cell nuclear antigen. Our results suggest amines as an important odor class for teleost fish., (© 2024. The Author(s).)

Many animal and plant species synthesize toxic compounds as deterrent; thus, detection of these compounds is of vital importance to avoid their ingestion. Often, such compounds are recognized by taste 2 receptors that mediate bitter taste in humans. Until now, bitter taste receptors have only been found in bony vertebrates, where they occur as a large family already in coelacanth, a "living fossil" and the earliest-diverging extant lobe-finned fish. Here, we have revisited the evolutionary origin of taste 2 receptors (T2Rs) making use of a multitude of recently available cartilaginous fish genomes. We have identified a singular T2R in 12 cartilaginous fish species (9 sharks, 1 sawfish, and 2 skates), which represents a sister clade to all bony fish T2Rs. We have examined its ligands for two shark species, a catshark and a bamboo shark. The ligand repertoire of bamboo shark represents a subset of that of the catshark, with roughly similar thresholds. Amarogentin, one of the most bitter natural substances for humans, also elicited the highest signal amplitudes with both shark receptors. Other subsets of ligands are shared with basal bony fish T2Rs indicating an astonishing degree of functional conservation over nearly 500 mya of separate evolution. Both shark receptors respond to endogenous steroids as well as xenobiotic compounds, whereas separate receptors exist for xenobiotics both in early- and late-derived bony vertebrates (coelacanth, zebrafish, and human), consistent with the shark T2R reflecting the original ligand repertoire of the ancestral bitter taste receptor at the evolutionary origin of this family., Competing Interests: Competing interests statement:The authors declare no competing interest.

Despite the important role of bitter taste for the rejection of potentially harmful food sources, birds have long been suspected to exhibit inferior bitter tasting abilities. Although more recent reports on the bitter recognition spectra of several bird species have cast doubt about the validity of this assumption, the bitter taste of avian species is still an understudied field. Previously, we reported the bitter activation profiles of three zebra finch receptors Tas2r5, -r6, and -r7, which represent orthologs of a single chicken bitter taste receptor, Tas2r1. In order to get a better understanding of the bitter tasting capabilities of zebra finches, we selected another Tas2r gene of this species that is similar to another chicken Tas2r. Using functional calcium mobilization experiments, we screened zebra finch Tas2r1 with 72 bitter compounds and observed responses for 7 substances. Interestingly, all but one of the newly identified bitter agonists were different from those previously identified for Tas2r5, -r6, and -r7 suggesting that the newly investigated receptor fills important gaps in the zebra finch bitter recognition profile. The most potent bitter agonist found in our study is cucurbitacin I, a highly toxic natural bitter substance. We conclude that zebra finch exhibits an exquisitely developed bitter taste with pronounced cucurbitacin I sensitivity suggesting a prominent ecological role of this compound for zebra finch., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kumar, Redel, Lang, Korsching and Behrens.)

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 pan-neuronal marker HuC in the olfactory organ suggests the same cell-type specificity of V2R/OlfC expression as for bony fishes, that is, in microvillous neurons. The relatively low number of olfactory receptors in cartilaginous fishes compared with 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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The senses of taste and smell detect overlapping sets of chemical compounds in fish, e.g. amino acids are detected by both senses. However, so far taste and smell organs appeared morphologically to be very distinct, with a specialized olfactory epithelium for detection of odors and taste buds located in the oral cavity and lip for detection of tastants. Here, we report dense clusters of cells expressing T1R and T2R receptors as well as their signal transduction molecule PLCβ2 in nostrils of zebrafish, i.e. on the entrance funnel through which odor molecules must pass to be detected by olfactory sensory neurons. Quantitative evaluation shows the density of these chemosensory cells in the nostrils to be as high or higher than that in the established taste organs oral cavity and lower lip. Hydrodynamic flow is maximal at the nostril rim enabling high throughput chemosensation in this organ. Taken together, our results suggest a sentinel function for these chemosensory cells in the nostril., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

During their maturation from horizontal basal stem cells, olfactory sensory neurons (OSNs) are known to select exactly one out of hundreds of olfactory receptors (ORs) and express it on their surface, a process called monogenic selection. Monogenic expression is preceded by a multigenic phase during which several OR genes are expressed in a single OSN. Here, we perform pseudotime analysis of a single cell RNA-Seq dataset of murine olfactory epithelium to precisely align the multigenic and monogenic expression phases with the cell types occurring during OSN differentiation. In combination with motif analysis of OR gene cluster-associated enhancer regions, we identify known and novel transcription (co-)factors (Ebf1, Lhx2, Ldb1, Fos and Ssbp2) and chromatin remodelers (Kdm1a, Eed and Zmynd8) associated with OR expression. The inferred temporal order of their activity suggests novel mechanisms contributing to multigenic OR expression and monogenic selection., (© 2022. The Author(s).)

The vertebrate sense of smell employs four main receptor families for detection of odors, among them the V1R/ORA family, which is unusually small and highly conserved in teleost fish. Zebrafish possess just seven ORA receptors, enabling a comprehensive analysis of the expression patterns of the entire family. The olfactory organ of zebrafish is representative for teleosts, cup-shaped, with lamella covered with sensory epithelium protruding into the cup from a median raphe. We have performed quantitative in situ hybridization on complete series of horizontal cryostat sections of adult zebrafish olfactory organ, and have analysed the location of ora-expressing cells in three dimensions, radial diameter, laminar height, and height-within-the-organ. We report broadly overlapping, but distinctly different distributions for all ora genes, even for ora3a and ora3b, the most recent gene duplication. Preferred positions in different dimensions are independent of each other. This spatial logic is very similar to previous reports for the much larger families of odorant receptor (or) and V2R-related olfC genes in zebrafish. Preferred positions for ora genes tend to be more central and more apical than those we observed for these other two families, consistent with expression in non-canonical sensory neuron types., (© 2022. The Author(s).)

