Vertebrate-wide transcriptomic screening identifies immune cell-specific expression of the conserved OR-κ gene

Vertebrate-wide transcriptomic screening identifies immune cell-specific expression of the conserved OR-κ gene | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Vertebrate-wide transcriptomic screening identifies immune cell-specific expression of the conserved OR-κ gene Kanoko Nishiura, Tatsuki Nagasawa, Masato Nikaido This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8198358/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Feb, 2026 Read the published version in Zoological Letters → Version 1 posted 4 You are reading this latest preprint version Abstract Vertebrate chemoreceptor genes play a central role in detecting environmental chemical compounds through the olfactory organs and taste buds, enabling the perception of odors and tastants that are critical for survival. Through evolutionary processes, these genes have repeatedly undergone duplication, divergence, and pseudogenization, giving rise to lineage-specific gene repertoires that reflect ecological and behavioral adaptation. While the canonical functions of these chemoreceptors are confined to the nasal and oral cavities, increasing evidence, particularly in mammals, indicates that some chemoreceptors are expressed and function in non-chemosensory (extra-nasal/oral) organs. However, such extra-nasal/oral expression has rarely been examined from a broad evolutionary perspective across vertebrate lineages. Here, we systematically investigated organ-wide expression patterns of chemoreceptor genes by conducting comprehensive bulk RNA-seq analysis across 13 organs in four representative species: mouse, Xenopus , Polypterus , and zebrafish. In all species, the majority (95–97%) of chemoreceptor genes were expressed in olfactory and gustatory organs, as expected. Remarkably, however, a subset (1–29%) showed expression in extra-nasal/oral organs, suggesting that such extra-nasal/oral expression may be a common phenomenon across vertebrates. In particular, the evolutionarily conserved OR-κ gene, with stable gene copy numbers, exhibited organ-independent expression across all analyzed species. Single-cell RNA-seq data further revealed that OR-κ is predominantly expressed in immune cells, implying potential function of chemoreception in immune systems. Furthermore, genomic context analysis showed that the OR-κ gene is isolated from canonical OR gene clusters, suggesting it may have distinct transcriptional regulatory mechanisms compared to typical olfactory receptors. Our findings expand the conventional view of chemoreceptors as sensory-specialized molecules, highlighting their unexpected functional diversity across vertebrate organs. Notably, the OR-κ gene appears to have an ancient evolutionary origin that likely traces back to the common ancestor of vertebrates. Taken together, this study compels us to reconsider the functions and evolutionary trajectories of chemoreceptor genes in vertebrates. Chemoreceptor Olfactory receptor Ectopic expression Evolution Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The accurate perception of the surrounding environment is essential for animal survival, allowing individuals to recognize nutritional sources, toxins, predators, and reproductive partners [ 1 ]. Among the five senses of vertebrates, olfaction and gustation are collectively referred to as chemosensation, because they detect chemical compounds derived from the external environment [ 2 ]. In vertebrates, chemoreception in the nose (olfactory organ/epithelium or vomeronasal organ) and mouth (including tongue) is mainly mediated by a subclass of chemoreceptors belonging to the G protein-coupled receptor (GPCR) superfamily [ 3 , 4 ]. These GPCRs comprise six different multigene families encoding seven-transmembrane-domain receptors: olfactory receptors (OR), trace amine-associated receptors (TAAR), vomeronasal receptor types 1 and 2 (V1R and V2R), and taste receptor types 1 and 2 (T1R and T2R) [ 5 ]. Chemoreceptor genes were initially discovered in mammalian nasal/oral organs as candidate molecules for odorant and gustatory perception [ 6 – 13 ]. Subsequently, homologous genes have been successively identified in non-mammalian vertebrates [ 14 – 18 ], and their expression in nasal/oral organs has been demonstrated in many species, such as channel catfish [ 14 ], zebrafish [ 16 , 17 , 19 – 21 ] and African clawed frog [ 22 , 23 ]. Recent advances in whole-genome sequencing technologies and the resulting expansion of genomic data have illuminated the evolutionary history of vertebrate chemoreceptor genes. OR, TAAR/V1R/V2R, and T1R/T2R genes were acquired before the divergence of cnidarians and bilaterians [ 24 ], jawless and jawed fish [ 25 – 29 ], and jawed vertebrates [ 30 – 32 ], respectively. These findings indicate that chemoreceptor genes, while first discovered in mammals, are evolutionarily ancient and have been conserved across diverse vertebrate lineages [ 27 , 33 – 35 ]. Vertebrate chemoreceptor genes are encoded as multigene families distributed across multiple genomic loci. During vertebrate evolution, these gene families have undergone repeated gene duplication and pseudogenization events —a process known as birth-and-death evolution— resulting in lineage- and species-specific variation in gene copy numbers. For example, the highest known copy numbers are reported for ORs (2,399 in echidna, Tachyglossus aculeatus ), TAARs (497 in reedfish, Erpetoichthys calabaricus ), and T2Rs (268 in frog, Glandirana rugosaa ) [ 36 – 40 ]. Such continuous turnover of chemoreceptor genes through the birth-and-death process generates extensive diversity in receptor-specific amino acid sequences, contributing to receptivity across a wide range of chemical compounds [ 41 ]. Thus, the diversity of chemoreceptor genes across lineages is thought to facilitate to the precise discrimination and perception of the myriad environmental chemical compounds encountered in each species' habitat [ 36 ]. Some of these multigene families encoding chemoreceptor genes are known to function in organs beyond the nasal and oral organs. For instance, human hOR17-4 and mouse MOR23 , both expressed in sperm, mediate chemotaxis in response to small chemical molecules or short peptides [ 42 , 43 ]. Additionally, human and mouse T1R2 and T1R3 have been associated with maintaining intestinal glucose homeostasis by regulating the sodium-glucose co-transporter SGLT1 [ 44 , 45 ]. Moreover, the secretion of gastrointestinal hormones can be modulated through the activation of mouse T2R108 receptor by luminal ligands [ 46 , 47 ]. Thus, chemoreceptor genes expressed in extra-nasal/oral organs appear to be used for monitoring the internal rather than the external environment [ 48 ]. In several studies, these receptors are referred to as “ectopic olfactory receptors” or “ectopic taste receptors”, terms generally implying abnormal expression in non-chemosensory organs [ 49 , 50 ]. However, chemoreceptors expressed in non-chemosensory organs are being increasingly discovered [ 44 , 51 – 58 ], and are now recognized as a widespread phenomenon. Therefore, in this study, we refer to these as “extra-nasal/oral chemoreceptors” rather than ectopic chemoreceptors. While expression patterns of chemoreceptor genes in extra-nasal/oral organs have been increasingly characterized in mammals, particularly humans and mice, knowledge from non-mammalian species remains limited, with only a few examples reported in zebrafish [ 59 , 60 ] and blind cavefish [ 61 ]. Consequently, a comprehensive evolutionary perspective has been lacking. Recently, bulk and single-cell RNA-seq data from various organs have become available, providing new opportunities to investigate the molecular evolution of chemoreceptor genes across a broad range of vertebrates. In this study, we conducted a comprehensive screening of chemoreceptors exhibiting extra-nasal/oral expression in representative vertebrates: mouse, Xenopus , Polypterus , and zebrafish. As a result, we detected the expression of members of all six chemoreceptor gene families in extra-nasal/oral organs, suggesting that such expression may represent a common feature shared across vertebrates, from basal ray-finned fishes to mammals. Furthermore, we found that OR-κ, which is evolutionarily stable compared to the expanded OR families, is predominantly expressed in immune cells in both teleosts and mammals. Given that OR-κ originated before the divergence of jawless fishes, these findings highlight the need to reconsider the function and evolutionary origins of vertebrate chemoreceptor genes. Results The exploration of extra-nasal/oral expression of chemosensory genes from bulk RNA-seq First, we conducted bulk RNA-seq analysis of 13 organs to obtain a comprehensive overview of chemoreceptor gene expression across vertebrate species (Fig. 1 and Table S2-5). We analyzed a mammal (mouse), amphibian ( Xenopus ), basal ray-finned fish ( Polypterus ), and teleost (zebrafish). The gene annotation dataset of chemoreceptors was compiled based on previous studies [ 36 ] (Fig. 1a). All bulk RNA-seq results in this study are summarized in Fig. S1 . The majority of chemoreceptor genes (97% in Polypterus , 96% in zebrafish, 97% in Xenopus , and 95% in mouse) were expressed in nasal/oral organ, which serve as canonical chemosensory tissues containing olfactory neurons and taste bud cells (Fig. 1b). In contrast, a subset of genes (ranging from 1% in the Polypterus pectoral fin and the Xenopus muscle to 29% in the zebrafish brain) also exhibited expression in extra-nasal/oral organs. Although most chemoreceptor genes were primarily expressed in chemosensory organs, all analyzed species possessed a subset of genes expressed in extra-nasal/oral organs, which may reflect that extra-nasal/oral expression is a widespread and evolutionarily conserved feature among vertebrates (Fig. 1c). Among the chemoreceptor genes showing extra-nasal/oral expression, some genes were detected in almost all analyzed organs (Fig. 1d). Some genes were not detected in several organs by bulk RNA-seq, but were detected by reverse transcription PCR (RT-PCR) validation (as indicated by the asterisks in Fig. 1d and S2). Each chemoreceptor gene family has been subdivided into several subfamilies based on molecular phylogenetic analysis (Fig. 1e; [ 32 , 37 , 62 ]). Conventional chemoreceptor genes have undergone repeated birth-and-death processes during evolution, forming species-specific gene repertoires. In contrast, the newly identified organ-independently expressed genes represent conserved subfamilies with stable gene numbers, mostly existing as single-copy genes across species, rather than the conventionally expanded genes. These results imply that while conventional-nasal/oral receptors have undergone evolutionary expansion to detect a wide range of chemical compounds, non-conventional-extra nasal/oral chemoreceptors have remained evolutionarily stable to detect specific chemical compounds. Among these, OR-κ was the only gene exhibiting multi-organ expression conserved across all four analyzed species, showing expression in all analyzed organs in three species (mouse, zebrafish and Polypterus ) as well as expression in most of the organs analyzed in Xenopus (Fig. 1d, S2a and b). Based on this high degree of conservation, we focused on OR-κ as a representative example of a conservative chemoreceptor gene in the following analysis. Evolutionary characteristics of organ-independently expressed OR-κ genes Next, we characterized the evolutionary features of OR-κ as a representative of organ-independently expressed chemoreceptor genes (Fig. 2). Olfactory receptors are known to be divided into two major clades through molecular phylogenetic analysis, Type 1 and Type 2. Type 1 ORs are composed of the expanded subfamily, exhibiting lineage-specific birth-and-death processes that enable the detection of diverse chemical substances. In contrast, Type 2 ORs, which include OR-κ, are composed of the conserved subfamilies with stable gene copy numbers, except for OR-η (Fig. 2a; [ 37 ]). It is known that conventional expanded ORs are organized genomically to be co-regulated by shared transcriptional regulatory sequences such as the H element [ 63 ], the P element [ 64 ], the J element [ 65 ] and Greek islands [ 66 ]. Since the OR-κ gene was more than 2.3 Mb apart from other OR genes in all analyzed species, it is likely to be transcribed independently of the regulatory mechanisms controlling expanded OR genes (Fig. 2b). A similar genomic arrangement was also observed for other conservative ORs, such as OR-θ (Fig. 2b). Single-cell RNA-seq analysis of olfactory epithelium revealed that expanded ORs were abundantly detected in both mouse and zebrafish, whereas OR-κ expression was absent in mouse olfactory sensory neurons and observed only in a minute fraction of zebrafish cells (8 /2,286 neurons) (Fig. S3). Analysis of genomic synteny revealed that the OR-κ genomic region is conserved within both fish lineages (zebrafish and Polypterus ) and tetrapod lineages ( Xenopus and mouse), indicating an ancient evolutionary origin (Fig. 2c). Expression of OR-κ genes at single-cell resolution Next, we identified the cell types expressing OR-κ genes (Fig. 3). We performed in situ hybridization and successfully visualized OR-κ-expressing cells in the intestine (Fig. 3a). The mRNA signal was concentrated in the basal region of intestinal folds and localized beneath the monolayered intestinal epithelium. To further characterize the OR-κ-expressing cells, we re-analyzed published single-cell RNA-seq datasets from zebrafish (Fig. 3b-e) and mouse (Fig. 3f-j). A comprehensive single-cell RNA-seq dataset of zebrafish encompassing whole-body tissues across developmental stages (pharyngula to adult) revealed OR-κ expression in multiple cell clusters (Fig. 3b), predominantly in immune cells (82%; 4,979/6,081 cells, Fig. 3c and Table S6). Focused analysis of the immune cell clusters confirmed that OR-κ is co-expressed with canonical immune cell markers, such as lcp1 , ctss1 , csf1ra , mpx and marco [ 67 – 71 ] (Fig. 3d, e and S4a). These findings suggest that the organ-independent expression of zebrafish OR-κ is likely derived from immune cells. Re-analysis of single-cell RNA-seq data from 29 organs in mouse similarly revealed OR-κ expression across multiple cell clusters (Fig. 3f), particularly in immune cells (36.6%; 1,336/3,653 cells) and endothelial cells (51%; 1,869/3,653 cells) (Fig. 3g and Table S7). Consistent with bulk RNA-seq results, OR-κ expression was detected across cells from multiple tissues (Table S8). Analysis restricted to immune and endothelial cells demonstrated co-expression of OR-κ with known cell-type-specific markers, such as Ptprb , Clec4g , Pecam1 , Tie1 and Cdh5 [ 72 – 74 ] (Fig. 3h-j and S4b). Discussion Ancient, conserved extra-nasal/oral chemoreceptor expression Among the five senses of vertebrates, olfaction and gustation are collectively referred to as ‘chemical senses’ because they detect chemical compounds originating from the external environment [ 2 ]. These chemical cues are received by GPCRs—including ORs, TAARs, V1Rs, V2Rs, T1Rs, and T2Rs—which are expressed in olfactory sensory neurons or taste bud cells. Upon activation, these receptors convert extracellular chemical signals into intracellular responses that are eventually perceived as odors or tastes in higher-order brain regions [ 5 ]. Because humans perceive smell and taste through the nose and tongue, chemoreceptor genes were initially isolated under the hypothesis that they are specifically expressed in olfactory and gustatory organs [ 6 , 8 , 9 , 12 , 75 – 77 ]. Owing to this empirical and historical background, their potential functions in non-chemosensory organs have often been overlooked. Although subsequent studies have repeatedly identified chemoreceptor expression in extra-nasal/oral tissues [ 78 ], most of these findings are limited to mammals, and their evolutionary context has remained largely unexplored [ 44 , 51 – 58 ]. In the present study, we performed a comprehensive expression analysis of 4,614 chemoreceptor genes across 13 organs from four representative vertebrate species. Consistent with previous reports, the vast majority of chemoreceptor genes (95–97%) were expressed in nasal/oral organs. However, a subset of these genes (1–29%) also exhibited expression in extra-nasal/oral organs (Fig. 1), suggesting that such expression may represent a conserved feature across vertebrates rather than a mammal-specific phenomenon. Evolutionary dynamics of conventional (expanded) and conservative (stable) chemoreceptor genes Vertebrate chemoreceptor genes constitute a multigene family that has undergone repeated gene duplication and pseudogenization (birth-and-death process) during evolution, resulting in lineage-specific diversity in gene copy number and sequence variation [ 36 , 41 ]. Such gene turnover is thought to contribute to the accurate discrimination of diverse chemical cues from the external environment [ 41 ]. Therefore, gene number expansion through frequent tandem duplication within genomic clusters is interpreted as a typical evolutionary pattern of chemoreceptors [ 25 , 79 – 81 ]. In contrast to these conventionally expanded genes, our analysis identified several evolutionarily stable chemoreceptor genes that exhibit organ-independent expression (Fig. 1d-e). Among them, the OR-κ gene was notably expressed in immune cells, and this expression pattern was shared between zebrafish and mouse (Fig. 3b-j), suggesting a conserved role of OR-κ gene vertebrate immune systems. Previous studies have reported that certain evolutionarily conserved ORs—such as OR51E1 / Olfr558 [ 51 , 82 ] and OR51E2 / Olfr78 [ 52 , 83 ] —function in extra-nasal organs. In addition, ORs conserved between human and chimpanzee also tend to be expressed extra-nasally [ 84 ]. Although the specific cell types expressing other evolutionary stable genes (e.g., OR-θ1, ancV1R , and T1R1 ) were not identified in this study, our findings suggest these receptors may serve functions distinct from odorant/gustatory perception. Further identification of their expressing cells will provide valuable insights into the molecular evolution and diversification of chemoreceptor gene functions. Distinct transcriptional regulation of the OR-κ gene The transcriptional regulatory mechanisms of conventional- (expanded-) chemoreceptor genes have been well characterized. These genes are often organized in genomic clusters, are controlled by shared enhancers [ 63 – 65 , 85 , 86 ], heterochromatin-mediated silencing [ 87 , 88 ] and dynamic three-dimensional chromosomal rearrangements [ 66 , 89 , 90 ]. It has been proposed that extra-nasal expression of ORs may result from the leakage or relaxation of these regulatory constraints within expanded gene clusters [ 50 ]. Many such ORs maintain their original olfactory function while also acquiring novel physiological roles in other tissues [ 42 , 50 , 91 – 95 ]. Interestingly, the OR-κ gene identified in this study has been evolutionarily isolated from other OR gene clusters since its origin, remaining more than 2.3Mb away from any other OR loci in all analyzed species (Fig. 2b and S4). This genomic isolation implies that OR-κ is transcribed under an independent regulatory mechanism distinct from that of conventional OR clusters. Such divergence in transcriptional control may underlie its unique expression in immune cells. Notably, similar genomic isolation was also observed for other conservative ORs (e.g., OR-θ), raising the possibility that these evolutionarily stable genes may likewise be expressed extra-nasally under distinct transcriptional control. Future studies focusing on the transcriptional regulation of OR-κ and other evolutionarily stable chemoreceptor genes will be crucial for elucidating the evolutionary origins of regulatory mechanisms governing chemoreceptor gene expression. Possible functions of the OR-κ gene in immune and endothelial cells Expression of OR genes in immune cells has been reported for several Type 1 ORs (conventional expanded ORs) in mouse [ 96 – 98 ], human, cattle [ 99 ], opossum and platypus [ 100 ]. In mice, these ORs have been implicated in cellular chemotaxis [ 96 – 98 ]. Such expression is thought to have arisen secondarily as a consequence of escaping the canonical transcriptional constraints during the birth-and-death evolution of expanded ORs. In contrast, the OR-κ gene copy numbers are evolutionarily stable from jawless fish to mammals [ 37 ], and its expression in immune cells is shared between zebrafish and mouse (Fig. 3), suggesting that immune-related expression may represent an ancestral and conserved feature across vertebrates. Our bulk RNA-seq analyses detected OR-κ expression in multiple tissues, likely reflecting its presence in circulating leukocytes distributed throughout the body. Although the precise immune function of OR-κ remains unclear, several results in this study provide intriguing clues. First, the evolutionary origin of OR-κ can be traced back to the common ancestor of vertebrates, including jawless fish (Fig. 1e), coinciding with the emergence of adaptive immunity [ 101 , 102 ]. Second, in mice, OR-κ was also expressed in a substantial population of endothelial cells in addition to immune cells (Fig. 3f-j). Given that, immune and endothelial cells are thought to share both evolutionary [ 103 – 105 ] and developmental origins [ 106 – 110 ], the shared expression of OR-κ in these two cell types is consistent with this finding and offers valuable insight into its potential role. Finally, although the OR-κ gene is shared across most vertebrates, it has been lost independently in several lineages, including human, chicken, and the coelacanth [ 33 , 37 ]. Whether this loss of OR-κ affects immune function or is compensated by other OR genes remains an important question. Determining the precise evolutionary timing of OR-κ gene loss will provide further insight into its ancestral role. Because our analyses were limited to representative species, expanding taxonomic coverage will be essential for future analyses. Taken together, the OR-κ gene appears to function differently from conventional olfactory receptor genes expressed in olfactory sensory neurons. Rather than contributing to the perception of external stimuli, OR-κ may play a role in sensing and regulating internal physiological or immune states. Comprehensive investigation of the evolutionarily history, expression patterns, and molecular functions of OR-κ and other evolutionary stable chemoreceptors will deepen our understanding of the origin and diversification of chemoreceptor systems. Conclusion In this study, we conducted a comprehensive survey of chemoreceptor gene expression across various extra-nasal and extra-oral organs in diverse vertebrate species, and evaluated their evolutionary dynamics. Notably, we identified the OR-κ, an olfactory receptor with stable gene numbers across vertebrates, as predominantly expressed in immune cell populations in both teleosts and mammals, unlike the conventional expanded OR families. Given that the OR-κ likely originated around the same evolutionary period as adaptive immunity—both emerging in the jawless vertebrate lineage—our results provide the first proposal for an evolutionarily conserved chemoreceptor expression in non-chemosensory organs across vertebrates. This work invites a reevaluation of chemoreceptor function, emphasizing their role beyond the classical olfactory and gustatory systems and implying an ancient link between chemosensation and the immune system conserved in vertebrates. Materials and methods Animals Polypterus ( Polypterus senegalus ) were obtained from a commercial supplier (Nettaigyo-tsuhan forest, Wakayama, Japan). The Xenopus ( Xenopus tropicalis ) strain Nigerian H (Xtr.NigerianH Huarc , RRID: HUARC_1002) and the zebrafish ( Danio rerio ) strain RIKEN WT (RW) were provided by National BioResource Project (NBRP) of MEXT through Hiroshima University Amphibian Research Center (RRID: SCR_019015) and RIKEN Center for Brain Science, respectively. All animals were maintained and bred at 27°C on a 12/12 h light/dark cycle. All experiments were conducted in accordance with the Institutional Animal Experiment Committee of the Institute of Science Tokyo. Bulk RNA-seq analysis RNA was extracted from Polypterus (intestine and pectoral fin) and Xenopus (nasal tissue, oral tissue, spinal cord, lung, heart, intestine, liver and kidney) using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA). The extracted total RNA was sequenced at 100 bp paired-end reads on a NovaSeq 6000 or Novaseq X by Macrogen Japan Corp., using a TruSeq stranded mRNA Library Prep Kit. Polypterus kidney sequence data were unpublished, provided by Y. Kimura. All other RNA sequence data were obtained from the NCBI SRA database (Table S1). SRA files were retrieved and converted to FASTQ format using the SRA Toolkit prefetch and fastq-dump v3.0.7 [111]. The quality control of raw sequence data was performed with fastp v0.23.4 [112] with the following options: -q 20 -l 25. The reads were mapped to the genome of Polypterus (ASM1683550v1), zebrafish (GRCz11), Xenopus (UCB_Xtro_10.0) or mouse ( Mus musculus ) (GRCm39) using STAR version 2.7.5c [113] and quantified using rsem-calculate-expression v1.3.3 [114]. The gene annotation data for the quantification were downloaded from Refseq and edited for the regions of known chemoreceptor genes [36]. NCBI database designations for the OR-κ genes are as follows: zgc:194312 in zebrafish; zgc:194312 (OR-κ2) and olfactory receptor 4K13-like (OR-κ3) in Polypterus ; Gm7582 (OR-κ2), Gm7609 (OR-κ3), Gm2666 (OR-κ4), Gm7592 (OR-κ5), and Csprs (OR-κ8) in mouse. Following previous studies with human, the threshold for classifying a gene as expressed was defined at 0.01 TPM [48]. Singe-cell RNA-seq reanalysis Public single-cell RNA-seq datasets reanalyzed in this study can be found on the Gene Expression Omnibus (GEO): GSE198832 [115]. Annotation of cell lineage and cell type for each cell followed Wang and colleagues [115]. Gene expression matrices from immune and endothelial cells in mice and immune cells in zebrafish were analyzed using Seurat v5 [116]. Twenty principal components (PCs) were selected as significant components for t-SNE analysis. The clustering parameter resolution was set to 0.8 for identifying cell clusters. Raw single-cell RNA sequencing data from zebrafish olfactory epithelium were retrieved from the NCBI SRA database (SRR31595086; [117]). Fastq files were processed using 10x Genomics Cell Ranger v6.0.2 with the GRCz11_v4.3.2_cellranger_v6 transcriptome reference [118]. Quality control filtering was applied using the following thresholds: nCount_RNA > 250 and < 5000, and mitochondrial gene percentage < 15%. Data normalization and identification of highly variable features were performed using default parameters with the NormalizeData and FindVariableFeatures functions in Seurat. UMAP dimensionality reduction was performed using seven PCs with a resolution parameter of 0.8. Initial clustering identified 19 cell clusters, which were annotated into seven cell types based on marker gene expression patterns consistent with those reported by Chen and colleagues. Single-cell RNA-seq data from mouse main olfactory epithelium were obtained from the GEO database (GSE185251; [119]). The filtered feature-barcode matrix was loaded into Seurat for downstream analysis. Data processing and cell type annotation were performed using identical parameters and marker genes as described in the original study. In situ hybridization (ISH) ISH was performed according to the method of Suzuki et al. [120] with several modifications. Briefly, the entire intestines were fixed in Davidson's solution overnight at 4°C. The fixed tissues were then immersed in 20% sucrose in PBS overnight and embedded in OCT compound before being frozen in liquid nitrogen. The embedded blocks were then sliced into sections 12 µm thick and placed on a coated glass slide. The frozen sections were then digested with 5 µg/ml proteinase K for 10 min at 37°C, after which they were hybridized with 5 ng/µl DIG-labelled riboprobes at 65°C overnight. The sections were washed, treated with 2 µg/ml RNase A in TNE buffer for 30 min at 37°C. Then, they were treated with a streptavidin/biotin blocking kit (Vector Laboratories, Newark, CA, USA). After that, they were treated with 1% blocking reagent (PerkinElmer, Waltham, MA, USA) in TBS buffer for 1 h. Signals were detected with a peroxidase-conjugated anti-DIG antibody (1:100, 11207733910, Roche), amplified by a TSA Plus Biotin kit (1:50, PerkinElmer), and visualized with an Alexa Fluor 488-conjugated streptavidin (1:200, Thermo Fisher Scientific, Waltham, MA, USA). The sections were mounted using Vectashield mounting medium containing DAPI (Vector Laboratories) and digitally captured using a Zeiss Axioplan SP fluorescence microscope and a Zeiss Axiocam 503 colour CCD camera (Carl Zeiss, Oberkochen, Germany). Reverse transcription-PCR (RT-PCR) Total RNA was extracted from a panel of Xenopus , mouse, Polypterus and zebrafish target tissues. After DNase I digestion (TaKaRa, Shiga, Japan), RNA samples were diluted to 10 ng/μl, and cDNA was synthesized from 2 μg of total RNA using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with oligo-dT18 primers. All PCRs were performed using KOD FX Neo polymerase or KOD One (TOYOBO, Osaka, Japan) with the following cycling conditions: 98°C for 10 sec, 55°C for 30 sec, and 68°C for 30 sec, repeated for 35 cycles. Primer sequences are listed in Table S9. β-actin was used as an internal control for RNA quality and expression normalization in the chemosensory organ. Declarations Acknowledgement We are grateful to Prof. Akira Kato, Takashi Suzuki, Junji Hirota, and Yuichi Hongoh for their invaluable advice throughout this study. We also thank Andrew Shedlock for improving the manuscript; Yuki Kimura for providing unpublished Polypterus kidney sequence data; the Integrative Bioscience Facility at Institute of Science Tokyo for DNA sequencing analysis; the Hongoh laboratory for helping with data production and analysis; and all laboratory members, with special thanks to Mitsuto Aibara and Ayumi Hirose for their invaluable support and collaboration. Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics. Ethical Approval and Consent to participate All experimental studies using the animals were approved by the Institutional Animal Experiment Committee of the Institute of Science Tokyo were performed in accordance with the institutional, governmental ARRIVE guidelines. Consent for publication Not applicable. Availability of data and material All sequence reads were deposited in the DDBJ Sequence Read Archive under accession no. PRJDB39629 and PRJDB39651. Competing interests The authors declare that they have no competing interests. Funding This study was supported by JSPS KAKENHI (20H03307, 24K02074 to M.N. and 23K14249 to T.N.), the Sasakawa Scientific Research Grant from the Japan Science Society to T.N. and JST SPRING (JPMJSP2180 to K.N.). Author contributions K.N., T.N., and M.N. conceived the research and wrote the manuscript. K.N. conducted the experiments. M.N. supervised the project. All authors read and approved the final manuscript. References Dangles O, Irschick D, Chittka L, Casas J. Variability in sensory ecology: expanding the bridge between physiology and evolutionary biology. Q Rev Biol. 2009;84:51–74. https://doi.org/10.1086/596463. The senses: A comprehensive reference. Elsevier Science & Technology; 2009. Fleischer J, Breer H, Strotmann J. Mammalian olfactory receptors. Front Cell Neurosci. 2009;3:9. https://doi.org/10.3389/neuro.03.009.2009. Roper SD, Chaudhari N. Processing umami and other tastes in mammalian taste buds. Ann N Y Acad Sci. 2009;1170:60–5. https://doi.org/10.1111/j.1749-6632.2009.04107.x. Baldwin MW, Ko M-C. Functional evolution of vertebrate sensory receptors. Horm Behav. 2020;124:104771. https://doi.org/10.1016/j.yhbeh.2020.104771. Buck L, Axel R. A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell. 1991;65:175–87. https://doi.org/10.1016/0092-8674(91)90418-x. Liberles SD, Buck LB. A second class of chemosensory receptors in the olfactory epithelium. Nature. 2006;442:645–50. https://doi.org/10.1038/nature05066. Dulac C, Axel R. A novel family of genes encoding putative pheromone receptors in mammals. Cell. 1995;83:195–206. https://doi.org/10.1016/0092-8674(95)90161-2. Herrada G, Dulac C. A novel family of putative pheromone receptors in mammals with a topographically organized and sexually dimorphic distribution. Cell. 1997;90:763–73. https://doi.org/10.1016/s0092-8674(00)80536-x. Matsunami H, Buck LB. A multigene family encoding a diverse array of putative pheromone receptors in mammals. Cell. 1997;90:775–84. https://doi.org/10.1016/s0092-8674(00)80537-1. Li X, Staszewski L, Xu H, Durick K, Zoller M, Adler E. Human receptors for sweet and umami taste. Proc Natl Acad Sci U S A. 2002;99:4692–6. https://doi.org/10.1073/pnas.072090199. Adler E, Hoon MA, Mueller KL, Chandrashekar J, Ryba NJ, Zuker CS. A novel family of mammalian taste receptors. Cell. 2000;100:693–702. https://doi.org/10.1016/s0092-8674(00)80705-9. Matsunami H, Montmayeur JP, Buck LB. A family of candidate taste receptors in human and mouse. Nature. 2000;404:601–4. https://doi.org/10.1038/35007072. Ngal J, Dowling M, Buck L, Axel R, Chess A. The family of genes encoding odorant receptors in the channel catfish. Cell. 1993;72:657–66. https://doi.org/10.1016/0092-8674(93)90395-7. Gloriam DEI, Bjarnadóttir TK, Yan Y-L, Postlethwait JH, Schiöth HB, Fredriksson R. The repertoire of trace amine G-protein-coupled receptors: large expansion in zebrafish. Mol Phylogenet Evol. 2005;35:470–82. https://doi.org/10.1016/j.ympev.2004.12.003. Ishimaru Y, Okada S, Naito H, Nagai T, Yasuoka A, Matsumoto I, et al. Two families of candidate taste receptors in fishes. Mech Dev. 2005;122:1310–21. https://doi.org/10.1016/j.mod.2005.07.005. Pfister P, Rodriguez I. Olfactory expression of a single and highly variable V1r pheromone receptor-like gene in fish species. Proc Natl Acad Sci U S A. 2005;102:5489–94. https://doi.org/10.1073/pnas.0402581102. Naito T, Saito Y, Yamamoto J, Nozaki Y, Tomura K, Hazama M, et al. Putative pheromone receptors related to the Ca2+-sensing receptor in Fugu. Proc Natl Acad Sci U S A. 1998;95:5178–81. https://doi.org/10.1073/pnas.95.9.5178. Weth F, Nadler W, Korsching S. Nested expression domains for odorant receptors in zebrafish olfactory epithelium. Proc Natl Acad Sci U S A. 1996;93:13321–6. https://doi.org/10.1073/pnas.93.23.13321. Hussain A, Saraiva LR, Korsching SI. Positive Darwinian selection and the birth of an olfactory receptor clade in teleosts. Proc Natl Acad Sci U S A. 2009;106:4313–8. https://doi.org/10.1073/pnas.0803229106. Sato Y, Miyasaka N, Yoshihara Y. Mutually exclusive glomerular innervation by two distinct types of olfactory sensory neurons revealed in transgenic zebrafish. J Neurosci. 2005;25:4889–97. https://doi.org/10.1523/JNEUROSCI.0679-05.2005. Date-Ito A, Ohara H, Ichikawa M, Mori Y, Hagino-Yamagishi K. Xenopus V1R vomeronasal receptor family is expressed in the main olfactory system. Chem Senses. 2008;33:339–46. https://doi.org/10.1093/chemse/bjm090. Syed AS, Sansone A, Röner S, Bozorg Nia S, Manzini I, Korsching SI. Different expression domains for two closely related amphibian TAARs generate a bimodal distribution similar to neuronal responses to amine odors. Sci Rep. 2015;5:13935. https://doi.org/10.1038/srep13935. Churcher AM, Taylor JS. The antiquity of chordate odorant receptors is revealed by the discovery of orthologs in the cnidarian Nematostella vectensis. Genome Biol Evol. 2011;3:36–43. https://doi.org/10.1093/gbe/evq079. Hashiguchi Y, Nishida M. Evolution of trace amine associated receptor (TAAR) gene family in vertebrates: lineage-specific expansions and degradations of a second class of vertebrate chemosensory receptors expressed in the olfactory epithelium. Mol Biol Evol. 2007;24:2099–107. https://doi.org/10.1093/molbev/msm140. Libants S, Carr K, Wu H, Teeter JH, Chung-Davidson Y-W, Zhang Z, et al. The sea lamprey Petromyzon marinus genome reveals the early origin of several chemosensory receptor families in the vertebrate lineage. BMC Evol Biol. 2009;9:180. https://doi.org/10.1186/1471-2148-9-180. Grus WE, Zhang J. Origin of the genetic components of the vomeronasal system in the common ancestor of all extant vertebrates. Mol Biol Evol. 2009;26:407–19. https://doi.org/10.1093/molbev/msn262. Sharma K, Syed AS, Ferrando S, Mazan S, Korsching SI. The Chemosensory Receptor Repertoire of a True Shark Is Dominated by a Single Olfactory Receptor Family. Genome Biol Evol. 2019;11:398–405. https://doi.org/10.1093/gbe/evz002. Kowatschew D, Korsching SI. Lamprey possess both V1R and V2R olfactory receptors, but only V1Rs are expressed in olfactory sensory neurons. Chem Senses. 