The sense of smell employs some of the largest gene families in the genome to detect and distinguish a multitude of different odors. Within vertebrates, 4 major olfactory receptor families have been described; of which, only 3 (OR, TAAR-like, and V1R) were found already in lamprey, a jawless vertebrate. The forth family (V2R) was believed to have originated later, in jawed vertebrates. Here we have delineated the entire vomeronasal receptor repertoire in 3 lamprey species. We report the presence of 6 v1r and 2 v2r genes in Lethenteron camtschaticum, arctic lamprey, and Lampetra fluviatilis, river lamprey (6 and 1, respectively, in sea lamprey, Petromyzon marinus). Three v1r genes but no v2r genes were found to be expressed in olfactory sensory neurons in the characteristic sparse expression pattern. Our results show the olfactory function of some V1Rs already in lamprey and, unexpectedly, an early origin of the V2R family in the shared ancestor of jawed and jawless vertebrates. However, lamprey v2r genes appear not to have acquired an olfactory function yet, thus dissociating the evolutionary origin of the family from the onset of a function as olfactory receptor., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Nucleotides are an important class of odorants for aquatic vertebrates such as frogs and fishes, but also have manifold signaling roles in other cellular processes. Recently, an adenosine receptor believed to belong to the adora2 clade has been identified as an olfactory receptor in zebrafish. Here, we set out to elucidate the evolutionary history of both this gene and its olfactory function. We have performed a thorough phylogenetic study in vertebrates, chordates and their sister group, ambulacraria, and show that the origin of the zebrafish olfactory receptor gene can be traced back to the most recent common ancestor of all three groups as a segregate sister clade (adorb) to the adora gene family. Eel, carp, and clawed frog all express adorb in a sparse and distributed pattern within their olfactory epithelium very similar to the pattern observed for zebrafish that is, consistent with a function as olfactory receptor. In sharp contrast, lamprey adorb-expressing cells are absent from the sensory region of the lamprey nose, but form a contiguous domain directly adjacent to the sensory region. Double-labeling experiments confirmed the expression of lamprey adorb in nonneuronal cells and are consistent with an expression in neuronal progenitor cells. Thus, adorb may have undergone a switch of function in the jawed lineage of vertebrates towards a role as olfactory receptor., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Olfactory receptor families have arisen independently several times during evolution. The origin of taar genes, one of the four major vertebrate olfactory receptor families, is disputed. We performed a phylogenetic analysis making use of 96 recently available genomes, and report that olfactory functionality has arisen twice independently within the TAAR family, once in jawed and once in jawless fish. In lamprey, an ancestral gene expanded to generate a large family of olfactory receptors, while the sister gene in jawed vertebrates did not expand and is not expressed in olfactory sensory neurons. Both clades do not exhibit the defining TAAR motif, and we suggest naming them taar-like receptors (tarl). We have identified the evolutionary origin of both taar and tarl genes in a duplication of the serotonergic receptor 4 that occurred in the most recent common ancestor of vertebrates. We infer two ancestral genes in bony fish (TAAR12, TAAR13) which gave rise to the complete repertoire of mammalian olfactory taar genes and to class II of the taar repertoire of teleost fish. We follow their evolution in seventy-one bony fish genomes and report a high evolutionary dynamic, with many late gene birth events and both early and late gene death events.

The careful evaluation of food is important for survival throughout the animal kingdom, and specialized chemoreceptors have evolved to recognize nutrients, minerals, acids, and many toxins. Vertebrate bitter taste, mediated by the taste receptor type 2 (T2R) family, warns against potentially toxic compounds. During evolution T2R receptors appear first in bony fish, but the functional properties of bony fish T2R receptors are mostly unknown. We performed a phylogenetic analysis showing the "living fossil" coelacanth (Latimeria chalumnae) and zebrafish (Danio rerio) to possess T2R repertoires typical for early-diverged species in the lobe-finned and the ray-finned clade, respectively. Receptors from these two species were selected for heterologous expression assays using a diverse panel of bitter substances. Remarkably, the ligand profile of the most basal coelacanth receptor, T2R01, is identical to that of its ortholog in zebrafish, consistent with functional conservation across >400 Myr of separate evolution. The second coelacanth receptor deorphaned, T2R02, is activated by steroid hormones and bile acids, evolutionary old molecules that are potentially endogenously synthesized agonists for extraoral T2Rs. For zebrafish, we report the presence of both specialized and promiscuous T2R receptors. Moreover, we identified an antagonist for one of the zebrafish receptors suggesting that bitter antagonism contributed to shape this receptor family throughout evolution., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

A sensory deficit both at the individual and at the species level can be compensated by increased acuity in other senses. The loss of vision in blind cavefish, Astyanax mexicanus, appears to be partially counterbalanced by enhanced chemosensory perception. Whether such improvement also involves adaptive changes in chemosensory receptor repertoires was unknown. The typical bitter taste receptor repertoire of teleost fish is reported as 3-5 genes, much smaller than that of many terrestrial species. Interestingly, several fish species, for example, mudskipper, have evolved a terrestrial lifestyle, but again it was unknown, whether this change in habitat is reflected in the size of gustatory receptor repertoires. We have searched the genomes of 15 fish species and performed a thorough phylogenetic analysis to delineate their bitter taste receptor repertoires. We report no adaptation for 4 mudskipper species, which exhibit 3-4 bitter taste receptor genes, and thus a typical teleost repertoire, shaped by few gene losses and minor gene duplications from an ancestral repertoire of 4 genes. However, and in sharp contrast to all other teleost fish species analyzed, blind cavefish possess more than 20 intact bitter taste receptors and several pseudogenes, rivaling the complexity of the human bitter taste receptor repertoire. The gene duplications giving rise to the current cavefish bitter taste receptor repertoire appear to have occurred well before the loss of vision, consistent with this increase in repertoire size constituting a preadaptive trait that conceivably could compensate to some extent for the lack of visual cues.