2022;47. https://doi.org/10.1093/chemse/bjac007. Behrens M, Lang T, Korsching SI. A singular shark bitter taste receptor provides insights into the evolution of bitter taste perception. Proc Natl Acad Sci U S A. 2023;120:e2310347120. https://doi.org/10.1073/pnas.2310347120. Itoigawa A, Toda Y, Kuraku S, Ishimaru Y. Evolutionary origins of bitter taste receptors in jawed vertebrates. Curr Biol. 2024;34:R271–2. https://doi.org/10.1016/j.cub.2024.02.024. Nishihara H, Toda Y, Kuramoto T, Kamohara K, Goto A, Hoshino K, et al. A vertebrate-wide catalogue of T1R receptors reveals diversity in taste perception. Nat Ecol Evol. 2024;8:111–20. https://doi.org/10.1038/s41559-023-02258-8. Niimura Y, Nei M. Evolutionary dynamics of olfactory receptor genes in fishes and tetrapods. Proc Natl Acad Sci U S A. 2005;102:6039–44. https://doi.org/10.1073/pnas.0501922102. Guo L, Dai W, Xu Z, Liang Q, Miller ET, Li S, et al. Evolution of Brain-Expressed Biogenic Amine Receptors into Olfactory Trace Amine-Associated Receptors. Mol Biol Evol. 2022;39. https://doi.org/10.1093/molbev/msac006. Shi P, Zhang J. Contrasting modes of evolution between vertebrate sweet/umami receptor genes and bitter receptor genes. Mol Biol Evol. 2006;23:292–300. https://doi.org/10.1093/molbev/msj028. Policarpo M, Baldwin MW, Casane D, Salzburger W. Diversity and evolution of the vertebrate chemoreceptor gene repertoire. Nat Commun. 2024;15:1421. https://doi.org/10.1038/s41467-024-45500-y. Niimura Y. On the origin and evolution of vertebrate olfactory receptor genes: comparative genome analysis among 23 chordate species. Genome Biol Evol. 2009;1:34–44. https://doi.org/10.1093/gbe/evp003. Policarpo M, Bemis KE, Laurenti P, Legendre L, Sandoz J-C, Rétaux S, et al. Coevolution of the olfactory organ and its receptor repertoire in ray-finned fishes. BMC Biol. 2022;20:195. https://doi.org/10.1186/s12915-022-01397-x. Zhou Y, Shearwin-Whyatt L, Li J, Song Z, Hayakawa T, Stevens D, et al. Platypus and echidna genomes reveal mammalian biology and evolution. Nature. 2021;592:756–62. https://doi.org/10.1038/s41586-020-03039-0. Zhong H, Huang J, Shang S, Yuan B. Evolutionary insights into umami, sweet, and bitter taste receptors in amphibians. Ecol Evol. 2021;11:18011–25. https://doi.org/10.1002/ece3.8398. Nei M, Rooney AP. Concerted and birth-and-death evolution of multigene families. Annu Rev Genet. 2005;39:121–52. https://doi.org/10.1146/annurev.genet.39.073003.112240. Spehr M, Gisselmann G, Poplawski A, Riffell JA, Wetzel CH, Zimmer RK, et al. Identification of a testicular odorant receptor mediating human sperm chemotaxis. Science. 2003;299:2054–8. https://doi.org/10.1126/science.1080376. Fukuda N, Yomogida K, Okabe M, Touhara K. Functional characterization of a mouse testicular olfactory receptor and its role in chemosensing and in regulation of sperm motility. J Cell Sci. 2004;117 Pt 24:5835–45. https://doi.org/10.1242/jcs.01507. Margolskee RF, Dyer J, Kokrashvili Z, Salmon KSH, Ilegems E, Daly K, et al. T1R3 and gustducin in gut sense sugars to regulate expression of Na + -glucose cotransporter 1. Proc Natl Acad Sci U S A. 2007;104:15075–80. https://doi.org/10.1073/pnas.0706678104. Young RL, Chia B, Isaacs NJ, Ma J, Khoo J, Wu T, et al. Disordered control of intestinal sweet taste receptor expression and glucose absorption in type 2 diabetes. Diabetes. 2013;62:3532–41. https://doi.org/10.2337/db13-0581. Kok BP, Galmozzi A, Littlejohn NK, Albert V, Godio C, Kim W, et al. Intestinal bitter taste receptor activation alters hormone secretion and imparts metabolic benefits. Mol Metab. 2018;16:76–87. https://doi.org/10.1016/j.molmet.2018.07.013. Chen MC, Wu SV, Reeve JR Jr, Rozengurt E. Bitter stimuli induce Ca2+ signaling and CCK release in enteroendocrine STC-1 cells: role of L-type voltage-sensitive Ca2+ channels. Am J Physiol Cell Physiol. 2006;291:C726-39. https://doi.org/10.1152/ajpcell.00003.2006. Flegel C, Manteniotis S, Osthold S, Hatt H, Gisselmann G. Expression profile of ectopic olfactory receptors determined by deep sequencing. PLoS One. 2013;8:e55368. https://doi.org/10.1371/journal.pone.0055368. Feldmesser E, Olender T, Khen M, Yanai I, Ophir R, Lancet D. Widespread ectopic expression of olfactory receptor genes. BMC Genomics. 2006;7:121. https://doi.org/10.1186/1471-2164-7-121. Maßberg D, Hatt H. Human Olfactory Receptors: Novel Cellular Functions Outside of the Nose. Physiol Rev. 2018;98:1739–63. https://doi.org/10.1152/physrev.00013.2017. Xu J, Choi R, Gupta K, Warren HR, Santhanam L, Pluznick JL. An evolutionarily conserved olfactory receptor is required for sex differences in blood pressure. Sci Adv. 2024;10:eadk1487. https://doi.org/10.1126/sciadv.adk1487. Chang AJ, Ortega FE, Riegler J, Madison DV, Krasnow MA. Oxygen regulation of breathing through an olfactory receptor activated by lactate. Nature. 2015;527:240–4. https://doi.org/10.1038/nature15721. Chéret J, Bertolini M, Ponce L, Lehmann J, Tsai T, Alam M, et al. Olfactory receptor OR2AT4 regulates human hair growth. Nat Commun. 2018;9:3624. https://doi.org/10.1038/s41467-018-05973-0. Griffin CA, Kafadar KA, Pavlath GK. MOR23 promotes muscle regeneration and regulates cell adhesion and migration. Dev Cell. 2009;17:649–61. https://doi.org/10.1016/j.devcel.2009.09.004. Janssen S, Laermans J, Verhulst P-J, Thijs T, Tack J, Depoortere I. Bitter taste receptors and α-gustducin regulate the secretion of ghrelin with functional effects on food intake and gastric emptying. Proc Natl Acad Sci U S A. 2011;108:2094–9. https://doi.org/10.1073/pnas.1011508108. Nakagawa Y, Nagasawa M, Yamada S, Hara A, Mogami H, Nikolaev VO, et al. Sweet taste receptor expressed in pancreatic beta-cells activates the calcium and cyclic AMP signaling systems and stimulates insulin secretion. PLoS One. 2009;4:e5106. https://doi.org/10.1371/journal.pone.0005106. Kyriazis GA, Soundarapandian MM, Tyrberg B. Sweet taste receptor signaling in beta cells mediates fructose-induced potentiation of glucose-stimulated insulin secretion. Proc Natl Acad Sci U S A. 2012;109:E524-32. https://doi.org/10.1073/pnas.1115183109. Weber L, Al-Refae K, Ebbert J, Jägers P, Altmüller J, Becker C, et al. Activation of odorant receptor in colorectal cancer cells leads to inhibition of cell proliferation and apoptosis. PLoS One. 2017;12:e0172491. https://doi.org/10.1371/journal.pone.0172491. Jundi D, Coutanceau J-P, Bullier E, Imarraine S, Fajloun Z, Hong E. Expression of olfactory receptor genes in non-olfactory tissues in the developing and adult zebrafish. Sci Rep. 2023;13:4651. https://doi.org/10.1038/s41598-023-30895-3. Birdal G, D’Gama PP, Jurisch-Yaksi N, Korsching SI. Expression of taste sentinels, T1R, T2R, and PLCβ2, on the passageway for olfactory signals in zebrafish. Chem Senses. 2023;48. https://doi.org/10.1093/chemse/bjad040. Bhatia V, de Jesus VC, Shaik FA, Jaggupilli A, Singh N, Chelikani P, et al. Extraoral expression and characterization of bitter taste receptors in Astyanax mexicanus (Mexican tetra fish). FASEB Bioadv. 2022;4:574–84. https://doi.org/10.1096/fba.2022-00032. Zhang Z, Sakuma A, Kuraku S, Nikaido M. Remarkable diversity of vomeronasal type 2 receptor (OlfC) genes of basal ray-finned fish and its evolutionary trajectory in jawed vertebrates. Sci Rep. 2022;12:6455. https://doi.org/10.1038/s41598-022-10428-0. Serizawa S, Miyamichi K, Nakatani H, Suzuki M, Saito M, Yoshihara Y, et al. Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse. Science. 2003;302:2088–94. https://doi.org/10.1126/science.1089122. Khan M, Vaes E, Mombaerts P. Regulation of the probability of mouse odorant receptor gene choice. Cell. 2011;147:907–21. https://doi.org/10.1016/j.cell.2011.09.049. Iwata T, Niimura Y, Kobayashi C, Shirakawa D, Suzuki H, Enomoto T, et al. A long-range cis-regulatory element for class I odorant receptor genes. Nat Commun. 2017;8:885. https://doi.org/10.1038/s41467-017-00870-4. Markenscoff-Papadimitriou E, Allen WE, Colquitt BM, Goh T, Murphy KK, Monahan K, et al. Enhancer interaction networks as a means for singular olfactory receptor expression. Cell. 2014;159:543–57. https://doi.org/10.1016/j.cell.2014.09.033. Shinomiya H. Plastin family of actin-bundling proteins: its functions in leukocytes, neurons, intestines, and cancer. Int J Cell Biol. 2012;2012:213492. https://doi.org/10.1155/2012/213492. Gao H, Zhang Z, Deng J, Song Y. Cathepsin S: molecular mechanisms in inflammatory and immunological processes. Front Immunol. 2025;16:1600206. https://doi.org/10.3389/fimmu.2025.1600206. Xiang C, Li H, Tang W. Targeting CSF-1R represents an effective strategy in modulating inflammatory diseases. Pharmacol Res. 2023;187:106566. https://doi.org/10.1016/j.phrs.2022.106566. Gurski CJ, Dittel BN. Myeloperoxidase as a marker to differentiate mouse monocyte/macrophage subsets. Int J Mol Sci. 2022;23:8246. https://doi.org/10.3390/ijms23158246. Arredouani MS. Is the scavenger receptor MARCO a new immune checkpoint? Oncoimmunology. 2014;3:e955709. https://doi.org/10.4161/21624011.2014.955709. Vestweber D. Vascular endothelial protein tyrosine phosphatase regulates endothelial function. Physiology (Bethesda). 2021;36:84–93. https://doi.org/10.1152/physiol.00026.2020. Feng W, Chen L, Nguyen PK, Wu SM, Li G. Single cell analysis of endothelial cells identified organ-specific molecular signatures and heart-specific cell populations and molecular features. Front Cardiovasc Med. 2019;6:165. https://doi.org/10.3389/fcvm.2019.00165. Korhonen EA, Lampinen A, Giri H, Anisimov A, Kim M, Allen B, et al. Tie1 controls angiopoietin function in vascular remodeling and inflammation. J Clin Invest. 2016;126:3495–510. https://doi.org/10.1172/JCI84923. Hoon MA, Adler E, Lindemeier J, Battey JF, Ryba NJ, Zuker CS. Putative mammalian taste receptors: a class of taste-specific GPCRs with distinct topographic selectivity. Cell. 1999;96:541–51. https://doi.org/10.1016/s0092-8674(00)80658-3. Borowsky B, Adham N, Jones KA, Raddatz R, Artymyshyn R, Ogozalek KL, et al. Trace amines: identification of a family of mammalian G protein-coupled receptors. Proc Natl Acad Sci U S A. 2001;98:8966–71. https://doi.org/10.1073/pnas.151105198. Bunzow JR, Sonders MS, Arttamangkul S, Harrison LM, Zhang G, Quigley DI, et al. Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor. Mol Pharmacol. 2001;60:1181–8. https://doi.org/10.1124/mol.60.6.1181. Parmentier M, Libert F, Schurmans S, Schiffmann S, Lefort A, Eggerickx D, et al. Expression of members of the putative olfactory receptor gene family in mammalian germ cells. Nature. 1992;355:453–5. https://doi.org/10.1038/355453a0. Nei M, Niimura Y, Nozawa M. The evolution of animal chemosensory receptor gene repertoires: roles of chance and necessity. Nat Rev Genet. 2008;9:951–63. https://doi.org/10.1038/nrg2480. Hashiguchi Y, Nishida M. Evolution and origin of vomeronasal-type odorant receptor gene repertoire in fishes. BMC Evol Biol. 2006;6:76. https://doi.org/10.1186/1471-2148-6-76. Higgins KW, Itoigawa A, Toda Y, Bellott DW, Anderson R, Márquez R, et al. Rapid expansion and specialization of the TAS2R bitter taste receptor family in amphibians. PLoS Genet. 2025;21:e1011533. https://doi.org/10.1371/journal.pgen.1011533. Bellono NW, Bayrer JR, Leitch DB, Castro J, Zhang C, O’Donnell TA, et al. Enterochromaffin cells are gut chemosensors that couple to sensory neural pathways. Cell. 2017;170:185-198.e16. https://doi.org/10.1016/j.cell.2017.05.034. Pluznick JL, Protzko RJ, Gevorgyan H, Peterlin Z, Sipos A, Han J, et al. Olfactory receptor responding to gut microbiota-derived signals plays a role in renin secretion and blood pressure regulation. Proc Natl Acad Sci U S A. 2013;110:4410–5. https://doi.org/10.1073/pnas.1215927110. De la Cruz O, Blekhman R, Zhang X, Nicolae D, Firestein S, Gilad Y. A signature of evolutionary constraint on a subset of ectopically expressed olfactory receptor genes. Mol Biol Evol. 2009;26:491–4. https://doi.org/10.1093/molbev/msn294. Shah A, Ratkowski M, Rosa A, Feinstein P, Bozza T. Olfactory expression of trace amine-associated receptors requires cooperative cis-acting enhancers. Nat Commun. 2021;12:3797. https://doi.org/10.1038/s41467-021-23824-3. Fei A, Wu W, Tan L, Tang C, Xu Z, Huo X, et al. Coordination of two enhancers drives expression of olfactory trace amine-associated receptors. Nat Commun. 2021;12:3798. https://doi.org/10.1038/s41467-021-23823-4. Magklara A, Yen A, Colquitt BM, Clowney EJ, Allen W, Markenscoff-Papadimitriou E, et al. An epigenetic signature for monoallelic olfactory receptor expression. Cell. 2011;145:555–70. https://doi.org/10.1016/j.cell.2011.03.040. Lyons DB, Magklara A, Goh T, Sampath SC, Schaefer A, Schotta G, et al. Heterochromatin-mediated gene silencing facilitates the diversification of olfactory neurons. Cell Rep. 2014;9:884–92. https://doi.org/10.1016/j.celrep.2014.10.001. Clowney EJ, LeGros MA, Mosley CP, Clowney FG, Markenskoff-Papadimitriou EC, Myllys M, et al. Nuclear aggregation of olfactory receptor genes governs their monogenic expression. Cell. 2012;151:724–37. https://doi.org/10.1016/j.cell.2012.09.043. Lomvardas S, Barnea G, Pisapia DJ, Mendelsohn M, Kirkland J, Axel R. Interchromosomal interactions and olfactory receptor choice. Cell. 2006;126:403–13. https://doi.org/10.1016/j.cell.2006.06.035. Bushdid C, Magnasco MO, Vosshall LB, Keller A. Humans can discriminate more than 1 trillion olfactory stimuli. Science. 2014;343:1370–2. https://doi.org/10.1126/science.1249168. Manteniotis S, Wojcik S, Brauhoff P, Möllmann M, Petersen L, Göthert JR, et al. Functional characterization of the ectopically expressed olfactory receptor 2AT4 in human myelogenous leukemia. Cell Death Discov. 2016;2:15070. https://doi.org/10.1038/cddiscovery.2015.70. Manteniotis S, Wojcik S, Göthert JR, Dürig J, Dührsen U, Gisselmann G, et al. Deorphanization and characterization of the ectopically expressed olfactory receptor OR51B5 in myelogenous leukemia cells. Cell Death Discov. 2016;2:16010. https://doi.org/10.1038/cddiscovery.2016.10. Tsai T, Veitinger S, Peek I, Busse D, Eckardt J, Vladimirova D, et al. Two olfactory receptors-OR2A4/7 and OR51B5-differentially affect epidermal proliferation and differentiation. Exp Dermatol. 2017;26:58–65. https://doi.org/10.1111/exd.13132. Fukuda N, Touhara K. Developmental expression patterns of testicular olfactory receptor genes during mouse spermatogenesis. Genes Cells. 2006;11:71–81. https://doi.org/10.1111/j.1365-2443.2005.00915.x. Li JJ, Tay HL, Plank M, Essilfie A-T, Hansbro PM, Foster PS, et al. Activation of olfactory receptors on mouse pulmonary macrophages promotes monocyte chemotactic protein-1 production. PLoS One. 2013;8:e80148. https://doi.org/10.1371/journal.pone.0080148. Clark AA, Nurmukhambetova S, Li X, Munger SD, Lees JR. Odorants specifically modulate chemotaxis and tissue retention of CD4+ T cells via cyclic adenosine monophosphate induction. J Leukoc Biol. 2016;100:699–709. https://doi.org/10.1189/jlb.1A0914-425RR. Orecchioni M, Kobiyama K, Winkels H, Ghosheh Y, McArdle S, Mikulski Z, et al. Olfactory receptor 2 in vascular macrophages drives atherosclerosis by NLRP3-dependent IL-1 production. Science. 2022;375:214–21. https://doi.org/10.1126/science.abg3067. Malki A, Fiedler J, Fricke K, Ballweg I, Pfaffl MW, Krautwurst D. Class I odorant receptors, TAS1R and TAS2R taste receptors, are markers for subpopulations of circulating leukocytes. J Leukoc Biol. 2015;97:533–45. https://doi.org/10.1189/jlb.2A0714-331RR. Sampson JM, Morrissey KA, Douek DC, Miller RD. A family of olfactory receptors uniquely expanded in marsupial and monotreme genomes are expressed by a T cell subset also unique to marsupials and monotremes. Dev Comp Immunol. 2024;154:105149. https://doi.org/10.1016/j.dci.2024.105149. Litman GW, Rast JP, Fugmann SD. The origins of vertebrate adaptive immunity. Nat Rev Immunol. 2010;10:543–53. https://doi.org/10.1038/nri2807. Hirano M, Das S, Guo P, Cooper MD. The evolution of adaptive immunity in vertebrates. Adv Immunol. 2011;109:125–57. https://doi.org/10.1016/B978-0-12-387664-5.00004-2. Amersfoort J, Eelen G, Carmeliet P. Immunomodulation by endothelial cells - partnering up with the immune system? Nat Rev Immunol. 2022;22:576–88. https://doi.org/10.1038/s41577-022-00694-4. Fauvarque M-O, Williams MJ. Drosophila cellular immunity: a story of migration and adhesion. J Cell Sci. 2011;124 Pt 9:1373–82. https://doi.org/10.1242/jcs.064592. Rotstein B, Paululat A. On the morphology of the Drosophila heart. J Cardiovasc Dev Dis. 2016;3:15. https://doi.org/10.3390/jcdd3020015. Bertrand JY, Chi NC, Santoso B, Teng S, Stainier DYR, Traver D. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature. 2010;464:108–11. https://doi.org/10.1038/nature08738. Boisset J-C, van Cappellen W, Andrieu-Soler C, Galjart N, Dzierzak E, Robin C. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature. 2010;464:116–20. https://doi.org/10.1038/nature08764. Kissa K, Herbomel P. Blood stem cells emerge from aortic endothelium by a novel type of cell transition. Nature. 2010;464:112–5. https://doi.org/10.1038/nature08761. Lam EYN, Hall CJ, Crosier PS, Crosier KE, Flores MV. Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells. Blood. 2010;116:909–14. https://doi.org/10.1182/blood-2010-01-264382. Williamson AE, Liyanage S, Hassanshahi M, Dona MSI, Toledo-Flores D, Tran DXA, et al. Discovery of an embryonically derived bipotent population of endothelial-macrophage progenitor cells in postnatal aorta. Nat Commun. 2024;15:7097. https://doi.org/10.1038/s41467-024-51637-7. Leinonen R, Sugawara H, Shumway M, International Nucleotide Sequence Database Collaboration. The sequence read archive. Nucleic Acids Res. 2011;39 Database issue:D19-21. https://doi.org/10.1093/nar/gkq1019. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884–90. https://doi.org/10.1093/bioinformatics/bty560. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. https://doi.org/10.1093/bioinformatics/bts635. Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323. https://doi.org/10.1186/1471-2105-12-323. Wang R, Zhang P, Wang J, Ma L, E W, Suo S, et al. Construction of a cross-species cell landscape at single-cell level. Nucleic Acids Res. 2023;51:501–16. https://doi.org/10.1093/nar/gkac633. Hao Y, Stuart T, Kowalski MH, Choudhary S, Hoffman P, Hartman A, et al. Dictionary learning for integrative, multimodal and scalable single-cell analysis. Nat Biotechnol. 2023. https://doi.org/10.1038/s41587-023-01767-y. Chen W, Jiang H, Wang C, Ding Z, Yu D, Liu Y, et al. Single-cell RNA sequencing of zebrafish olfactory epithelium reveals cellular heterogeneity and responses to a conspecific alarm substance. Water Biology and Security. 2024;4:100324. https://doi.org/10.1016/j.watbs.2024.100324. Lawson ND, Li R, Shin M, Grosse A, Yukselen O, Stone OA, et al. An improved zebrafish transcriptome annotation for sensitive and comprehensive detection of cell type-specific genes. Elife. 2020;9. https://doi.org/10.7554/eLife.55792. Horgue LF, Assens A, Fodoulian L, Marconi L, Tuberosa J, Haider A, et al. Transcriptional adaptation of olfactory sensory neurons to GPCR identity and activity. Nat Commun. 2022;13:2929. https://doi.org/10.1038/s41467-022-30511-4. Suzuki H, Nikaido M, Hagino-Yamagishi K, Okada N. Distinct functions of two olfactory marker protein genes derived from teleost-specific whole genome duplication. BMC Evol Biol. 2015;15:245. https://doi.org/10.1186/s12862-015-0530-y. Vassar R, Ngai J, Axel R. Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell. 1993;74:309–18. https://doi.org/10.1016/0092-8674(93)90422-m. Ressler KJ, Sullivan SL, Buck LB. A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell. 1993;73:597–609. https://doi.org/10.1016/0092-8674(93)90145-g. Supplementary Files NishiuraetalFigures5.png Fig. S1 Heatmap showing gene expression profiles of all chemoreceptor genes analyzed in this study. Each row represents a chemoreceptor gene, and each column corresponds to an organ analyzed. Normalized expression levels (TPM) are shown as a heatmap. The legend provided in the lower right corner indicates expression values, color codes for chemoreceptor gene families, and the list of analyzed organs. NishiuraetalFigures6.png Fig. S2 Reverse transcription PCR (RT-PCR) detection of chemoreceptor genes undetected by bulk RNA-seq RT-PCR analysis detecting chemoreceptor gene expression in tissues where transcripts were not identified by bulk RNA-seq analysis. OR-κ expression in various tissues of (a) Xenopus and (b) mouse. (c) T1R2A expression in Polypterus intestine. (d) T1R1 expression in zebrafish muscle. Genomic DNA (g.DNA) was used as a positive control. Because OR-κ, T1R2A and T1R1 genes consist of a single exon, RT-PCR was performed with and without reverse transcriptase (+/–) to distinguish cDNA-derived amplification products. NishiuraetalFigures7.png Fig. S3 Expression profile of OR-κ genes revealed by single-cell RNA-seq analysis of the olfactory organ. Expression patterns of OR-κ genes and representative canonical ORs in the zebrafish olfactory epithelium (a–c) and mouse main olfactory organ (d–f). (a) Number of cells expressing each OR gene in zebrafish ciliated olfactory sensory neurons (cOSNs). The top 30 canonical ORs with the highest number of expressing cells, together with OR-κ, are shown. As reported previously [21], canonical ORs are abundantly expressed in cOSNs. Consistent with this pattern, OR-κ was also detected in a small population of cOSNs (8 cells). (b, c) Cellular distribution of OR-κ–expressing cells (b) and a representative canonical OR (or132-4) (c). Similar to or132-4, OR-κ was detected in sOSNs (8 cells). In addition, OR-κ showed expression in immune cells, which was not observed for the canonical OR. cOSN: ciliated olfactory sensory neuron, GBC: globose basal cells, HBC: horizontal basal cells, iOSN: immature olfactory sensory neuron, MvOSN: microvillous olfactory sensory neuron, Sus: sustentacular cells. (d) Number of cells expressing each OR gene in mouse mature olfactory sensory neurons (mOSNs). The top 50 canonical ORs and all genome-annotatedOR-κ genes are shown. As established in previous studies [121, 122], canonical ORs are broadly expressed in mOSNs, whereas none of the OR-κ genes were detected in mOSNs. (e, f) Cellular distribution of cells expressing OR-κ (OR-κ3 and OR-κ8) (e) and representative canonical ORs (Olfr365 and Olfr1266) (f). Canonical ORs showed robust expression in mOSNs (125 and 120 cells), whereas OR-κ expression was absent from this neuronal population. INP: immediate neuronal precursors, Mv: microvillar cells. NishiuraetalFigures8.png Fig. S4 Co-expression of OR-κ and immune cell marker genes. Single-cell RNA-seq datasets were re-analyzed to visualize the co-expression of OR-κ and immune cell marker genes in (a) zebrafish and (b) mouse. Using the immune cell populations extracted from the datasets analyzed in Fig. 3, OR-κ expression (green) and the expression of each immune cell marker gene (orange) were mapped onto t-SNE plots. Scatter plots show the co-expression relationships between OR-κ and the corresponding marker genes. Each dot represents a single cell, with OR-κ expression on the x-axis and marker gene expression on the y-axis. The linear distribution of points indicates strong co-expression of OR-κ with each marker gene. Cell-type identities are indicated by the color scheme shown in the box on the left. In zebrafish, OR-κ expression was exclusively detected in immune cells, whereas in mouse, it was observed not only in immune cells but also in a substantial number of endothelial cells. NishiuraetalSuppleTable.xlsx Cite Share Download PDF Status: Published Journal Publication published 07 Feb, 2026 Read the published version in Zoological Letters → Version 1 posted Reviewers agreed at journal 25 Nov, 2025 Reviewers invited by journal 25 Nov, 2025 Editor assigned by journal 25 Nov, 2025 First submitted to journal 24 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8198358","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":550834580,"identity":"93a88f2e-d84d-4f03-8fc9-c84ba8ff6592","order_by":0,"name":"Kanoko Nishiura","email":"data:image/png;base64,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","orcid":"https://orcid.org/0009-0007-4832-8717","institution":"Institute of Science Tokyo","correspondingAuthor":true,"prefix":"","firstName":"Kanoko","middleName":"","lastName":"Nishiura","suffix":""},{"id":550834581,"identity":"05ff78b2-7996-4039-acd1-993a2994e231","order_by":1,"name":"Tatsuki Nagasawa","email":"","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Tatsuki","middleName":"","lastName":"Nagasawa","suffix":""},{"id":550834582,"identity":"8dbce82a-527f-4244-a285-f6769610eb76","order_by":2,"name":"Masato Nikaido","email":"","orcid":"https://orcid.org/0000-0002-2430-6974","institution":"Institute of Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Masato","middleName":"","lastName":"Nikaido","suffix":""}],"badges":[],"createdAt":"2025-11-25 03:24:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8198358/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8198358/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40851-026-00260-z","type":"published","date":"2026-02-07T15:57:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":97175506,"identity":"0dcfbda2-aea2-4b76-a3b6-8240e2239b22","added_by":"auto","created_at":"2025-12-01 15:27:16","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8348,"visible":true,"origin":"","legend":"","description":"","filename":"zletZLETD2500041.xml","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/55057bc5b8af67fd2e289472.xml"},{"id":97175513,"identity":"1427317a-87ae-45a7-bb3f-57a95fe843d3","added_by":"auto","created_at":"2025-12-01 15:27:17","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1029,"visible":true,"origin":"","legend":"","description":"","filename":"ZLETD2500041835.go.xml","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/b3fe82fa8bf562c277a3c145.xml"},{"id":97175514,"identity":"8a978f9e-b939-4d8f-a4d3-ee39e1db2957","added_by":"auto","created_at":"2025-12-01 15:27:17","extension":"xml","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":827,"visible":true,"origin":"","legend":"","description":"","filename":"ZLETD2500041Import.xml","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/dbfe9c1aaf1a9437e53fcc0d.xml"},{"id":97175521,"identity":"73f24745-b60a-4c1f-a1a6-0b2a3c547b7d","added_by":"auto","created_at":"2025-12-01 15:27:17","extension":"xml","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":233682,"visible":true,"origin":"","legend":"","description":"","filename":"ZLETD25000410enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/fadafe7e6d2587b629311a0d.xml"},{"id":97175520,"identity":"e04d93b5-5332-4793-baf1-b179dacdf83e","added_by":"auto","created_at":"2025-12-01 15:27:17","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17928555,"visible":true,"origin":"","legend":"","description":"","filename":"NishiuraetalFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/41736f2e6984ecc427533e0e.pdf"},{"id":97175519,"identity":"bf550eb7-4500-4379-a4f7-605d683bebd4","added_by":"auto","created_at":"2025-12-01 15:27:17","extension":"xml","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":231472,"visible":true,"origin":"","legend":"","description":"","filename":"ZLETD25000410structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/5735dc4928c9a2a0977915da.xml"},{"id":97175518,"identity":"b7089cc4-74cf-4fb7-a2c9-8163b31030ba","added_by":"auto","created_at":"2025-12-01 15:27:17","extension":"html","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":248280,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/c7f7a766c3ba63dcfc6d034c.html"},{"id":97175505,"identity":"10314936-8fd4-4a82-8f35-08f0891d876b","added_by":"auto","created_at":"2025-12-01 15:27:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":93440,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic detection of chemoreceptor genes from bulk RNA-seq data.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Number of chemoreceptor genes used in this study. All sequence information for chemoreceptor genes was obtained from previous studies [36, 62]. Numbers at the nodes in the species tree indicate divergence dates (million years ago). (b) Number, proportions (relative to total chemoreceptor genes), and total expression levels of genes detected in chemosensory organs (nasal/oral organs). Bar colors correspond to those in Fig. 1a. (c) Numbers, proportions, and total expression levels of genes detected in extra-nasal/-oral organs. Bar colors correspond to those in Fig. 1a. (d) List of genes expressed in multiple organs. While conventional chemoreceptor genes showed chemosensory organ-specific expression, several genes were detected with organ-independent expression. The asterisk indicates that expression was detected by RT-PCR rather than bulk RNA-seq. (e) Organ-independently expressed genes were not members of expanded chemoreceptor subfamilies. Subfamily classification of each chemoreceptor gene (above phylogenetic tree) followed previous studies [32, 37, 62]. The number of genes contained in each subfamily is indicated by a bubble chart labeled numerically. Genes that exhibited organ-independent expression were not conventional expanded chemoreceptor genes but rather evolutionarily conserved across species (red boxes). Species analyzed by bulk RNA-seq in this study are highlighted in red.\u003c/p\u003e","description":"","filename":"NishiuraetalFigures1.png","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/6ff6e5be70775b0f079e1e51.png"},{"id":97249210,"identity":"988875eb-cafc-4c52-9bd1-80648747367e","added_by":"auto","created_at":"2025-12-02 13:11:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":50721,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic location of OR-κ genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Classification overview of each olfactory receptor (OR) gene subfamily. The classification scheme follows previous studies [37]. The OR-κ investigated in this study belongs to the Type 2 group, representing an evolutionarily conserved OR subfamily. (b) Isolated genomic location of OR-κ genes. The genomic positions of OR-κ and other OR genes located on the same chromosome are shown for each species. Even in \u003cem\u003eXenopus\u003c/em\u003e, where OR-κ is closest to another OR (OR-θ), these two genes are separated more than 2.3 Mb, indicating that OR-κ is genomically isolated from other ORs. (c) Genomic synteny of OR-κ genes. The syntenic relationships surrounding OR-κ (red pentagon) are conserved within fishes (zebrafish and \u003cem\u003ePolypterus\u003c/em\u003e) and within tetrapods (\u003cem\u003eXenopus\u003c/em\u003e and mouse).\u003c/p\u003e","description":"","filename":"NishiuraetalFigures2.png","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/fc66e4d1edba282c91fce2c1.png"},{"id":97175511,"identity":"f541125a-6223-4a58-8f9e-525bb36a1e04","added_by":"auto","created_at":"2025-12-01 15:27:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":750245,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of OR-κ genes in zebrafish and mouse at single cell resolution.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) \u003cem\u003ein situ \u003c/em\u003ehybridization analysis of OR-κ expression in the zebrafish intestine. Left panel shows the OR-κ expression pattern in the small intestine. The detected signals were concentrated in the intestinal crypts (arrowheads). Middle panel shows a higher-magnification view of the white dotted box area in the left panel. The asterisk marks the intestinal lumen, and the dotted line marks the tissue boundary. Right panel shows the result of negative control using sense probe. (b–e) OR-κ gene expression revealed by re-analysis of single-cell RNA-seq data in zebrafish. (b) OR-κ expression pattern based on whole-body single-cell RNA-seq data. (c) Cell types of detected OR-κ-expressing cells and their proportions; bar colors follow Fig. 3b. OR-κ expression was predominantly detected in immune cells. (d–e) Re-analysis focusing exclusively on cell types extracted from the dataset used in Fig. 3c. (d) Expression levels of OR-κ genes in immune cells. (e) Co-expression of OR-κ and marker genes in immune cells; the marker gene expression shown in orange, OR-κ expression in green and co-expression in yellow. (f–j) OR-κ gene expression revealed by re-analysis of single-cell RNA-seq data in mouse. (f) OR-κ expression pattern based on single-cell RNA-seq data from 29 tissues. Results for genome-annotated OR-κ2, 3, 4, 5 and 8 are collectively displayed. (g) Cell types of detected OR-κ-expressing cells and their proportions; bar colors follow Fig. 3f. OR-κ expression was mainly detected in immune and endothelial cells. (h–j) Re-analysis focusing exclusively on immune and endothelial cells extracted from the dataset used in Fig. 3g. (h) Expression levels of OR-κ genes in immune and endothelial cells (representative result shown). (i-j) Co-expression of marker genes and OR-κ3 in immune (i) and endothelial (j) cells. Marker gene expression shown in orange, OR-κ3 expression in green and co-expression in yellow.\u003c/p\u003e","description":"","filename":"NishiuraetalFigures3.png","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/304b0d2c39bdc651ee86ba07.png"},{"id":97249249,"identity":"4b1a5f25-bc16-4f56-9e80-47c4f95c7330","added_by":"auto","created_at":"2025-12-02 13:11:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":106287,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"NishiuraetalFigures4.png","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/b909392b0f7ae7c42461a8e9.png"},{"id":102235255,"identity":"044eb13c-1c96-4af2-9fb7-b77f5f77e86c","added_by":"auto","created_at":"2026-02-09 16:16:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1748350,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/891cdaff-bac9-4863-9c21-9ef94b69494e.pdf"},{"id":97248965,"identity":"5bd79a9d-6128-41a6-9e0f-a8019bc57a96","added_by":"auto","created_at":"2025-12-02 13:08:57","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":108164,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1 Heatmap showing gene expression profiles of all chemoreceptor genes analyzed in this study.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach row represents a chemoreceptor gene, and each column corresponds to an organ analyzed. Normalized expression levels (TPM) are shown as a heatmap. The legend provided in the lower right corner indicates expression values, color codes for chemoreceptor gene families, and the list of analyzed organs.\u003c/p\u003e","description":"","filename":"NishiuraetalFigures5.png","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/7daefe1196efa79c63e22b9d.png"},{"id":97175509,"identity":"fc0f0071-a793-455d-b2b6-1b0b9303f0c7","added_by":"auto","created_at":"2025-12-01 15:27:17","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":128285,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2 Reverse transcription PCR (RT-PCR) detection of chemoreceptor genes undetected by bulk RNA-seq\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT-PCR analysis detecting chemoreceptor gene expression in tissues where transcripts were not identified by bulk RNA-seq analysis. OR-κ expression in various tissues of (a) \u003cem\u003eXenopus\u003c/em\u003e and (b) mouse. (c) \u003cem\u003eT1R2A\u003c/em\u003eexpression in \u003cem\u003ePolypterus \u003c/em\u003eintestine. (d) T1R1 expression in zebrafish muscle. Genomic DNA (g.DNA) was used as a positive control. Because OR-κ, \u003cem\u003eT1R2A\u003c/em\u003eand \u003cem\u003eT1R1\u003c/em\u003e genes consist of a single exon, RT-PCR was performed with and without reverse transcriptase (+/–) to distinguish cDNA-derived amplification products.\u003c/p\u003e","description":"","filename":"NishiuraetalFigures6.png","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/16d51f3e5c730b5d95f4f51a.png"},{"id":97175507,"identity":"7ac71e37-65d8-453b-a036-ac58c6c27335","added_by":"auto","created_at":"2025-12-01 15:27:16","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":59242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S3 Expression profile of OR-κ genes revealed by single-cell RNA-seq analysis of the olfactory organ.