Background: The sense of smell is unrivaled in terms of molecular complexity of its input channels. Even zebrafish, a model vertebrate system in many research fields including olfaction, possesses several hundred different olfactory receptor genes, organized in four different gene families. For one of these families, the initially discovered odorant receptors proper, segregation of expression into distinct spatial subdomains within a common sensory surface has been observed both in teleost fish and in mammals. However, for the remaining three families, little to nothing was known about their spatial coding logic. Here we wished to investigate, whether the principle of spatial segregation observed for odorant receptors extends to another olfactory receptor family, the V2R-related OlfC genes. Furthermore we thought to examine, how expression of OlfC genes is integrated into expression zones of odorant receptor genes, which in fish share a single sensory surface with OlfC genes., Results: To select representative genes, we performed a comprehensive phylogenetic study of the zebrafish OlfC family, which identified a novel OlfC gene, reduced the number of pseudogenes to 1, and brought the total family size to 60 intact OlfC receptors. We analyzed the spatial pattern of OlfC-expressing cells for seven representative receptors in three dimensions (height within the epithelial layer, horizontal distance from the center of the olfactory organ, and height within the olfactory organ). We report non-random distributions of labeled neurons for all OlfC genes analysed. Distributions for sparsely expressed OlfC genes are significantly different from each other in nearly all cases, broad overlap notwithstanding. For two of the three coordinates analyzed, OlfC expression zones are intercalated with those of odorant receptor zones, whereas in the third dimension some segregation is observed., Conclusion: Our results show that V2R-related OlfC genes follow the same spatial logic of expression as odorant receptors and their expression zones intermingle with those of odorant receptor genes. Thus, distinctly different expression zones for individual receptor genes constitute a general feature shared by teleost and tetrapod V2R/OlfC and odorant receptor families alike.

All olfactory receptors identified in teleost fish are expressed in a single sensory surface, whereas mammalian olfactory receptor gene families segregate into different olfactory organs, chief among them the main olfactory epithelium expressing ORs and TAARs, and the vomeronasal organ expressing V1Rs and V2Rs. A transitional stage is embodied by amphibians, with their vomeronasal organ expressing more 'modern', later diverging V2Rs, whereas more 'ancient', earlier diverging V2Rs are expressed in the main olfactory epithelium. During metamorphosis, the main olfactory epithelium of Xenopus tadpoles transforms into an air-filled cavity (principal cavity, air nose), whereas a newly formed cavity (middle cavity) takes over the function of a water nose. We report here that larval expression of ancient V2Rs is gradually lost from the main olfactory epithelium as it transforms into the air nose. Concomitantly, ancient v2r gene expression begins to appear in the basal layers of the newly forming water nose. We observe the same transition for responses to amino acid odorants, consistent with the hypothesis that amino acid responses may be mediated by V2R receptors.

The death-associated odor cadaverine, generated by bacteria-mediated decarboxylation of lysine, has been described as the principal activator of a particular olfactory receptor in zebrafish, TAAR13c. Low concentrations of cadaverine activated mainly TAAR13c-expressing olfactory sensory neurons, suggesting TAAR13c as an important element of the neuronal processing pathway linking cadaverine stimulation to a strongly aversive innate behavioral response. Here, we characterized the initial steps of this neuronal pathway. First we identified TAAR13c-expressing cells as ciliated neurons, equivalent to the situation for mammalian taar genes, which shows a high degree of conservation despite the large evolutionary distance between teleost fishes and mammals. Next we identified the target area of cadaverine-responsive OSNs in the olfactory bulb. We report that cadaverine dose-dependently activates a group of dorsolateral glomeruli, at the lowest concentration down to a single invariant glomerulus, situated at the medial border of the dorsolateral cluster. This is the first demonstration of a single stereotyped target glomerulus in the fish olfactory system for a non-pheromone odor. A mix of different amines activates many glomeruli within the same dorsolateral cluster, suggesting this area to function as a general amine response region.

Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors.

Background: Chemical senses serve a multitude of essential functions across the animal kingdom. Vertebrates employ four GPCR families to detect odors, among them the v1r/ora gene family. The V1R family is known to evolve rapidly in the lobe-finned lineage giving rise to tetrapods, but the homologous ORA family consists of just six highly conserved genes in teleost fish, with direct orthologs in the lobe-finned fish coelacanth. Thus, the teleost repertoire of six canonical ora genes was assumed to be the ancestral feature before the divergence of ray-finned and lobe-finned fish. So far, this hypothesis has not been tested with earlier diverging ray-finned fish., Results: We have newly identified the complete ora gene repertoires of five teleost species, and of spotted gar, a basal ray-finned fish, using thorough data mining and extensive phylogenetic analysis. The genomes of eight further teleost species were re-analyzed for their ORA repertoires. We report that direct orthologs of the six canonical ora genes (ora1-6) were present in all newly analyzed species, with faithfully preserved exon/intron structure and mostly preserved genomic arrangement in symmetric pairs for ora1-4. In four teleost species including medaka and cave fish we observe species-specific gene duplication events. Thus, the ora gene repertoire in teleost fish is not quite as strictly conserved as previously assumed. In fact, the examination of non-synonymous vs. synonymous substitution rates (dN/dS) shows pronounced negative selection in five of the six ora genes, but also rare occurrence of positive selection in ora3 and ora6. Surprisingly, spotted gar possesses beyond the six canonical genes three additional genes, ora7-8b, orthologous to coelacanth genes v1r07-10. No orthologs for these genes were found in teleosts and cartilaginous fish., Conclusions: Early diverging ray-finned fish such as the spotted gar possess several v1r-like genes previously assumed to be restricted to the lobe-finned lineage, but now found to be already present in the most recent common ancestor of lobe- and ray-finned fish. Thus, the presence of just six canonical ora genes in many teleost species is not the ancestral feature of the ray-finned lineage, but caused by loss of two ancestral genes in teleosts.