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression patterns of OR-κ genes and representative canonical ORs in the zebrafish olfactory epithelium (a–c) and mouse main olfactory organ (d–f). (a) Number of cells expressing each OR gene in zebrafish ciliated olfactory sensory neurons (cOSNs). The top 30 canonical ORs with the highest number of expressing cells, together with OR-κ, are shown. As reported previously [21], canonical ORs are abundantly expressed in cOSNs. Consistent with this pattern, OR-κ was also detected in a small population of cOSNs (8 cells). (b, c) Cellular distribution of OR-κ–expressing cells (b) and a representative canonical OR (or132-4) (c). Similar to or132-4, OR-κ was detected in sOSNs (8 cells). In addition, OR-κ showed expression in immune cells, which was not observed for the canonical OR. cOSN: ciliated olfactory sensory neuron, GBC: globose basal cells, HBC: horizontal basal cells, iOSN: immature olfactory sensory neuron, MvOSN: microvillous olfactory sensory neuron, Sus: sustentacular cells. (d) Number of cells expressing each OR gene in mouse mature olfactory sensory neurons (mOSNs). The top 50 canonical ORs and all genome-annotatedOR-κ genes are shown. As established in previous studies [121, 122], canonical ORs are broadly expressed in mOSNs, whereas none of the OR-κ genes were detected in mOSNs. (e, f) Cellular distribution of cells expressing OR-κ (OR-κ3 and OR-κ8) (e) and representative canonical ORs (Olfr365 and Olfr1266) (f). Canonical ORs showed robust expression in mOSNs (125 and 120 cells), whereas OR-κ expression was absent from this neuronal population. INP: immediate neuronal precursors, Mv: microvillar cells.\u003c/p\u003e","description":"","filename":"NishiuraetalFigures7.png","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/855e37b5304e0e61953e81b2.png"},{"id":97249383,"identity":"c57527eb-b392-456b-b19e-5516731f5c44","added_by":"auto","created_at":"2025-12-02 13:12:24","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":437204,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S4 Co-expression of OR-κ and immune cell marker genes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle-cell RNA-seq datasets were re-analyzed to visualize the co-expression of OR-κ and immune cell marker genes in (a) zebrafish and (b) mouse. Using the immune cell populations extracted from the datasets analyzed in Fig. 3, OR-κ expression (green) and the expression of each immune cell marker gene (orange) were mapped onto t-SNE plots. Scatter plots show the co-expression relationships between OR-κ and the corresponding marker genes. Each dot represents a single cell, with OR-κ expression on the x-axis and marker gene expression on the y-axis. The linear distribution of points indicates strong co-expression of OR-κ with each marker gene. Cell-type identities are indicated by the color scheme shown in the box on the left. In zebrafish, OR-κ expression was exclusively detected in immune cells, whereas in mouse, it was observed not only in immune cells but also in a substantial number of endothelial cells.\u003c/p\u003e","description":"","filename":"NishiuraetalFigures8.png","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/480c232e04a3423bf3d083b7.png"},{"id":97248899,"identity":"674ee54b-8143-41dd-ad21-0e4ab4e67939","added_by":"auto","created_at":"2025-12-02 13:08:02","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1322837,"visible":true,"origin":"","legend":"","description":"","filename":"NishiuraetalSuppleTable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8198358/v1/da2ade21589705d5d0fb0d6d.xlsx"}],"financialInterests":"","formattedTitle":"Vertebrate-wide transcriptomic screening identifies immune cell-specific expression of the conserved OR-κ gene","fulltext":[{"header":"Background","content":"\u003cp\u003eThe accurate perception of the surrounding environment is essential for animal survival, allowing individuals to recognize nutritional sources, toxins, predators, and reproductive partners [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among the five senses of vertebrates, olfaction and gustation are collectively referred to as chemosensation, because they detect chemical compounds derived from the external environment [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In vertebrates, chemoreception in the nose (olfactory organ/epithelium or vomeronasal organ) and mouth (including tongue) is mainly mediated by a subclass of chemoreceptors belonging to the G protein-coupled receptor (GPCR) superfamily [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These GPCRs comprise six different multigene families encoding seven-transmembrane-domain receptors: olfactory receptors (OR), trace amine-associated receptors (TAAR), vomeronasal receptor types 1 and 2 (V1R and V2R), and taste receptor types 1 and 2 (T1R and T2R) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eChemoreceptor genes were initially discovered in mammalian nasal/oral organs as candidate molecules for odorant and gustatory perception [\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Subsequently, homologous genes have been successively identified in non-mammalian vertebrates [\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and their expression in nasal/oral organs has been demonstrated in many species, such as channel catfish [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], zebrafish [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and African clawed frog [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Recent advances in whole-genome sequencing technologies and the resulting expansion of genomic data have illuminated the evolutionary history of vertebrate chemoreceptor genes. OR, TAAR/V1R/V2R, and T1R/T2R genes were acquired before the divergence of cnidarians and bilaterians [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], jawless and jawed fish [\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and jawed vertebrates [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], respectively. These findings indicate that chemoreceptor genes, while first discovered in mammals, are evolutionarily ancient and have been conserved across diverse vertebrate lineages [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eVertebrate chemoreceptor genes are encoded as multigene families distributed across multiple genomic loci. During vertebrate evolution, these gene families have undergone repeated gene duplication and pseudogenization events \u0026mdash;a process known as birth-and-death evolution\u0026mdash; resulting in lineage- and species-specific variation in gene copy numbers. For example, the highest known copy numbers are reported for ORs (2,399 in echidna, \u003cem\u003eTachyglossus aculeatus\u003c/em\u003e), TAARs (497 in reedfish, \u003cem\u003eErpetoichthys calabaricus\u003c/em\u003e), and T2Rs (268 in frog, \u003cem\u003eGlandirana rugosaa\u003c/em\u003e) [\u003cspan additionalcitationids=\"CR37 CR38 CR39\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Such continuous turnover of chemoreceptor genes through the birth-and-death process generates extensive diversity in receptor-specific amino acid sequences, contributing to receptivity across a wide range of chemical compounds [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Thus, the diversity of chemoreceptor genes across lineages is thought to facilitate to the precise discrimination and perception of the myriad environmental chemical compounds encountered in each species' habitat [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSome of these multigene families encoding chemoreceptor genes are known to function in organs beyond the nasal and oral organs. For instance, human \u003cem\u003ehOR17-4\u003c/em\u003e and mouse \u003cem\u003eMOR23\u003c/em\u003e, both expressed in sperm, mediate chemotaxis in response to small chemical molecules or short peptides [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Additionally, human and mouse \u003cem\u003eT1R2\u003c/em\u003e and \u003cem\u003eT1R3\u003c/em\u003e have been associated with maintaining intestinal glucose homeostasis by regulating the sodium-glucose co-transporter SGLT1 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Moreover, the secretion of gastrointestinal hormones can be modulated through the activation of mouse \u003cem\u003eT2R108\u003c/em\u003e receptor by luminal ligands [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Thus, chemoreceptor genes expressed in extra-nasal/oral organs appear to be used for monitoring the internal rather than the external environment [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In several studies, these receptors are referred to as \u0026ldquo;ectopic olfactory receptors\u0026rdquo; or \u0026ldquo;ectopic taste receptors\u0026rdquo;, terms generally implying abnormal expression in non-chemosensory organs [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. However, chemoreceptors expressed in non-chemosensory organs are being increasingly discovered [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan additionalcitationids=\"CR52 CR53 CR54 CR55 CR56 CR57\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], and are now recognized as a widespread phenomenon. Therefore, in this study, we refer to these as \u0026ldquo;extra-nasal/oral chemoreceptors\u0026rdquo; rather than ectopic chemoreceptors. While expression patterns of chemoreceptor genes in extra-nasal/oral organs have been increasingly characterized in mammals, particularly humans and mice, knowledge from non-mammalian species remains limited, with only a few examples reported in zebrafish [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] and blind cavefish [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Consequently, a comprehensive evolutionary perspective has been lacking.\u003c/p\u003e\u003cp\u003eRecently, bulk and single-cell RNA-seq data from various organs have become available, providing new opportunities to investigate the molecular evolution of chemoreceptor genes across a broad range of vertebrates. In this study, we conducted a comprehensive screening of chemoreceptors exhibiting extra-nasal/oral expression in representative vertebrates: mouse, \u003cem\u003eXenopus\u003c/em\u003e, \u003cem\u003ePolypterus\u003c/em\u003e, and zebrafish. As a result, we detected the expression of members of all six chemoreceptor gene families in extra-nasal/oral organs, suggesting that such expression may represent a common feature shared across vertebrates, from basal ray-finned fishes to mammals. Furthermore, we found that OR-κ, which is evolutionarily stable compared to the expanded OR families, is predominantly expressed in immune cells in both teleosts and mammals. Given that OR-κ originated before the divergence of jawless fishes, these findings highlight the need to reconsider the function and evolutionary origins of vertebrate chemoreceptor genes.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eThe exploration of extra-nasal/oral expression of chemosensory genes from bulk RNA-seq\u003c/h2\u003e\u003cp\u003eFirst, we conducted bulk RNA-seq analysis of 13 organs to obtain a comprehensive overview of chemoreceptor gene expression across vertebrate species (Fig.\u0026nbsp;1 and Table S2-5). We analyzed a mammal (mouse), amphibian (\u003cem\u003eXenopus\u003c/em\u003e), basal ray-finned fish (\u003cem\u003ePolypterus\u003c/em\u003e), and teleost (zebrafish). The gene annotation dataset of chemoreceptors was compiled based on previous studies [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] (Fig.\u0026nbsp;1a). All bulk RNA-seq results in this study are summarized in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The majority of chemoreceptor genes (97% in \u003cem\u003ePolypterus\u003c/em\u003e, 96% in zebrafish, 97% in \u003cem\u003eXenopus\u003c/em\u003e, and 95% in mouse) were expressed in nasal/oral organ, which serve as canonical chemosensory tissues containing olfactory neurons and taste bud cells (Fig.\u0026nbsp;1b). In contrast, a subset of genes (ranging from 1% in the \u003cem\u003ePolypterus\u003c/em\u003e pectoral fin and the \u003cem\u003eXenopus\u003c/em\u003e muscle to 29% in the zebrafish brain) also exhibited expression in extra-nasal/oral organs. Although most chemoreceptor genes were primarily expressed in chemosensory organs, all analyzed species possessed a subset of genes expressed in extra-nasal/oral organs, which may reflect that extra-nasal/oral expression is a widespread and evolutionarily conserved feature among vertebrates (Fig.\u0026nbsp;1c).\u003c/p\u003e\u003cp\u003eAmong the chemoreceptor genes showing extra-nasal/oral expression, some genes were detected in almost all analyzed organs (Fig.\u0026nbsp;1d). Some genes were not detected in several organs by bulk RNA-seq, but were detected by reverse transcription PCR (RT-PCR) validation (as indicated by the asterisks in Fig.\u0026nbsp;1d and S2). Each chemoreceptor gene family has been subdivided into several subfamilies based on molecular phylogenetic analysis (Fig.\u0026nbsp;1e; [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]). Conventional chemoreceptor genes have undergone repeated birth-and-death processes during evolution, forming species-specific gene repertoires. In contrast, the newly identified organ-independently expressed genes represent conserved subfamilies with stable gene numbers, mostly existing as single-copy genes across species, rather than the conventionally expanded genes. These results imply that while conventional-nasal/oral receptors have undergone evolutionary expansion to detect a wide range of chemical compounds, non-conventional-extra nasal/oral chemoreceptors have remained evolutionarily stable to detect specific chemical compounds. Among these, OR-κ was the only gene exhibiting multi-organ expression conserved across all four analyzed species, showing expression in all analyzed organs in three species (mouse, zebrafish and \u003cem\u003ePolypterus\u003c/em\u003e) as well as expression in most of the organs analyzed in \u003cem\u003eXenopus\u003c/em\u003e (Fig.\u0026nbsp;1d, S2a and b). Based on this high degree of conservation, we focused on OR-κ as a representative example of a conservative chemoreceptor gene in the following analysis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEvolutionary characteristics of organ-independently expressed OR-κ genes\u003c/h3\u003e\n\u003cp\u003eNext, we characterized the evolutionary features of OR-κ as a representative of organ-independently expressed chemoreceptor genes (Fig.\u0026nbsp;2). Olfactory receptors are known to be divided into two major clades through molecular phylogenetic analysis, Type 1 and Type 2. Type 1 ORs are composed of the expanded subfamily, exhibiting lineage-specific birth-and-death processes that enable the detection of diverse chemical substances. In contrast, Type 2 ORs, which include OR-κ, are composed of the conserved subfamilies with stable gene copy numbers, except for OR-η (Fig.\u0026nbsp;2a; [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]). It is known that conventional expanded ORs are organized genomically to be co-regulated by shared transcriptional regulatory sequences such as the H element [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], the P element [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], the J element [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] and Greek islands [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Since the OR-κ gene was more than 2.3 Mb apart from other OR genes in all analyzed species, it is likely to be transcribed independently of the regulatory mechanisms controlling expanded OR genes (Fig.\u0026nbsp;2b). A similar genomic arrangement was also observed for other conservative ORs, such as OR-θ (Fig.\u0026nbsp;2b). Single-cell RNA-seq analysis of olfactory epithelium revealed that expanded ORs were abundantly detected in both mouse and zebrafish, whereas OR-κ expression was absent in mouse olfactory sensory neurons and observed only in a minute fraction of zebrafish cells (8 /2,286 neurons) (Fig. S3). Analysis of genomic synteny revealed that the OR-κ genomic region is conserved within both fish lineages (zebrafish and \u003cem\u003ePolypterus\u003c/em\u003e) and tetrapod lineages (\u003cem\u003eXenopus\u003c/em\u003e and mouse), indicating an ancient evolutionary origin (Fig.\u0026nbsp;2c).\u003c/p\u003e\n\u003ch3\u003eExpression of OR-κ genes at single-cell resolution\u003c/h3\u003e\n\u003cp\u003eNext, we identified the cell types expressing OR-κ genes (Fig.\u0026nbsp;3). We performed \u003cem\u003ein situ\u003c/em\u003e hybridization and successfully visualized OR-κ-expressing cells in the intestine (Fig.\u0026nbsp;3a). The mRNA signal was concentrated in the basal region of intestinal folds and localized beneath the monolayered intestinal epithelium. To further characterize the OR-κ-expressing cells, we re-analyzed published single-cell RNA-seq datasets from zebrafish (Fig.\u0026nbsp;3b-e) and mouse (Fig.\u0026nbsp;3f-j). A comprehensive single-cell RNA-seq dataset of zebrafish encompassing whole-body tissues across developmental stages (pharyngula to adult) revealed OR-κ expression in multiple cell clusters (Fig.\u0026nbsp;3b), predominantly in immune cells (82%; 4,979/6,081 cells, Fig.\u0026nbsp;3c and Table S6). Focused analysis of the immune cell clusters confirmed that OR-κ is co-expressed with canonical immune cell markers, such as \u003cem\u003elcp1\u003c/em\u003e, \u003cem\u003ectss1\u003c/em\u003e, \u003cem\u003ecsf1ra\u003c/em\u003e, \u003cem\u003empx\u003c/em\u003e and \u003cem\u003emarco\u003c/em\u003e [\u003cspan additionalcitationids=\"CR68 CR69 CR70\" citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e] (Fig.\u0026nbsp;3d, e and S4a). These findings suggest that the organ-independent expression of zebrafish OR-κ is likely derived from immune cells. Re-analysis of single-cell RNA-seq data from 29 organs in mouse similarly revealed OR-κ expression across multiple cell clusters (Fig.\u0026nbsp;3f), particularly in immune cells (36.6%; 1,336/3,653 cells) and endothelial cells (51%; 1,869/3,653 cells) (Fig.