Olfactory perception is mediated by a multitude of olfactory receptors, whose expression in the sensory surface, the olfactory epithelium, is spatially regulated. A common theme is the segregation of different olfactory receptors in different expression domains, which in turn leads to corresponding segregation in the neuronal responses to different odor groups. The amphibian olfactory receptor gene family of trace amine associated receptors, in short TAARs, is exceedingly small and allows a comprehensive analysis of spatial expression patterns, as well as a comparison with neuronal responses to the expected ligands for this receptor family, amines. Here we report that TAAR4b exhibits a spatial expression pattern characteristically different in two dimensions from that of TAAR4a, its close homolog. Together, these two genes result in a bimodal distribution resembling that of amine responses as visualized by calcium imaging. A stringent quantitative analysis suggests the involvement of additional olfactory receptors in amphibian responses to amine odors.

Studies of the two major olfactory organs of rodents, the olfactory mucosa (OM) and the vomeronasal organ (VNO), unraveled the molecular basis of smell in vertebrates. However, some vertebrates lack a VNO. Here we generated and analyzed the olfactory transcriptome of the zebrafish and compared it to the olfactory transcriptomes of mouse to investigate the evolutionary and molecular relationship between single and dual olfactory systems. Our analyses revealed a high degree of molecular conservation, with orthologs of mouse olfactory cell-specific markers and all but one of their chemosensory receptor classes expressed in the single zebrafish olfactory organ. Zebrafish chemosensory receptor genes are expressed across a large dynamic range and their RNA abundance correlates positively with the number of neurons expressing that RNA. Thus we estimate the relative proportions of neuronal sub-types expressing different chemosensory receptors. Receptor repertoire size drives the absolute abundance of different classes of neurons, but we find similar underlying patterns in both species. Finally, we identified novel marker genes that characterize rare neuronal populations in both mouse and zebrafish. In sum, we find that the molecular and cellular mechanisms underpinning olfaction in teleosts and mammals are similar despite 430 million years of evolutionary divergence.

Bitter taste perception in vertebrates relies on a variable number of bitter taste receptor (Tas2r) genes, ranging from only three functional genes in chicken to as many as approximately 50 in frogs. Humans possess a medium-sized Tas2r repertoire encoding three broadly and several narrowly tuned receptors plus receptors with intermediate tuning properties. Such tuning information is not available for bitter taste receptors of other vertebrate species. In particular it is not known, whether a small Tas2r repertoire may be compensated for by broad tuning of these receptors, and on the other side, whether a large repertoire might entail a preponderance of narrowly tuned receptors. To elucidate this question, we cloned all three chicken Tas2rs, the two turkey Tas2rs, three zebra finch Tas2rs, and six Tas2rs of the Western clawed frog representative of major branches of the phylogenetic tree, and screened them with 46 different bitter compounds. All chicken and turkey Tas2rs were broadly tuned, the zebra finch Tas2rs were narrowly tuned, and frog Tas2rs ranged from broadly to narrowly tuned receptors. We conclude that a low number of functional Tas2r genes does not imply a reduced importance of bitter taste per se, as it can be compensated by large tuning width. A high number of functional Tas2r genes appears to allow the evolution of specialized receptors, possibly for toxins with species-specific relevance. In sum, we show that variability in tuning breadth, overlapping agonist profiles, and staggered effective agonist concentration ranges are shared features of human and other vertebrate Tas2rs., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Chemical senses are one of the foremost means by which organisms make sense of their environment, among them the olfactory and gustatory sense of vertebrates and arthropods. Both senses use large repertoires of receptors to achieve perception of complex chemosensory stimuli. High evolutionary dynamics of some olfactory and gustatory receptor gene families result in considerable variance of chemosensory perception between species. Interestingly, both ora/v1r genes and the closely related t2r genes constitute small and rather conserved families in teleost fish, but show rapid evolution and large species differences in tetrapods. To understand this transition, chemosensory gene repertoires of earlier diverging members of the tetrapod lineage, i.e. lobe-finned fish such as Latimeria would be of high interest., Results: We report here the complete T2R repertoire of Latimeria chalumnae, using thorough data mining and extensive phylogenetic analysis. Eighty t2r genes were identified, by far the largest family reported for any species so far. The genomic neighborhood of t2r genes is enriched in repeat elements, which may have facilitated the extensive gene duplication events resulting in such a large family. Examination of non-synonymous vs. synonymous substitution rates (dN/dS) suggests pronounced positive Darwinian selection in Latimeria T2Rs, conceivably ensuring efficient neo-functionalization of newly born t2r genes. Notably, both traits, positive selection and enrichment of repeat elements in the genomic neighborhood, are absent in the twenty v1r genes of Latimeria. Sequence divergence in Latimeria T2Rs and V1Rs is high, reminescent of the corresponding teleost families. Some conserved sequence motifs of Latimeria T2Rs and V1Rs are shared with the respective teleost but not tetrapod genes, consistent with a potential role of such motifs in detection of aquatic chemosensory stimuli., Conclusions: The singularly large T2R repertoire of Latimeria may have been generated by facilitating local gene duplication via increased density of repeat elements, and efficient neofunctionalization via positive Darwinian selection.The high evolutionary dynamics of tetrapod t2r gene families precedes the emergence of tetrapods, i.e. the water-to-land transition, and thus constitutes a basal feature of the lobe-finned lineage of vertebrates.

Complete segregation of the main olfactory epithelium (MOE) and the vomeronasal epithelium is first observed in amphibians. In contrast, teleost fishes possess a single olfactory surface, in which genetic components of the main and vomeronasal olfactory systems are intermingled. The transient receptor potential channel TRPC2, a marker of vomeronasal neurons, is present in the single fish sensory surface, but is already restricted to the vomeronasal epithelium in a terrestrial amphibian, the red-legged salamander (Plethodon shermani). Here we examined the localization of TRPC2 in an aquatic amphibian and cloned the Xenopus laevis trpc2 gene. We show that it is expressed in both the MOE and the vomeronasal epithelium. This is the first description of a broad trpc2 expression in the MOE of a tetrapod. The expression pattern of trpc2 in the MOE is virtually undistinguishable from that of MOE-specific v2rs, indicating that they are co-expressed in the same neuronal subpopulation., (© 2014. Published by The Company of Biologists Ltd.)

Perception of olfactory stimuli is mediated by distinct populations of olfactory sensory neurons, each with a characteristic set of morphological as well as functional parameters. Beyond two large populations of ciliated and microvillous neurons, a third population, crypt neurons, has been identified in teleost and cartilaginous fishes. We report here a novel, fourth olfactory sensory neuron population in zebrafish, which we named kappe neurons for their characteristic shape. Kappe neurons are identified by their Go-like immunoreactivity, and show a distinct spatial distribution within the olfactory epithelium, similar to, but significantly different from that of crypt neurons. Furthermore, kappe neurons project to a single identified target glomerulus within the olfactory bulb, mdg5 of the mediodorsal cluster, whereas crypt neurons are known to project exclusively to the mdg2 glomerulus. Kappe neurons are negative for established markers of ciliated, microvillous and crypt neurons, but appear to have microvilli. Kappe neurons constitute the fourth type of olfactory sensory neurons reported in teleost fishes and their existence suggests that encoding of olfactory stimuli may require a higher complexity than hitherto assumed already in the peripheral olfactory system.

The semi-aquatic lifestyle of amphibians represents a unique opportunity to study the molecular driving forces involved in the transition of aquatic to terrestrial olfaction in vertebrates. Most amphibians have anatomically segregated main and vomeronasal olfactory systems, but at the cellular and molecular level the segregation differs from that found in mammals. We have recently shown that amino acid responses in the main olfactory epithelium (MOE) of larval Xenopus laevis segregate into a lateral and a medial processing stream, and that the former is part of a vomeronasal type 2 receptor expression zone in the MOE. We hypothesized that the lateral amino acid responses might be mediated via a vomeronasal-like transduction machinery. Here we report that amino acid-responsive receptor neurons in the lateral MOE employ a phospholipase C (PLC) and diacylglycerol-mediated transduction cascade that is independent of Ca(2+) store depletion. Furthermore, we found that putative transient receptor potential (TRP) channel blockers inhibit most amino acid-evoked responses in the lateral MOE, suggesting that ion channels belonging to the TRP family may be involved in the signaling pathway. Our data show, for the first time, a widespread PLC- and diacylglycerol-dependent transduction cascade in the MOE of a vertebrate already possessing a vomeronasal organ.

Carrion smell is strongly repugnant to humans and triggers distinct innate behaviors in many other species. This smell is mainly carried by two small aliphatic diamines, putrescine and cadaverine, which are generated by bacterial decarboxylation of the basic amino acids ornithine and lysine. Depending on the species, these diamines may also serve as feeding attractants, oviposition attractants, or social cues. Behavioral responses to diamines have not been investigated in zebrafish, a powerful model system for studying vertebrate olfaction. Furthermore, olfactory receptors that detect cadaverine and putrescine have not been identified in any species so far. Here, we show robust olfactory-mediated avoidance behavior of zebrafish to cadaverine and related diamines, and concomitant activation of sparse olfactory sensory neurons by these diamines. The large majority of neurons activated by low concentrations of cadaverine expresses a particular olfactory receptor, trace amine-associated receptor 13c (TAAR13c). Structure-activity analysis indicates TAAR13c to be a general diamine sensor, with pronounced selectivity for odd chains of medium length. This receptor can also be activated by decaying fish extracts, a physiologically relevant source of diamines. The identification of a sensitive zebrafish olfactory receptor for these diamines provides a molecular basis for studying neural circuits connecting sensation, perception, and innate behavior.

In contrast to the single sensory surface present in teleost fishes, several spatially segregated subsystems with distinct molecular and functional characteristics define the mammalian olfactory system. However, the evolutionary steps of that transition remain unknown. Here we analyzed the olfactory system of an early diverging tetrapod, the amphibian Xenopus laevis, and report for the first time the existence of two odor-processing streams, sharply segregated in the main olfactory bulb and partially segregated in the olfactory epithelium of pre-metamorphic larvae. A lateral odor-processing stream is formed by microvillous receptor neurons and is characterized by amino acid responses and Gαo/Gαi as probable signal transducers, whereas a medial stream formed by ciliated receptor neurons is characterized by responses to alcohols, aldehydes, and ketones, and Gαolf/cAMP as probable signal transducers. To reveal candidates for the olfactory receptors underlying these two streams, the spatial distribution of 12 genes from four olfactory receptor gene families was determined. Several class II and some class I odorant receptors (ORs) mimic the spatial distribution observed for the medial stream, whereas a trace amine-associated receptor closely parallels the spatial pattern of the lateral odor-processing stream. Other olfactory receptors (some class I odorant receptors and vomeronasal type 1 receptors) and odor responses (to bile acids, amines) were not lateralized, the latter not even in the olfactory bulb, suggesting an incomplete segregation. Thus, the olfactory system of X. laevis exhibits an intermediate stage of segregation and as such appears well suited to investigate the molecular driving forces behind olfactory regionalization.

Mammalian olfactory receptor families are segregated into different olfactory organs, with type 2 vomeronasal receptor (v2r) genes expressed in a basal layer of the vomeronasal epithelium. In contrast, teleost fish v2r genes are intermingled with all other olfactory receptor genes in a single sensory surface. We report here that, strikingly different from both lineages, the v2r gene family of the amphibian Xenopus laevis is expressed in the main olfactory as well as the vomeronasal epithelium. Interestingly, late diverging v2r genes are expressed exclusively in the vomeronasal epithelium, whereas "ancestral" v2r genes, including the single member of v2r family C, are restricted to the main olfactory epithelium. Moreover, within the main olfactory epithelium, v2r genes are expressed in a basal zone, partially overlapping, but clearly distinct from an apical zone of olfactory marker protein and odorant receptor-expressing cells. These zones are also apparent in the spatial distribution of odor responses, enabling a tentative assignment of odor responses to olfactory receptor gene families. Responses to alcohols, aldehydes, and ketones show an apical localization, consistent with being mediated by odorant receptors, whereas amino acid responses overlap extensively with the basal v2r-expressing zone. The unique bimodal v2r expression pattern in main and accessory olfactory system of amphibians presents an excellent opportunity to study the transition of v2r gene expression during evolution of higher vertebrates.