\u0026nbsp;3g and Table S7). Consistent with bulk RNA-seq results, OR-κ expression was detected across cells from multiple tissues (Table S8). Analysis restricted to immune and endothelial cells demonstrated co-expression of OR-κ with known cell-type-specific markers, such as \u003cem\u003ePtprb\u003c/em\u003e, \u003cem\u003eClec4g\u003c/em\u003e, \u003cem\u003ePecam1\u003c/em\u003e, \u003cem\u003eTie1\u003c/em\u003e and \u003cem\u003eCdh5\u003c/em\u003e [\u003cspan additionalcitationids=\"CR73\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e] (Fig.\u0026nbsp;3h-j and S4b).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eAncient, conserved extra-nasal/oral chemoreceptor expression\u003c/h2\u003e\u003cp\u003eAmong the five senses of vertebrates, olfaction and gustation are collectively referred to as \u0026lsquo;chemical senses\u0026rsquo; because they detect chemical compounds originating from the external environment [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These chemical cues are received by GPCRs\u0026mdash;including ORs, TAARs, V1Rs, V2Rs, T1Rs, and T2Rs\u0026mdash;which are expressed in olfactory sensory neurons or taste bud cells. Upon activation, these receptors convert extracellular chemical signals into intracellular responses that are eventually perceived as odors or tastes in higher-order brain regions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Because humans perceive smell and taste through the nose and tongue, chemoreceptor genes were initially isolated under the hypothesis that they are specifically expressed in olfactory and gustatory organs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR76\" citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Owing to this empirical and historical background, their potential functions in non-chemosensory organs have often been overlooked. Although subsequent studies have repeatedly identified chemoreceptor expression in extra-nasal/oral tissues [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e], most of these findings are limited to mammals, and their evolutionary context has remained largely unexplored [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan additionalcitationids=\"CR52 CR53 CR54 CR55 CR56 CR57\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. In the present study, we performed a comprehensive expression analysis of 4,614 chemoreceptor genes across 13 organs from four representative vertebrate species. Consistent with previous reports, the vast majority of chemoreceptor genes (95\u0026ndash;97%) were expressed in nasal/oral organs. However, a subset of these genes (1\u0026ndash;29%) also exhibited expression in extra-nasal/oral organs (Fig.\u0026nbsp;1), suggesting that such expression may represent a conserved feature across vertebrates rather than a mammal-specific phenomenon.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEvolutionary dynamics of conventional (expanded) and conservative (stable) chemoreceptor genes\u003c/h2\u003e\u003cp\u003eVertebrate chemoreceptor genes constitute a multigene family that has undergone repeated gene duplication and pseudogenization (birth-and-death process) during evolution, resulting in lineage-specific diversity in gene copy number and sequence variation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Such gene turnover is thought to contribute to the accurate discrimination of diverse chemical cues from the external environment [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, gene number expansion through frequent tandem duplication within genomic clusters is interpreted as a typical evolutionary pattern of chemoreceptors [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR80\" citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. In contrast to these conventionally expanded genes, our analysis identified several evolutionarily stable chemoreceptor genes that exhibit organ-independent expression (Fig.\u0026nbsp;1d-e). Among them, the OR-κ gene was notably expressed in immune cells, and this expression pattern was shared between zebrafish and mouse (Fig.\u0026nbsp;3b-j), suggesting a conserved role of OR-κ gene vertebrate immune systems. Previous studies have reported that certain evolutionarily conserved ORs\u0026mdash;such as \u003cem\u003eOR51E1\u003c/em\u003e/\u003cem\u003eOlfr558\u003c/em\u003e [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e] and \u003cem\u003eOR51E2\u003c/em\u003e/\u003cem\u003eOlfr78\u003c/em\u003e [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e] \u0026mdash;function in extra-nasal organs. In addition, ORs conserved between human and chimpanzee also tend to be expressed extra-nasally [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. Although the specific cell types expressing other evolutionary stable genes (e.g., OR-θ1, \u003cem\u003eancV1R\u003c/em\u003e, and \u003cem\u003eT1R1\u003c/em\u003e) were not identified in this study, our findings suggest these receptors may serve functions distinct from odorant/gustatory perception. Further identification of their expressing cells will provide valuable insights into the molecular evolution and diversification of chemoreceptor gene functions.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDistinct transcriptional regulation of the OR-κ gene\u003c/h3\u003e\n\u003cp\u003eThe transcriptional regulatory mechanisms of conventional- (expanded-) chemoreceptor genes have been well characterized. These genes are often organized in genomic clusters, are controlled by shared enhancers [\u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e], heterochromatin-mediated silencing [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e] and dynamic three-dimensional chromosomal rearrangements [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. It has been proposed that extra-nasal expression of ORs may result from the leakage or relaxation of these regulatory constraints within expanded gene clusters [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Many such ORs maintain their original olfactory function while also acquiring novel physiological roles in other tissues [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan additionalcitationids=\"CR92 CR93 CR94\" citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e]. Interestingly, the OR-κ gene identified in this study has been evolutionarily isolated from other OR gene clusters since its origin, remaining more than 2.3Mb away from any other OR loci in all analyzed species (Fig.\u0026nbsp;2b and S4). This genomic isolation implies that OR-κ is transcribed under an independent regulatory mechanism distinct from that of conventional OR clusters. Such divergence in transcriptional control may underlie its unique expression in immune cells. Notably, similar genomic isolation was also observed for other conservative ORs (e.g., OR-θ), raising the possibility that these evolutionarily stable genes may likewise be expressed extra-nasally under distinct transcriptional control. Future studies focusing on the transcriptional regulation of OR-κ and other evolutionarily stable chemoreceptor genes will be crucial for elucidating the evolutionary origins of regulatory mechanisms governing chemoreceptor gene expression.\u003c/p\u003e\n\u003ch3\u003ePossible functions of the OR-κ gene in immune and endothelial cells\u003c/h3\u003e\n\u003cp\u003eExpression of OR genes in immune cells has been reported for several Type 1 ORs (conventional expanded ORs) in mouse [\u003cspan additionalcitationids=\"CR97\" citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e], human, cattle [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e], opossum and platypus [\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e]. In mice, these ORs have been implicated in cellular chemotaxis [\u003cspan additionalcitationids=\"CR97\" citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. Such expression is thought to have arisen secondarily as a consequence of escaping the canonical transcriptional constraints during the birth-and-death evolution of expanded ORs. In contrast, the OR-κ gene copy numbers are evolutionarily stable from jawless fish to mammals [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and its expression in immune cells is shared between zebrafish and mouse (Fig.\u0026nbsp;3), suggesting that immune-related expression may represent an ancestral and conserved feature across vertebrates. Our bulk RNA-seq analyses detected OR-κ expression in multiple tissues, likely reflecting its presence in circulating leukocytes distributed throughout the body.\u003c/p\u003e\u003cp\u003eAlthough the precise immune function of OR-κ remains unclear, several results in this study provide intriguing clues. First, the evolutionary origin of OR-κ can be traced back to the common ancestor of vertebrates, including jawless fish (Fig.\u0026nbsp;1e), coinciding with the emergence of adaptive immunity [\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e, \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e]. Second, in mice, OR-κ was also expressed in a substantial population of endothelial cells in addition to immune cells (Fig.\u0026nbsp;3f-j). Given that, immune and endothelial cells are thought to share both evolutionary [\u003cspan additionalcitationids=\"CR104\" citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e] and developmental origins [\u003cspan additionalcitationids=\"CR107 CR108 CR109\" citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e], the shared expression of OR-κ in these two cell types is consistent with this finding and offers valuable insight into its potential role. Finally, although the OR-κ gene is shared across most vertebrates, it has been lost independently in several lineages, including human, chicken, and the coelacanth [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Whether this loss of OR-κ affects immune function or is compensated by other OR genes remains an important question. Determining the precise evolutionary timing of OR-κ gene loss will provide further insight into its ancestral role. Because our analyses were limited to representative species, expanding taxonomic coverage will be essential for future analyses.\u003c/p\u003e\u003cp\u003eTaken together, the OR-κ gene appears to function differently from conventional olfactory receptor genes expressed in olfactory sensory neurons. Rather than contributing to the perception of external stimuli, OR-κ may play a role in sensing and regulating internal physiological or immune states. Comprehensive investigation of the evolutionarily history, expression patterns, and molecular functions of OR-κ and other evolutionary stable chemoreceptors will deepen our understanding of the origin and diversification of chemoreceptor systems.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we conducted a comprehensive survey of chemoreceptor gene expression across various extra-nasal and extra-oral organs in diverse vertebrate species, and evaluated their evolutionary dynamics. Notably, we identified the OR-κ, an olfactory receptor with stable gene numbers across vertebrates, as predominantly expressed in immune cell populations in both teleosts and mammals, unlike the conventional expanded OR families. Given that the OR-κ likely originated around the same evolutionary period as adaptive immunity\u0026mdash;both emerging in the jawless vertebrate lineage\u0026mdash;our results provide the first proposal for an evolutionarily conserved chemoreceptor expression in non-chemosensory organs across vertebrates. This work invites a reevaluation of chemoreceptor function, emphasizing their role beyond the classical olfactory and gustatory systems and implying an ancient link between chemosensation and the immune system conserved in vertebrates.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePolypterus\u003c/em\u003e (\u003cem\u003ePolypterus senegalus\u003c/em\u003e)\u003cem\u003e\u0026nbsp;\u003c/em\u003ewere obtained from a commercial supplier (Nettaigyo-tsuhan forest, Wakayama, Japan). The \u003cem\u003eXenopus\u0026nbsp;\u003c/em\u003e(\u003cem\u003eXenopus tropicalis\u003c/em\u003e) strain Nigerian H (Xtr.NigerianH\u003csup\u003eHuarc\u003c/sup\u003e, RRID: HUARC_1002) and the zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) strain RIKEN WT (RW) were provided by National BioResource Project (NBRP) of MEXT through Hiroshima University Amphibian Research Center (RRID: SCR_019015) and RIKEN Center for Brain Science, respectively. All animals were maintained and bred at 27\u0026deg;C on a 12/12 h light/dark cycle. All experiments were conducted in accordance with the Institutional Animal Experiment Committee of the Institute of Science Tokyo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBulk RNA-seq analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was extracted from \u003cem\u003ePolypterus\u003c/em\u003e (intestine and pectoral fin) and \u003cem\u003eXenopus\u003c/em\u003e (nasal tissue, oral tissue, spinal cord, lung, heart, intestine, liver and kidney) using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA). The extracted total RNA was sequenced at 100\u0026thinsp;bp paired-end reads on a NovaSeq 6000 or Novaseq X by Macrogen Japan Corp., using a TruSeq stranded mRNA Library Prep Kit. \u003cem\u003ePolypterus\u003c/em\u003e kidney sequence data were unpublished, provided by Y. Kimura. All other RNA sequence data were obtained from the NCBI SRA database (Table S1). SRA files were retrieved and converted to FASTQ format using the SRA Toolkit prefetch and fastq-dump v3.0.7 [111]. The quality control of raw sequence data was performed with fastp v0.23.4 [112] with the following options: -q 20 -l 25. The reads were mapped to the genome of \u003cem\u003ePolypterus\u003c/em\u003e (ASM1683550v1), zebrafish (GRCz11), \u003cem\u003eXenopus\u003c/em\u003e (UCB_Xtro_10.0) or mouse (\u003cem\u003eMus musculus\u003c/em\u003e) (GRCm39) using STAR version 2.7.5c [113] and quantified using rsem-calculate-expression v1.3.3 [114]. The gene annotation data for the quantification were downloaded from Refseq and edited for the regions of known chemoreceptor genes [36]. NCBI database designations for the OR-\u0026kappa; genes are as follows: \u003cem\u003ezgc:194312\u003c/em\u003e in zebrafish; \u003cem\u003ezgc:194312\u003c/em\u003e (OR-\u0026kappa;2) and \u003cem\u003eolfactory receptor 4K13-like\u003c/em\u003e (OR-\u0026kappa;3) in \u003cem\u003ePolypterus\u003c/em\u003e; \u003cem\u003eGm7582\u003c/em\u003e (OR-\u0026kappa;2), \u003cem\u003eGm7609\u003c/em\u003e (OR-\u0026kappa;3), \u003cem\u003eGm2666\u003c/em\u003e (OR-\u0026kappa;4), \u003cem\u003eGm7592\u003c/em\u003e (OR-\u0026kappa;5), and \u003cem\u003eCsprs\u003c/em\u003e (OR-\u0026kappa;8) in mouse. Following previous studies with human, the threshold for classifying a gene as expressed was defined at 0.01 TPM [48].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSinge-cell RNA-seq reanalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePublic single-cell RNA-seq datasets reanalyzed in this study can be found on the Gene Expression Omnibus (GEO): GSE198832 [115]. Annotation of cell lineage and cell type for each cell followed Wang and colleagues [115]. Gene expression matrices from immune and endothelial cells in mice and immune cells in zebrafish were analyzed using Seurat v5 [116]. Twenty principal components (PCs) were selected as significant components for t-SNE analysis. The clustering parameter resolution was set to 0.8 for identifying cell clusters.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRaw single-cell RNA sequencing data from zebrafish olfactory epithelium were retrieved from the NCBI SRA database (SRR31595086; [117]). Fastq files were processed using 10x Genomics Cell Ranger v6.0.2 with the GRCz11_v4.3.2_cellranger_v6 transcriptome reference [118]. Quality control filtering was applied using the following thresholds: nCount_RNA \u0026gt; 250 and \u0026lt; 5000, and mitochondrial gene percentage \u0026lt; 15%. Data normalization and identification of highly variable features were performed using default parameters with the NormalizeData and FindVariableFeatures functions in Seurat. UMAP dimensionality reduction was performed using seven PCs with a resolution parameter of 0.8. Initial clustering identified 19 cell clusters, which were annotated into seven cell types based on marker gene expression patterns consistent with those reported by Chen and colleagues. Single-cell RNA-seq data from mouse main olfactory epithelium were obtained from the GEO database (GSE185251; [119]). The filtered feature-barcode matrix was loaded into Seurat for downstream analysis. Data processing and cell type annotation were performed using identical parameters and marker genes as described in the original study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn situ\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;hybridization (ISH)\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eISH was performed according to the method of Suzuki \u003cem\u003eet al.\u003c/em\u003e [120] with several modifications. Briefly, the entire intestines were fixed in Davidson\u0026apos;s solution overnight at 4\u0026deg;C. The fixed tissues were then immersed in 20% sucrose in PBS overnight and embedded in OCT compound before being frozen in liquid nitrogen. The embedded blocks were then sliced into sections 12 \u0026micro;m thick and placed on a coated glass slide. The frozen sections were then digested with 5 \u0026micro;g/ml proteinase K for 10 min at 37\u0026deg;C, after which they were hybridized with 5 ng/\u0026micro;l DIG-labelled riboprobes at 65\u0026deg;C overnight. The sections were washed, treated with 2 \u0026micro;g/ml RNase A in TNE buffer for 30 min at 37\u0026deg;C. Then, they were treated with a streptavidin/biotin blocking kit (Vector Laboratories, Newark, CA, USA). After that, they were treated with 1% blocking reagent (PerkinElmer, Waltham, MA, USA) in TBS buffer for 1 h. Signals were detected with a peroxidase-conjugated anti-DIG antibody (1:100, 11207733910, Roche), amplified by a TSA Plus Biotin kit (1:50, PerkinElmer), and visualized with an Alexa Fluor 488-conjugated streptavidin (1:200, Thermo Fisher Scientific, Waltham, MA, USA). The sections were mounted using Vectashield mounting medium containing DAPI (Vector Laboratories) and digitally captured using a Zeiss Axioplan SP fluorescence microscope and a Zeiss Axiocam 503 colour CCD camera (Carl Zeiss, Oberkochen, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse transcription-PCR (RT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from a panel of \u003cem\u003eXenopus\u003c/em\u003e, mouse, \u003cem\u003ePolypterus\u003c/em\u003e and zebrafish target tissues. After DNase I digestion (TaKaRa, Shiga, Japan), RNA samples were diluted to 10 ng/\u0026mu;l, and cDNA was synthesized from 2 \u0026mu;g of total RNA using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) with oligo-dT18 primers. All PCRs were performed using KOD FX Neo polymerase or KOD One (TOYOBO, Osaka, Japan) with the following cycling conditions: 98\u0026deg;C for 10 sec, 55\u0026deg;C for 30 sec, and 68\u0026deg;C for 30 sec, repeated for 35 cycles. Primer sequences are listed in Table S9. \u0026beta;-actin was used as an internal control for RNA quality and expression normalization in the chemosensory organ.