Crypt neurons are a third type of olfactory receptor neurons with a highly unusual "one cell type--one receptor" mode of expression, the same receptor being expressed by the entire population of crypt neurons. Attempts to identify the target region(s) of crypt neurons have been inconclusive so far. We report that TrkA-like immunoreactivity specifically labeled somata, axons, and terminals of zebrafish crypt neurons and reveal a single glomerulus, mdg2 of the dorsomedial group, as target glomerulus of crypt neurons. Injection of a fluorescent tracing dye into the mdg2 glomerulus retrogradely labeled mostly crypt neurons, as assessed by quantitative morphometry, whereas no crypt neurons were found after injections in neighboring glomeruli. Our data provide strong evidence that crypt neurons converge onto a single glomerulus, and thus form a labeled line consisting of a single sensory cell type, a single olfactory receptor and a single target glomerulus.

Both ciliated and microvillous olfactory sensory neuron populations express large families of olfactory receptor genes. However, individual neurons generally express only a single receptor gene according to the "one neuron-one receptor" rule. We report here that crypt neurons, the third type of olfactory neurons in fish species, use an even more restricted mode of expression. We recently identified a novel olfactory receptor family of 6 highly conserved G protein-coupled receptors, the v1r-like ora genes. We show now that a single member of this family, ora4 is expressed in nearly all crypt neurons, whereas the other 5 ora genes are not found in this cell type. Consistent with these findings, ora4 is never coexpressed with any of the remaining 5 ora genes. Furthermore, several lines of evidence indicate the absence of any other olfactory receptor families in crypt neurons. These results suggest that the vast majority of the crypt neuron population may select one and the same olfactory receptor gene, a "one cell type-one receptor" mode of expression. Such an expression pattern is familiar in the visual system, with rhodopsin as the sole light receptor of rod photoreceptor cells, but unexpected in the sense of smell.

Chemosensory systems in vertebrates employ G protein-coupled receptors as sensors. In mammals, several families of olfactory and gustatory receptors as well as specific G alpha proteins coupling to them have been identified, for example, gustducin for taste. Orthologous receptor families have been characterized in fish, but the corresponding G alpha genes have not been well investigated so far. We have performed a comprehensive search of several lower vertebrate genomes to establish the G alpha protein family in these taxa and to identify those genes that may be involved in chemosensory signal transduction in fish. We report that gustducin is absent from the genomes of all teleost and amphibian species analyzed, presumably due to independent gene losses in these lineages. However, 2 other G alpha genes, Gi1b and G14a, are expressed in zebrafish taste buds and 4 G proteins, Go1, Go2, Gi1b, and Golf2, were detected in the olfactory epithelium. Golf2, Gi1b, and G14a are expressed already shortly after hatching, consistent with the physiological and behavioral responses of larvae to odorants and tastants. Our results show general similarity to the mammalian situation but also clear-cut differences and as such are essential for using the zebrafish model system to study chemosensory perception.

Heterotrimeric G proteins are key molecules regulating cellular responses to extracellular stimuli, and are composed of alpha, beta and gamma subunits. All alpha subunits in vertebrates belong to four major classes, Gs, Gi, Gq and G12, which are conserved throughout the animal kingdom. Unexpectedly, now a fifth class of Galpha protein, Gv, has been discovered. Gv is conserved across the animal kingdom and present in vertebrates, arthropods, mollusks, annelids and even sponges. Presumably, Gv has been missed so far, because it has been lost in many lineages in the major model organisms such as nematodes, fruit fly and mammals. On the other hand, gene gains are also observed for Gv, with at least two independent gene duplications, one in sponges and the other in the teleost lineage. Such frequent gene gains and losses fit to a birth-and-death mode of evolution, which is unusual for a well-conserved and ancient gene family like the Galpha proteins. The discovery of a novel major class of Galpha proteins provides new insights in the evolution of the Galpha protein family and opens new possibilities in G protein signaling research.

Trace amine-associated receptors (TAARs) in mammals recently have been shown to function as olfactory receptors. We have delineated the taar gene family in jawless, cartilaginous, and bony fish (zero, 2, and >100 genes, respectively). We conclude that taar genes are evolutionary much younger than the related OR and ORA/V1R olfactory receptor families, which are present already in lamprey, a jawless vertebrate. The 2 cartilaginous fish genes appear to be ancestral for 2 taar classes, each with mammalian and bony fish (teleost) representatives. Unexpectedly, a whole new clade, class III, of taar genes originated even later, within the teleost lineage. Taar genes from all 3 classes are expressed in subsets of zebrafish olfactory receptor neurons, supporting their function as olfactory receptors. The highly conserved TAAR1 (shark, mammalian, and teleost orthologs) is not expressed in the olfactory epithelium and may constitute the sole remnant of a primordial, nonolfactory function of this family. Class III comprises three-fourths of all teleost taar genes and is characterized by the complete loss of the aminergic ligand-binding motif, stringently conserved in the other 2 classes. Two independent intron gains in class III taar genes represent extraordinary evolutionary dynamics, considering the virtual absence of intron gains during vertebrate evolution. The d(N)/d(S) analysis suggests both minimal global negative selection and an unparalleled degree of local positive selection as another hallmark of class III genes. The accelerated evolution of class III teleost taar genes conceivably might mark the birth of another olfactory receptor gene family.

All alpha-subunits of vertebrate heterotrimeric G proteins have been classified into 4 major classes, Gs, Gi, Gq, and G12, which possess orthologs already in sponges, one of the earliest animal phyla to evolve. Here we report the discovery of the fifth class of Galpha protein, Gv, ancient like the other 4 classes, with members already in sponges, and encoded by 1-2 gnav genes per species. Gv is conserved across the animal kingdom including vertebrates, arthropods, mollusks, and annelids, but has been lost in many lineages such as nematodes, fruit fly, jawless fish, and tetrapods, concordant with a birth-and-death mode of evolution. All Gv proteins contain 5 G-box motifs characteristic of GTP-binding proteins and the expected acylation consensus sites in the N-terminal region. Sixty amino acid residues are conserved only among Gv, suggesting that they may constitute interaction sites for Gv-specific partner molecules. Overall Gv homology is high, on average 70% amino acid identity among vertebrate family members. The d(N)/d(S) analysis of teleost gnav genes reveals evolution under stringent negative selection. Genomic structure of vertebrate gnav genes is well conserved and different from those of the other 4 classes. The predicted full ORF of zebrafish gnav1 was confirmed by isolation from cDNA. RT-PCR analysis showed broad expression of gnav1 in adult zebrafish and in situ hybridization demonstrated a more restricted expression in larval tissues including the developing inner ear. The discovery of this fifth class of Galpha proteins changes our understanding of G protein evolution.

While for two of three mammalian olfactory receptor families (OR and V2R) ortholog teleost families have been identified, the third family (V1R) has been thought to be represented by a single, closely linked gene pair. We identified four further V1R-like genes in every teleost species analyzed (Danio rerio, Gasterosteus aculeatus, Oryzias latipes, Tetraodon nigroviridis, Takifugu rubripes). In the phylogenetic analysis these ora genes (olfactory receptor class A-related) form a single clade, which includes the entire mammalian V1R superfamily. Homologies are much lower in paralogs than in orthologs, indicating that all six family members are evolutionarily much older than the speciation events in the teleost lineage analyzed here. These ora genes are under strong negative selection, as evidenced by very small d(N)/d(S) values in comparisons between orthologs. A pairwise configuration in the phylogenetic tree suggests the existence of three ancestral Ora subclades, one of which has been lost in amphibia, and a further one in mammals. Unexpectedly, two ora genes exhibit a highly conserved multi-exonic structure and four ora genes are organized in closely linked gene pairs across all fish species studied. All ora genes are expressed specifically in the olfactory epithelium of zebrafish, in sparse cells within the sensory surface, consistent with the expectation for olfactory receptors. The ora gene repertoire is highly conserved across teleosts, in striking contrast to the frequent species-specific expansions observed in tetrapod, especially mammalian V1Rs, possibly reflecting a major shift in gene regulation as well as gene function upon the transition to tetrapods.

Tubulin, the building block of microtubules, consists of an alpha and beta subunit, each in itself a family of several highly homologous isotypes. Abundance, tissue specificity, developmental regulation, and possibly function vary between isotypes. Six isotypes of beta tubulin (class I to class VI) have been cloned from several vertebrate species. Class I beta tubulin is believed to be widely expressed, but has not been studied by in situ hybridization in any vertebrate species so far. We have cloned a beta tubulin from zebrafish that appears most similar to other vertebrate class I tubulins and name it zbeta1 tubulin, accordingly. We report a distinct expression pattern of zbeta1 tubulin in the zebrafish embryo in restricted regions of the peripheral and central nervous system that comprise early-differentiating neurons. The expression pattern changes during development and in the adult zebrafish expression mostly is confined to a subset of proliferative zones that include the subependymal zone around the telencephalic ventricle, zones in the preoptic and hypothalamic area and in the olfactory epithelium. Thus, zbeta1 tubulin is expressed with remarkable selectivity during neuronal differentiation and neurogenesis in the embryonic and adult nervous system, respectively.

Olfactory receptor neurons select a single odourant receptor gene for expression out of a large gene family. The mechanisms of this extreme selectivity are largely unknown. We have determined in detail the developmental expression dynamics of a representative subset of the zebrafish odourant receptor repertoire, using in situ hybridization analysis. We have thus generated a dataset, which allows us to test hypotheses of odourant receptor gene regulation. The receptors chosen belong to four different groups with respect to ontogenetic onset of expression (onset groups). Statistical analysis of the data supports a model in which the final choice of an individual odourant receptor gene occurs stochastically from within a group of genes sharing a deterministically defined onset of expression. Genomic mapping revealed a pronounced correlation of onset of expression with genomic neighbourhood. During a protracted juvenile developmental period individual regulatory influences seem to modify the expression of odourant receptor genes, a notable example being a transient decrease in expressivity of two odourant receptor genes.

RFamides constitute a large family of neuromodulatory peptides. We have cloned a zebrafish gene, which is presumably a homologue to the mammalian PQRF subfamily of RFamides, and named it zfPQRF for its species and subfamily allocation. We report that in contrast to its mammalian counterparts zfPQRF is expressed in the olfactory bulb and the nucleus olfactoretinalis in the telencephalon, but absent in more caudal regions, including hypothalamus, brain stem and spinal cord. zfPQRF-expressing neurons originate in the vicinity of the olfactory placode and populate the nuclei of the terminal nerve during later development, as demonstrated by co-expression of zebrafish salmon-type gonadotropin releasing hormone, which was found to exclusively label terminal nerve neurons., (Copyright 2002 Elsevier Science Ireland Ltd.)

More chemicals can be smelled than there are olfactory receptors for them, necessitating a combinatorial representation by somewhat broadly tuned receptors. To understand the perception of odor quality and concentration, it is essential to establish the nature of the receptor repertoires that are activated by particular odorants at particular concentrations. We have taken advantage of the one-to-one correspondence of glomeruli and olfactory receptor molecules in the mouse olfactory bulb to analyze the tuning properties of a major receptor population by high resolution calcium imaging of odor responses selectively in the presynaptic compartment of glomeruli. We show that eighty different olfactory receptors projecting to the dorsal olfactory bulb respond to high concentrations of aldehydes with limited specificity. Varying ensembles of about 10 to 20 receptors encode any particular aldehyde at low stimulus concentrations with high specificity. Even normalized odor response patterns are markedly concentration dependent, caused by pronounced differences in affinity within the aldehyde receptor repertoire.