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Prof. Akira Kato, Takashi Suzuki, Junji Hirota, and Yuichi Hongoh for their invaluable advice throughout this study. We also thank Andrew Shedlock for improving the manuscript; Yuki Kimura for providing unpublished \u003cem\u003ePolypterus\u003c/em\u003e kidney sequence data; the Integrative Bioscience Facility at Institute of Science Tokyo for DNA sequencing analysis; the Hongoh laboratory for helping with data production and analysis; and all laboratory members, with special thanks to Mitsuto Aibara and Ayumi Hirose for their invaluable support and collaboration. Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental studies using the animals were approved by the Institutional Animal Experiment Committee of the Institute of Science Tokyo were performed in accordance with the institutional, governmental ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll sequence reads were deposited in the DDBJ Sequence Read Archive under accession no. PRJDB39629 and PRJDB39651.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by JSPS KAKENHI (20H03307, 24K02074 to M.N. and 23K14249 to T.N.), the Sasakawa Scientific Research Grant from the Japan Science Society to T.N. and JST SPRING (JPMJSP2180 to K.N.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.N., T.N., and M.N. conceived the research and wrote the manuscript. K.N. conducted the experiments. M.N. supervised the project. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDangles O, Irschick D, Chittka L, Casas J. Variability in sensory ecology: expanding the bridge between physiology and evolutionary biology. Q Rev Biol. 2009;84:51\u0026ndash;74. https://doi.org/10.1086/596463.\u003c/li\u003e\n\u003cli\u003eThe senses: A comprehensive reference. Elsevier Science \u0026amp; Technology; 2009.\u003c/li\u003e\n\u003cli\u003eFleischer J, Breer H, Strotmann J. Mammalian olfactory receptors. Front Cell Neurosci. 2009;3:9. https://doi.org/10.3389/neuro.03.009.2009.\u003c/li\u003e\n\u003cli\u003eRoper SD, Chaudhari N. Processing umami and other tastes in mammalian taste buds. Ann N Y Acad Sci. 2009;1170:60\u0026ndash;5. https://doi.org/10.1111/j.1749-6632.2009.04107.x.\u003c/li\u003e\n\u003cli\u003eBaldwin MW, Ko M-C. Functional evolution of vertebrate sensory receptors. Horm Behav. 2020;124:104771. https://doi.org/10.1016/j.yhbeh.2020.104771.\u003c/li\u003e\n\u003cli\u003eBuck L, Axel R. A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell. 1991;65:175\u0026ndash;87. https://doi.org/10.1016/0092-8674(91)90418-x.\u003c/li\u003e\n\u003cli\u003eLiberles SD, Buck LB. A second class of chemosensory receptors in the olfactory epithelium. Nature. 2006;442:645\u0026ndash;50. https://doi.org/10.1038/nature05066.\u003c/li\u003e\n\u003cli\u003eDulac C, Axel R. A novel family of genes encoding putative pheromone receptors in mammals. Cell. 1995;83:195\u0026ndash;206. https://doi.org/10.1016/0092-8674(95)90161-2.\u003c/li\u003e\n\u003cli\u003eHerrada G, Dulac C. A novel family of putative pheromone receptors in mammals with a topographically organized and sexually dimorphic distribution. Cell. 1997;90:763\u0026ndash;73. https://doi.org/10.1016/s0092-8674(00)80536-x.\u003c/li\u003e\n\u003cli\u003eMatsunami H, Buck LB. A multigene family encoding a diverse array of putative pheromone receptors in mammals. Cell. 1997;90:775\u0026ndash;84. https://doi.org/10.1016/s0092-8674(00)80537-1.\u003c/li\u003e\n\u003cli\u003eLi X, Staszewski L, Xu H, Durick K, Zoller M, Adler E. Human receptors for sweet and umami taste. Proc Natl Acad Sci U S A. 2002;99:4692\u0026ndash;6. https://doi.org/10.1073/pnas.072090199.\u003c/li\u003e\n\u003cli\u003eAdler E, Hoon MA, Mueller KL, Chandrashekar J, Ryba NJ, Zuker CS. A novel family of mammalian taste receptors. Cell. 2000;100:693\u0026ndash;702. https://doi.org/10.1016/s0092-8674(00)80705-9.\u003c/li\u003e\n\u003cli\u003eMatsunami H, Montmayeur JP, Buck LB. A family of candidate taste receptors in human and mouse. Nature. 2000;404:601\u0026ndash;4. https://doi.org/10.1038/35007072.\u003c/li\u003e\n\u003cli\u003eNgal J, Dowling M, Buck L, Axel R, Chess A. The family of genes encoding odorant receptors in the channel catfish. Cell. 1993;72:657\u0026ndash;66. https://doi.org/10.1016/0092-8674(93)90395-7.\u003c/li\u003e\n\u003cli\u003eGloriam DEI, Bjarnad\u0026oacute;ttir TK, Yan Y-L, Postlethwait JH, Schi\u0026ouml;th HB, Fredriksson R. The repertoire of trace amine G-protein-coupled receptors: large expansion in zebrafish. Mol Phylogenet Evol. 2005;35:470\u0026ndash;82. https://doi.org/10.1016/j.ympev.2004.12.003.\u003c/li\u003e\n\u003cli\u003eIshimaru Y, Okada S, Naito H, Nagai T, Yasuoka A, Matsumoto I, et al. Two families of candidate taste receptors in fishes. Mech Dev. 2005;122:1310\u0026ndash;21. https://doi.org/10.1016/j.mod.2005.07.005.\u003c/li\u003e\n\u003cli\u003ePfister P, Rodriguez I. Olfactory expression of a single and highly variable V1r pheromone receptor-like gene in fish species. Proc Natl Acad Sci U S A. 2005;102:5489\u0026ndash;94. https://doi.org/10.1073/pnas.0402581102.\u003c/li\u003e\n\u003cli\u003eNaito T, Saito Y, Yamamoto J, Nozaki Y, Tomura K, Hazama M, et al. Putative pheromone receptors related to the Ca2+-sensing receptor in Fugu. Proc Natl Acad Sci U S A. 1998;95:5178\u0026ndash;81. https://doi.org/10.1073/pnas.95.9.5178.\u003c/li\u003e\n\u003cli\u003eWeth F, Nadler W, Korsching S. Nested expression domains for odorant receptors in zebrafish olfactory epithelium. Proc Natl Acad Sci U S A. 1996;93:13321\u0026ndash;6. https://doi.org/10.1073/pnas.93.23.13321.\u003c/li\u003e\n\u003cli\u003eHussain A, Saraiva LR, Korsching SI. Positive Darwinian selection and the birth of an olfactory receptor clade in teleosts. Proc Natl Acad Sci U S A. 2009;106:4313\u0026ndash;8. https://doi.org/10.1073/pnas.0803229106.\u003c/li\u003e\n\u003cli\u003eSato Y, Miyasaka N, Yoshihara Y. Mutually exclusive glomerular innervation by two distinct types of olfactory sensory neurons revealed in transgenic zebrafish. J Neurosci. 2005;25:4889\u0026ndash;97. https://doi.org/10.1523/JNEUROSCI.0679-05.2005.\u003c/li\u003e\n\u003cli\u003eDate-Ito A, Ohara H, Ichikawa M, Mori Y, Hagino-Yamagishi K. Xenopus V1R vomeronasal receptor family is expressed in the main olfactory system. Chem Senses. 2008;33:339\u0026ndash;46. https://doi.org/10.1093/chemse/bjm090.\u003c/li\u003e\n\u003cli\u003eSyed AS, Sansone A, R\u0026ouml;ner S, Bozorg Nia S, Manzini I, Korsching SI. Different expression domains for two closely related amphibian TAARs generate a bimodal distribution similar to neuronal responses to amine odors. Sci Rep. 2015;5:13935. https://doi.org/10.1038/srep13935.\u003c/li\u003e\n\u003cli\u003eChurcher AM, Taylor JS. The antiquity of chordate odorant receptors is revealed by the discovery of orthologs in the cnidarian Nematostella vectensis. Genome Biol Evol. 2011;3:36\u0026ndash;43. https://doi.org/10.1093/gbe/evq079.\u003c/li\u003e\n\u003cli\u003eHashiguchi Y, Nishida M. Evolution of trace amine associated receptor (TAAR) gene family in vertebrates: lineage-specific expansions and degradations of a second class of vertebrate chemosensory receptors expressed in the olfactory epithelium. Mol Biol Evol. 2007;24:2099\u0026ndash;107. https://doi.org/10.1093/molbev/msm140.\u003c/li\u003e\n\u003cli\u003eLibants S, Carr K, Wu H, Teeter JH, Chung-Davidson Y-W, Zhang Z, et al. The sea lamprey Petromyzon marinus genome reveals the early origin of several chemosensory receptor families in the vertebrate lineage. BMC Evol Biol. 2009;9:180. https://doi.org/10.1186/1471-2148-9-180.\u003c/li\u003e\n\u003cli\u003eGrus WE, Zhang J. Origin of the genetic components of the vomeronasal system in the common ancestor of all extant vertebrates. Mol Biol Evol. 2009;26:407\u0026ndash;19. https://doi.org/10.1093/molbev/msn262.\u003c/li\u003e\n\u003cli\u003eSharma K, Syed AS, Ferrando S, Mazan S, Korsching SI. The Chemosensory Receptor Repertoire of a True Shark Is Dominated by a Single Olfactory Receptor Family. Genome Biol Evol. 2019;11:398\u0026ndash;405. https://doi.org/10.1093/gbe/evz002.\u003c/li\u003e\n\u003cli\u003eKowatschew D, Korsching SI. Lamprey possess both V1R and V2R olfactory receptors, but only V1Rs are expressed in olfactory sensory neurons. Chem Senses. 2022;47. https://doi.org/10.1093/chemse/bjac007.\u003c/li\u003e\n\u003cli\u003eBehrens M, Lang T, Korsching SI. A singular shark bitter taste receptor provides insights into the evolution of bitter taste perception. Proc Natl Acad Sci U S A. 2023;120:e2310347120. https://doi.org/10.1073/pnas.2310347120.\u003c/li\u003e\n\u003cli\u003eItoigawa A, Toda Y, Kuraku S, Ishimaru Y. Evolutionary origins of bitter taste receptors in jawed vertebrates. Curr Biol. 2024;34:R271\u0026ndash;2. https://doi.org/10.1016/j.cub.2024.02.024.\u003c/li\u003e\n\u003cli\u003eNishihara H, Toda Y, Kuramoto T, Kamohara K, Goto A, Hoshino K, et al. A vertebrate-wide catalogue of T1R receptors reveals diversity in taste perception. Nat Ecol Evol. 2024;8:111\u0026ndash;20. https://doi.org/10.1038/s41559-023-02258-8.\u003c/li\u003e\n\u003cli\u003eNiimura Y, Nei M. Evolutionary dynamics of olfactory receptor genes in fishes and tetrapods. Proc Natl Acad Sci U S A. 2005;102:6039\u0026ndash;44. https://doi.org/10.1073/pnas.0501922102.\u003c/li\u003e\n\u003cli\u003eGuo L, Dai W, Xu Z, Liang Q, Miller ET, Li S, et al. Evolution of Brain-Expressed Biogenic Amine Receptors into Olfactory Trace Amine-Associated Receptors. Mol Biol Evol. 2022;39. https://doi.org/10.1093/molbev/msac006.\u003c/li\u003e\n\u003cli\u003eShi P, Zhang J. Contrasting modes of evolution between vertebrate sweet/umami receptor genes and bitter receptor genes. Mol Biol Evol. 2006;23:292\u0026ndash;300. https://doi.org/10.1093/molbev/msj028.\u003c/li\u003e\n\u003cli\u003ePolicarpo M, Baldwin MW, Casane D, Salzburger W. Diversity and evolution of the vertebrate chemoreceptor gene repertoire. Nat Commun. 2024;15:1421. https://doi.org/10.1038/s41467-024-45500-y.\u003c/li\u003e\n\u003cli\u003eNiimura Y. On the origin and evolution of vertebrate olfactory receptor genes: comparative genome analysis among 23 chordate species. Genome Biol Evol. 2009;1:34\u0026ndash;44. https://doi.org/10.1093/gbe/evp003.\u003c/li\u003e\n\u003cli\u003ePolicarpo M, Bemis KE, Laurenti P, Legendre L, Sandoz J-C, R\u0026eacute;taux S, et al. Coevolution of the olfactory organ and its receptor repertoire in ray-finned fishes. BMC Biol. 2022;20:195. https://doi.org/10.1186/s12915-022-01397-x.\u003c/li\u003e\n\u003cli\u003eZhou Y, Shearwin-Whyatt L, Li J, Song Z, Hayakawa T, Stevens D, et al. Platypus and echidna genomes reveal mammalian biology and evolution. Nature. 2021;592:756\u0026ndash;62. https://doi.org/10.1038/s41586-020-03039-0.\u003c/li\u003e\n\u003cli\u003eZhong H, Huang J, Shang S, Yuan B. Evolutionary insights into umami, sweet, and bitter taste receptors in amphibians. Ecol Evol. 2021;11:18011\u0026ndash;25. https://doi.org/10.1002/ece3.8398.\u003c/li\u003e\n\u003cli\u003eNei M, Rooney AP. Concerted and birth-and-death evolution of multigene families. Annu Rev Genet. 2005;39:121\u0026ndash;52. https://doi.org/10.1146/annurev.genet.39.073003.112240.\u003c/li\u003e\n\u003cli\u003eSpehr M, Gisselmann G, Poplawski A, Riffell JA, Wetzel CH, Zimmer RK, et al. Identification of a testicular odorant receptor mediating human sperm chemotaxis. Science. 2003;299:2054\u0026ndash;8. https://doi.org/10.1126/science.1080376.\u003c/li\u003e\n\u003cli\u003eFukuda N, Yomogida K, Okabe M, Touhara K. Functional characterization of a mouse testicular olfactory receptor and its role in chemosensing and in regulation of sperm motility. J Cell Sci. 2004;117 Pt 24:5835\u0026ndash;45. https://doi.org/10.1242/jcs.01507.\u003c/li\u003e\n\u003cli\u003eMargolskee RF, Dyer J, Kokrashvili Z, Salmon KSH, Ilegems E, Daly K, et al. T1R3 and gustducin in gut sense sugars to regulate expression of Na\u003csup\u003e+\u003c/sup\u003e-glucose cotransporter 1. Proc Natl Acad Sci U S A. 2007;104:15075\u0026ndash;80. https://doi.org/10.1073/pnas.0706678104.\u003c/li\u003e\n\u003cli\u003eYoung RL, Chia B, Isaacs NJ, Ma J, Khoo J, Wu T, et al. Disordered control of intestinal sweet taste receptor expression and glucose absorption in type 2 diabetes. Diabetes. 2013;62:3532\u0026ndash;41. https://doi.org/10.2337/db13-0581.\u003c/li\u003e\n\u003cli\u003eKok BP, Galmozzi A, Littlejohn NK, Albert V, Godio C, Kim W, et al. Intestinal bitter taste receptor activation alters hormone secretion and imparts metabolic benefits. Mol Metab. 2018;16:76\u0026ndash;87. https://doi.org/10.1016/j.molmet.2018.07.013.\u003c/li\u003e\n\u003cli\u003eChen MC, Wu SV, Reeve JR Jr, Rozengurt E. Bitter stimuli induce Ca2+ signaling and CCK release in enteroendocrine STC-1 cells: role of L-type voltage-sensitive Ca2+ channels. Am J Physiol Cell Physiol. 2006;291:C726-39. https://doi.org/10.1152/ajpcell.00003.2006.\u003c/li\u003e\n\u003cli\u003eFlegel C, Manteniotis S, Osthold S, Hatt H, Gisselmann G. Expression profile of ectopic olfactory receptors determined by deep sequencing. PLoS One. 2013;8:e55368. https://doi.org/10.1371/journal.pone.0055368.\u003c/li\u003e\n\u003cli\u003eFeldmesser E, Olender T, Khen M, Yanai I, Ophir R, Lancet D. Widespread ectopic expression of olfactory receptor genes. BMC Genomics. 2006;7:121. https://doi.org/10.1186/1471-2164-7-121.\u003c/li\u003e\n\u003cli\u003eMa\u0026szlig;berg D, Hatt H. Human Olfactory Receptors: Novel Cellular Functions Outside of the Nose. Physiol Rev. 2018;98:1739\u0026ndash;63. https://doi.org/10.1152/physrev.00013.2017.\u003c/li\u003e\n\u003cli\u003eXu J, Choi R, Gupta K, Warren HR, Santhanam L, Pluznick JL. An evolutionarily conserved olfactory receptor is required for sex differences in blood pressure. Sci Adv. 2024;10:eadk1487. https://doi.org/10.1126/sciadv.adk1487.\u003c/li\u003e\n\u003cli\u003eChang AJ, Ortega FE, Riegler J, Madison DV, Krasnow MA. Oxygen regulation of breathing through an olfactory receptor activated by lactate. Nature. 2015;527:240\u0026ndash;4. https://doi.org/10.1038/nature15721.\u003c/li\u003e\n\u003cli\u003eCh\u0026eacute;ret J, Bertolini M, Ponce L, Lehmann J, Tsai T, Alam M, et al. Olfactory receptor OR2AT4 regulates human hair growth. Nat Commun. 2018;9:3624. https://doi.org/10.1038/s41467-018-05973-0.\u003c/li\u003e\n\u003cli\u003eGriffin CA, Kafadar KA, Pavlath GK. MOR23 promotes muscle regeneration and regulates cell adhesion and migration. Dev Cell. 2009;17:649\u0026ndash;61. https://doi.org/10.1016/j.devcel.2009.09.004.\u003c/li\u003e\n\u003cli\u003eJanssen S, Laermans J, Verhulst P-J, Thijs T, Tack J, Depoortere I. Bitter taste receptors and \u0026alpha;-gustducin regulate the secretion of ghrelin with functional effects on food intake and gastric emptying. Proc Natl Acad Sci U S A. 2011;108:2094\u0026ndash;9. https://doi.org/10.1073/pnas.1011508108.\u003c/li\u003e\n\u003cli\u003eNakagawa Y, Nagasawa M, Yamada S, Hara A, Mogami H, Nikolaev VO, et al. Sweet taste receptor expressed in pancreatic beta-cells activates the calcium and cyclic AMP signaling systems and stimulates insulin secretion. PLoS One. 2009;4:e5106. https://doi.org/10.1371/journal.pone.0005106.\u003c/li\u003e\n\u003cli\u003eKyriazis GA, Soundarapandian MM, Tyrberg B. Sweet taste receptor signaling in beta cells mediates fructose-induced potentiation of glucose-stimulated insulin secretion. Proc Natl Acad Sci U S A. 2012;109:E524-32. https://doi.org/10.1073/pnas.1115183109.\u003c/li\u003e\n\u003cli\u003eWeber L, Al-Refae K, Ebbert J, J\u0026auml;gers P, Altm\u0026uuml;ller J, Becker C, et al. Activation of odorant receptor in colorectal cancer cells leads to inhibition of cell proliferation and apoptosis. PLoS One. 2017;12:e0172491. https://doi.org/10.1371/journal.pone.0172491.\u003c/li\u003e\n\u003cli\u003eJundi D, Coutanceau J-P, Bullier E, Imarraine S, Fajloun Z, Hong E. Expression of olfactory receptor genes in non-olfactory tissues in the developing and adult zebrafish. Sci Rep. 2023;13:4651. https://doi.org/10.1038/s41598-023-30895-3.\u003c/li\u003e\n\u003cli\u003eBirdal G, D\u0026rsquo;Gama PP, Jurisch-Yaksi N, Korsching SI. Expression of taste sentinels, T1R, T2R, and PLC\u0026beta;2, on the passageway for olfactory signals in zebrafish. Chem Senses. 2023;48. https://doi.org/10.1093/chemse/bjad040.\u003c/li\u003e\n\u003cli\u003eBhatia V, de Jesus VC, Shaik FA, Jaggupilli A, Singh N, Chelikani P, et al. Extraoral expression and characterization of bitter taste receptors in Astyanax mexicanus (Mexican tetra fish). FASEB Bioadv. 2022;4:574\u0026ndash;84. https://doi.org/10.1096/fba.2022-00032.\u003c/li\u003e\n\u003cli\u003eZhang Z, Sakuma A, Kuraku S, Nikaido M. Remarkable diversity of vomeronasal type 2 receptor (OlfC) genes of basal ray-finned fish and its evolutionary trajectory in jawed vertebrates. Sci Rep. 2022;12:6455. https://doi.org/10.1038/s41598-022-10428-0.\u003c/li\u003e\n\u003cli\u003eSerizawa S, Miyamichi K, Nakatani H, Suzuki M, Saito M, Yoshihara Y, et al. Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse. Science. 2003;302:2088\u0026ndash;94. https://doi.org/10.1126/science.1089122.\u003c/li\u003e\n\u003cli\u003eKhan M, Vaes E, Mombaerts P. Regulation of the probability of mouse odorant receptor gene choice. Cell. 2011;147:907\u0026ndash;21. https://doi.org/10.1016/j.cell.2011.09.049.