The olfactory nervous system of fish, in particular zebrafish, has become a valid model for that of higher vertebrates. However, no genetic markers for olfactory specific cell types, e.g. the olfactory receptor neurons, have been established in this species. Olfactory marker protein (OMP) is a reliable marker for olfactory receptor neurons in several other vertebrates. We have cloned zOMP, the zebrafish homologue of olfactory marker protein. During development, zOMP is expressed exclusively in the olfactory placode, presumably in olfactory receptor neurons, as shown by in situ hybridization. In the adult nasal epithelium zOMP is found restricted to the sensory region. zOMP appears to be a single gene, without close family members. The 5'-flanking region lacks most of the expected regulatory sequence motifs, both general and cell type-specific ones. Nevertheless, it drives reporter gene expression strongly and specifically in olfactory receptor neurons during the whole developmental period examined. Thus the zOMP promoter constitutes a powerful tool which should be useful to selectively introduce a wide variety of genetic modifications into olfactory receptor neurons.

The structural determinants of an odor molecule necessary and/or sufficient for interaction with the cognate olfactory receptor(s) are not known. Olfactory receptor neurons expressing the same olfactory receptor converge in the olfactory bulb. Thus, optical imaging of neuronal activity in the olfactory bulb can visualize at once the contributions by all the different olfactory receptors responsive to a particular odorant. We have used this technique to derive estimates about the structural requirements and minimal number of different zebrafish olfactory receptors that respond to a series of naturally occurring amino acids and some structurally related compounds. We report that the alpha-carboxyl group, the alpha-amino group, and l-conformation of the amino acid are all required for activation of amino acid-responsive receptors. Increasing carbon chain length recruits successively more receptors. With increasing concentrations, the activity patterns induced by a homolog series of amino acids became more similar to each other. At intermediate concentrations patterns were unique across substances and across concentrations. The introduction of a terminal amino group (charged) both recruits additional receptors and prevents binding to some of the receptors that were responsive to the unsubstituted analog. In contrast, the introduction of a beta-hydroxyl group (polar) excluded the odorants from some of the receptors that are capable of binding the unsubstituted analog. Cross-adaptation experiments independently confirmed these results. Thus, odorant detection requires several different receptors even for relatively simple odorants such as amino acids, and individual receptors require the presence of some molecular features, the absence of others, and tolerate still other molecular features.

An inexpensive headholder for mice ranging from 3-week-old animals to adults was designed to provide reliable long-term head fixation. Its shape allows the direct access to the dorsal and ventral sides of the head which makes the headholder ideal for the use with both an upright and an inverted microscope. Because the headholder does not use a nose clamp, the apparatus allows surgery, stimulation of the olfactory system and concomitant optical recording of neuronal activity.

The olfactory sense detects and distinguishes a multitude of different odors. Recent progress in molecular as well as physiological approaches has elucidated basic principles of neuronal encoding of odorants, common to insects and vertebrates. The construction of neuronal representations for odors begins with the task of mapping the multidimensional odor space onto the two-dimensional sensory surface, and subsequently onto the olfactory bulb or antennal lobe. A distributed expression of odorant receptors, albeit restricted to subregions of the sensory surface (large, intermediate or small for zebrafish, mouse or drosophila, respectively), ensures a robust representation, insensitive to mechanical insult. Olfactory receptor neurons expressing the same odorant receptors converge to form a receptotopic map in the olfactory bulb or antennal lobe. The emerging coding principle is a chemotopic representation of odorants at the first brain level, realized either as combinatorial or as monospecific representation, depending on the odorant.

Odor information is first represented in the brain by patterns of input activity across the glomeruli of the olfactory bulb (OB). To examine how odorants are represented at this stage of olfactory processing, we labeled anterogradely the axons of olfactory receptor neurons with the voltage-sensitive dye Di8-ANEPPQ in zebrafish. The activity induced by diverse natural odorants in afferent axons and across the array of glomeruli was then recorded optically. The results show that certain subregions of the OB are preferentially activated by defined chemical odorant classes. Within these subregions, "ordinary" odorants (amino acids, bile acids, and nucleotides) induce overlapping activity patterns involving multiple glomeruli, indicating that they are represented by combinatorial activity patterns. In contrast, two putative pheromone components (prostaglandin F2alpha and 17alpha, 20beta-dihydroxy-4-pregnene-3-one-20-sulfate) each induce a single focus of activity, at least one of which comes from a single, highly specific and sensitive glomerulus. These results indicate that the OB is organized into functional subregions processing classes of odorants. Furthermore, they suggest that individual odorants can be represented by "combinatorial" or "noncombinatorial" (focal) activity patterns and that the latter may serve to process odorants triggering distinct responses such as that of pheromones.

Odors are thought to be represented by a distributed code across the glomerular modules in the olfactory bulb (OB). Here, we optically imaged presynaptic activity in glomerular modules of the zebrafish OB induced by a class of natural odorants (amino acids [AAs]) after labeling of primary afferents with a calcium-sensitive dye. AAs induce complex combinatorial patterns of active glomerular modules that are unique for different stimuli and concentrations. Quantitative analysis shows that defined molecular features of stimuli are correlated with activity in spatially confined groups of glomerular modules. These results provide direct evidence that identity and concentration of odorants are encoded by glomerular activity patterns and reveal a coarse chemotopic organization of the array of glomerular modules.

Zebrafish, Danio rerio, possess a well-developed sense of smell which governs a variety of behaviors. Both the number of odorant receptor genes and the number of modules in the olfactory bulb (glomeruli) are about an order of magnitude smaller than those of mammals. Nevertheless, the spatial organization of functional properties within the sensory surface and the olfactory bulb are comparable to those of mammals. The quantitatively reduced olfactory system of zebrafish, together with the suitability of this species for developmental and genetic studies, make zebrafish an interesting model system to study olfactory differentiation and neuronal representation of olfactory information.