\u003c/li\u003e\n\u003cli\u003eIwata T, Niimura Y, Kobayashi C, Shirakawa D, Suzuki H, Enomoto T, et al. A long-range cis-regulatory element for class I odorant receptor genes. Nat Commun. 2017;8:885. https://doi.org/10.1038/s41467-017-00870-4.\u003c/li\u003e\n\u003cli\u003eMarkenscoff-Papadimitriou E, Allen WE, Colquitt BM, Goh T, Murphy KK, Monahan K, et al. Enhancer interaction networks as a means for singular olfactory receptor expression. Cell. 2014;159:543\u0026ndash;57. https://doi.org/10.1016/j.cell.2014.09.033.\u003c/li\u003e\n\u003cli\u003eShinomiya H. Plastin family of actin-bundling proteins: its functions in leukocytes, neurons, intestines, and cancer. Int J Cell Biol. 2012;2012:213492. https://doi.org/10.1155/2012/213492.\u003c/li\u003e\n\u003cli\u003eGao H, Zhang Z, Deng J, Song Y. Cathepsin S: molecular mechanisms in inflammatory and immunological processes. Front Immunol. 2025;16:1600206. https://doi.org/10.3389/fimmu.2025.1600206.\u003c/li\u003e\n\u003cli\u003eXiang C, Li H, Tang W. Targeting CSF-1R represents an effective strategy in modulating inflammatory diseases. Pharmacol Res. 2023;187:106566. https://doi.org/10.1016/j.phrs.2022.106566.\u003c/li\u003e\n\u003cli\u003eGurski CJ, Dittel BN. Myeloperoxidase as a marker to differentiate mouse monocyte/macrophage subsets. Int J Mol Sci. 2022;23:8246. https://doi.org/10.3390/ijms23158246.\u003c/li\u003e\n\u003cli\u003eArredouani MS. Is the scavenger receptor MARCO a new immune checkpoint? Oncoimmunology. 2014;3:e955709. https://doi.org/10.4161/21624011.2014.955709.\u003c/li\u003e\n\u003cli\u003eVestweber D. Vascular endothelial protein tyrosine phosphatase regulates endothelial function. Physiology (Bethesda). 2021;36:84\u0026ndash;93. https://doi.org/10.1152/physiol.00026.2020.\u003c/li\u003e\n\u003cli\u003eFeng W, Chen L, Nguyen PK, Wu SM, Li G. Single cell analysis of endothelial cells identified organ-specific molecular signatures and heart-specific cell populations and molecular features. Front Cardiovasc Med. 2019;6:165. https://doi.org/10.3389/fcvm.2019.00165.\u003c/li\u003e\n\u003cli\u003eKorhonen EA, Lampinen A, Giri H, Anisimov A, Kim M, Allen B, et al. Tie1 controls angiopoietin function in vascular remodeling and inflammation. J Clin Invest. 2016;126:3495\u0026ndash;510. https://doi.org/10.1172/JCI84923.\u003c/li\u003e\n\u003cli\u003eHoon MA, Adler E, Lindemeier J, Battey JF, Ryba NJ, Zuker CS. Putative mammalian taste receptors: a class of taste-specific GPCRs with distinct topographic selectivity. Cell. 1999;96:541\u0026ndash;51. https://doi.org/10.1016/s0092-8674(00)80658-3.\u003c/li\u003e\n\u003cli\u003eBorowsky B, Adham N, Jones KA, Raddatz R, Artymyshyn R, Ogozalek KL, et al. Trace amines: identification of a family of mammalian G protein-coupled receptors. Proc Natl Acad Sci U S A. 2001;98:8966\u0026ndash;71. https://doi.org/10.1073/pnas.151105198.\u003c/li\u003e\n\u003cli\u003eBunzow JR, Sonders MS, Arttamangkul S, Harrison LM, Zhang G, Quigley DI, et al. Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor. Mol Pharmacol. 2001;60:1181\u0026ndash;8. https://doi.org/10.1124/mol.60.6.1181.\u003c/li\u003e\n\u003cli\u003eParmentier M, Libert F, Schurmans S, Schiffmann S, Lefort A, Eggerickx D, et al. Expression of members of the putative olfactory receptor gene family in mammalian germ cells. Nature. 1992;355:453\u0026ndash;5. https://doi.org/10.1038/355453a0.\u003c/li\u003e\n\u003cli\u003eNei M, Niimura Y, Nozawa M. The evolution of animal chemosensory receptor gene repertoires: roles of chance and necessity. Nat Rev Genet. 2008;9:951\u0026ndash;63. https://doi.org/10.1038/nrg2480.\u003c/li\u003e\n\u003cli\u003eHashiguchi Y, Nishida M. Evolution and origin of vomeronasal-type odorant receptor gene repertoire in fishes. BMC Evol Biol. 2006;6:76. https://doi.org/10.1186/1471-2148-6-76.\u003c/li\u003e\n\u003cli\u003eHiggins KW, Itoigawa A, Toda Y, Bellott DW, Anderson R, M\u0026aacute;rquez R, et al. Rapid expansion and specialization of the TAS2R bitter taste receptor family in amphibians. PLoS Genet. 2025;21:e1011533. https://doi.org/10.1371/journal.pgen.1011533.\u003c/li\u003e\n\u003cli\u003eBellono NW, Bayrer JR, Leitch DB, Castro J, Zhang C, O\u0026rsquo;Donnell TA, et al. Enterochromaffin cells are gut chemosensors that couple to sensory neural pathways. Cell. 2017;170:185-198.e16. https://doi.org/10.1016/j.cell.2017.05.034.\u003c/li\u003e\n\u003cli\u003ePluznick JL, Protzko RJ, Gevorgyan H, Peterlin Z, Sipos A, Han J, et al. Olfactory receptor responding to gut microbiota-derived signals plays a role in renin secretion and blood pressure regulation. Proc Natl Acad Sci U S A. 2013;110:4410\u0026ndash;5. https://doi.org/10.1073/pnas.1215927110.\u003c/li\u003e\n\u003cli\u003eDe la Cruz O, Blekhman R, Zhang X, Nicolae D, Firestein S, Gilad Y. A signature of evolutionary constraint on a subset of ectopically expressed olfactory receptor genes. Mol Biol Evol. 2009;26:491\u0026ndash;4. https://doi.org/10.1093/molbev/msn294.\u003c/li\u003e\n\u003cli\u003eShah A, Ratkowski M, Rosa A, Feinstein P, Bozza T. Olfactory expression of trace amine-associated receptors requires cooperative cis-acting enhancers. Nat Commun. 2021;12:3797. https://doi.org/10.1038/s41467-021-23824-3.\u003c/li\u003e\n\u003cli\u003eFei A, Wu W, Tan L, Tang C, Xu Z, Huo X, et al. Coordination of two enhancers drives expression of olfactory trace amine-associated receptors. Nat Commun. 2021;12:3798. https://doi.org/10.1038/s41467-021-23823-4.\u003c/li\u003e\n\u003cli\u003eMagklara A, Yen A, Colquitt BM, Clowney EJ, Allen W, Markenscoff-Papadimitriou E, et al. An epigenetic signature for monoallelic olfactory receptor expression. Cell. 2011;145:555\u0026ndash;70. https://doi.org/10.1016/j.cell.2011.03.040.\u003c/li\u003e\n\u003cli\u003eLyons DB, Magklara A, Goh T, Sampath SC, Schaefer A, Schotta G, et al. Heterochromatin-mediated gene silencing facilitates the diversification of olfactory neurons. Cell Rep. 2014;9:884\u0026ndash;92. https://doi.org/10.1016/j.celrep.2014.10.001.\u003c/li\u003e\n\u003cli\u003eClowney EJ, LeGros MA, Mosley CP, Clowney FG, Markenskoff-Papadimitriou EC, Myllys M, et al. Nuclear aggregation of olfactory receptor genes governs their monogenic expression. Cell. 2012;151:724\u0026ndash;37. https://doi.org/10.1016/j.cell.2012.09.043.\u003c/li\u003e\n\u003cli\u003eLomvardas S, Barnea G, Pisapia DJ, Mendelsohn M, Kirkland J, Axel R. Interchromosomal interactions and olfactory receptor choice. Cell. 2006;126:403\u0026ndash;13. https://doi.org/10.1016/j.cell.2006.06.035.\u003c/li\u003e\n\u003cli\u003eBushdid C, Magnasco MO, Vosshall LB, Keller A. Humans can discriminate more than 1 trillion olfactory stimuli. Science. 2014;343:1370\u0026ndash;2. https://doi.org/10.1126/science.1249168.\u003c/li\u003e\n\u003cli\u003eManteniotis S, Wojcik S, Brauhoff P, M\u0026ouml;llmann M, Petersen L, G\u0026ouml;thert JR, et al. Functional characterization of the ectopically expressed olfactory receptor 2AT4 in human myelogenous leukemia. Cell Death Discov. 2016;2:15070. https://doi.org/10.1038/cddiscovery.2015.70.\u003c/li\u003e\n\u003cli\u003eManteniotis S, Wojcik S, G\u0026ouml;thert JR, D\u0026uuml;rig J, D\u0026uuml;hrsen U, Gisselmann G, et al. Deorphanization and characterization of the ectopically expressed olfactory receptor OR51B5 in myelogenous leukemia cells. Cell Death Discov. 2016;2:16010. https://doi.org/10.1038/cddiscovery.2016.10.\u003c/li\u003e\n\u003cli\u003eTsai T, Veitinger S, Peek I, Busse D, Eckardt J, Vladimirova D, et al. Two olfactory receptors-OR2A4/7 and OR51B5-differentially affect epidermal proliferation and differentiation. Exp Dermatol. 2017;26:58\u0026ndash;65. https://doi.org/10.1111/exd.13132.\u003c/li\u003e\n\u003cli\u003eFukuda N, Touhara K. Developmental expression patterns of testicular olfactory receptor genes during mouse spermatogenesis. Genes Cells. 2006;11:71\u0026ndash;81. https://doi.org/10.1111/j.1365-2443.2005.00915.x.\u003c/li\u003e\n\u003cli\u003eLi JJ, Tay HL, Plank M, Essilfie A-T, Hansbro PM, Foster PS, et al. Activation of olfactory receptors on mouse pulmonary macrophages promotes monocyte chemotactic protein-1 production. PLoS One. 2013;8:e80148. https://doi.org/10.1371/journal.pone.0080148.\u003c/li\u003e\n\u003cli\u003eClark AA, Nurmukhambetova S, Li X, Munger SD, Lees JR. Odorants specifically modulate chemotaxis and tissue retention of CD4+ T cells via cyclic adenosine monophosphate induction. J Leukoc Biol. 2016;100:699\u0026ndash;709. https://doi.org/10.1189/jlb.1A0914-425RR.\u003c/li\u003e\n\u003cli\u003eOrecchioni M, Kobiyama K, Winkels H, Ghosheh Y, McArdle S, Mikulski Z, et al. Olfactory receptor 2 in vascular macrophages drives atherosclerosis by NLRP3-dependent IL-1 production. Science. 2022;375:214\u0026ndash;21. https://doi.org/10.1126/science.abg3067.\u003c/li\u003e\n\u003cli\u003eMalki A, Fiedler J, Fricke K, Ballweg I, Pfaffl MW, Krautwurst D. Class I odorant receptors, TAS1R and TAS2R taste receptors, are markers for subpopulations of circulating leukocytes. J Leukoc Biol. 2015;97:533\u0026ndash;45. https://doi.org/10.1189/jlb.2A0714-331RR.\u003c/li\u003e\n\u003cli\u003eSampson JM, Morrissey KA, Douek DC, Miller RD. A family of olfactory receptors uniquely expanded in marsupial and monotreme genomes are expressed by a T cell subset also unique to marsupials and monotremes. Dev Comp Immunol. 2024;154:105149. https://doi.org/10.1016/j.dci.2024.105149.\u003c/li\u003e\n\u003cli\u003eLitman GW, Rast JP, Fugmann SD. The origins of vertebrate adaptive immunity. Nat Rev Immunol. 2010;10:543\u0026ndash;53. https://doi.org/10.1038/nri2807.\u003c/li\u003e\n\u003cli\u003eHirano M, Das S, Guo P, Cooper MD. The evolution of adaptive immunity in vertebrates. Adv Immunol. 2011;109:125\u0026ndash;57. https://doi.org/10.1016/B978-0-12-387664-5.00004-2.\u003c/li\u003e\n\u003cli\u003eAmersfoort J, Eelen G, Carmeliet P. Immunomodulation by endothelial cells - partnering up with the immune system? Nat Rev Immunol. 2022;22:576\u0026ndash;88. https://doi.org/10.1038/s41577-022-00694-4.\u003c/li\u003e\n\u003cli\u003eFauvarque M-O, Williams MJ. Drosophila cellular immunity: a story of migration and adhesion. J Cell Sci. 2011;124 Pt 9:1373\u0026ndash;82. https://doi.org/10.1242/jcs.064592.\u003c/li\u003e\n\u003cli\u003eRotstein B, Paululat A. On the morphology of the Drosophila heart. J Cardiovasc Dev Dis. 2016;3:15. https://doi.org/10.3390/jcdd3020015.\u003c/li\u003e\n\u003cli\u003eBertrand JY, Chi NC, Santoso B, Teng S, Stainier DYR, Traver D. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature. 2010;464:108\u0026ndash;11. https://doi.org/10.1038/nature08738.\u003c/li\u003e\n\u003cli\u003eBoisset J-C, van Cappellen W, Andrieu-Soler C, Galjart N, Dzierzak E, Robin C. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature. 2010;464:116\u0026ndash;20. https://doi.org/10.1038/nature08764.\u003c/li\u003e\n\u003cli\u003eKissa K, Herbomel P. Blood stem cells emerge from aortic endothelium by a novel type of cell transition. Nature. 2010;464:112\u0026ndash;5. https://doi.org/10.1038/nature08761.\u003c/li\u003e\n\u003cli\u003eLam EYN, Hall CJ, Crosier PS, Crosier KE, Flores MV. Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells. Blood. 2010;116:909\u0026ndash;14. https://doi.org/10.1182/blood-2010-01-264382.\u003c/li\u003e\n\u003cli\u003eWilliamson AE, Liyanage S, Hassanshahi M, Dona MSI, Toledo-Flores D, Tran DXA, et al. Discovery of an embryonically derived bipotent population of endothelial-macrophage progenitor cells in postnatal aorta. Nat Commun. 2024;15:7097. https://doi.org/10.1038/s41467-024-51637-7.\u003c/li\u003e\n\u003cli\u003eLeinonen R, Sugawara H, Shumway M, International Nucleotide Sequence Database Collaboration. The sequence read archive. Nucleic Acids Res. 2011;39 Database issue:D19-21. https://doi.org/10.1093/nar/gkq1019.\u003c/li\u003e\n\u003cli\u003eChen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884\u0026ndash;90. https://doi.org/10.1093/bioinformatics/bty560.\u003c/li\u003e\n\u003cli\u003eDobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15\u0026ndash;21. https://doi.org/10.1093/bioinformatics/bts635.\u003c/li\u003e\n\u003cli\u003eLi B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323. https://doi.org/10.1186/1471-2105-12-323.\u003c/li\u003e\n\u003cli\u003eWang R, Zhang P, Wang J, Ma L, E W, Suo S, et al. Construction of a cross-species cell landscape at single-cell level. Nucleic Acids Res. 2023;51:501\u0026ndash;16. https://doi.org/10.1093/nar/gkac633.\u003c/li\u003e\n\u003cli\u003eHao Y, Stuart T, Kowalski MH, Choudhary S, Hoffman P, Hartman A, et al. Dictionary learning for integrative, multimodal and scalable single-cell analysis. Nat Biotechnol. 2023. https://doi.org/10.1038/s41587-023-01767-y.\u003c/li\u003e\n\u003cli\u003eChen W, Jiang H, Wang C, Ding Z, Yu D, Liu Y, et al. Single-cell RNA sequencing of zebrafish olfactory epithelium reveals cellular heterogeneity and responses to a conspecific alarm substance. Water Biology and Security. 2024;4:100324. https://doi.org/10.1016/j.watbs.2024.100324.\u003c/li\u003e\n\u003cli\u003eLawson ND, Li R, Shin M, Grosse A, Yukselen O, Stone OA, et al. An improved zebrafish transcriptome annotation for sensitive and comprehensive detection of cell type-specific genes. Elife. 2020;9. https://doi.org/10.7554/eLife.55792.\u003c/li\u003e\n\u003cli\u003eHorgue LF, Assens A, Fodoulian L, Marconi L, Tuberosa J, Haider A, et al. Transcriptional adaptation of olfactory sensory neurons to GPCR identity and activity. Nat Commun. 2022;13:2929. https://doi.org/10.1038/s41467-022-30511-4.\u003c/li\u003e\n\u003cli\u003eSuzuki H, Nikaido M, Hagino-Yamagishi K, Okada N. Distinct functions of two olfactory marker protein genes derived from teleost-specific whole genome duplication. BMC Evol Biol. 2015;15:245. https://doi.org/10.1186/s12862-015-0530-y.\u003c/li\u003e\n\u003cli\u003eVassar R, Ngai J, Axel R. Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell. 1993;74:309\u0026ndash;18. https://doi.org/10.1016/0092-8674(93)90422-m.\u003c/li\u003e\n\u003cli\u003eRessler KJ, Sullivan SL, Buck LB. A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell. 1993;73:597\u0026ndash;609. https://doi.org/10.1016/0092-8674(93)90145-g.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"zoological-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"zlet","sideBox":"Learn more about [Zoological Letters](https://www.springer.com/journal/40851) ","snPcode":"40851","submissionUrl":"https://www.editorialmanager.com/zlet/default2.aspx","title":"Zoological Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Chemoreceptor, Olfactory receptor, Ectopic expression, Evolution","lastPublishedDoi":"10.21203/rs.3.rs-8198358/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8198358/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVertebrate chemoreceptor genes play a central role in detecting environmental chemical compounds through the olfactory organs and taste buds, enabling the perception of odors and tastants that are critical for survival. Through evolutionary processes, these genes have repeatedly undergone duplication, divergence, and pseudogenization, giving rise to lineage-specific gene repertoires that reflect ecological and behavioral adaptation. While the canonical functions of these chemoreceptors are confined to the nasal and oral cavities, increasing evidence, particularly in mammals, indicates that some chemoreceptors are expressed and function in non-chemosensory (extra-nasal/oral) organs. However, such extra-nasal/oral expression has rarely been examined from a broad evolutionary perspective across vertebrate lineages. Here, we systematically investigated organ-wide expression patterns of chemoreceptor genes by conducting comprehensive bulk RNA-seq analysis across 13 organs in four representative species: mouse, \u003cem\u003eXenopus\u003c/em\u003e, \u003cem\u003ePolypterus\u003c/em\u003e, and zebrafish. In all species, the majority (95\u0026ndash;97%) of chemoreceptor genes were expressed in olfactory and gustatory organs, as expected. Remarkably, however, a subset (1\u0026ndash;29%) showed expression in extra-nasal/oral organs, suggesting that such extra-nasal/oral expression may be a common phenomenon across vertebrates. In particular, the evolutionarily conserved OR-κ gene, with stable gene copy numbers, exhibited organ-independent expression across all analyzed species. Single-cell RNA-seq data further revealed that OR-κ is predominantly expressed in immune cells, implying potential function of chemoreception in immune systems. Furthermore, genomic context analysis showed that the OR-κ gene is isolated from canonical OR gene clusters, suggesting it may have distinct transcriptional regulatory mechanisms compared to typical olfactory receptors. Our findings expand the conventional view of chemoreceptors as sensory-specialized molecules, highlighting their unexpected functional diversity across vertebrate organs. Notably, the OR-κ gene appears to have an ancient evolutionary origin that likely traces back to the common ancestor of vertebrates. Taken together, this study compels us to reconsider the functions and evolutionary trajectories of chemoreceptor genes in vertebrates.\u003c/p\u003e","manuscriptTitle":"Vertebrate-wide transcriptomic screening identifies immune cell-specific expression of the conserved OR-κ gene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 15:27:12","doi":"10.21203/rs.3.rs-8198358/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-11-26T01:06:52+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-26T00:36:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-25T12:39:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Zoological Letters","date":"2025-11-24T22:23:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"zoological-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"zlet","sideBox":"Learn more about [Zoological Letters](https://www.springer.com/journal/40851) ","snPcode":"40851","submissionUrl":"https://www.editorialmanager.com/zlet/default2.aspx","title":"Zoological Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c5917050-8e6a-44fb-8978-113109fe5b6c","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:12:00+00:00","versionOfRecord":{"articleIdentity":"rs-8198358","link":"https://doi.org/10.1186/s40851-026-00260-z","journal":{"identity":"zoological-letters","isVorOnly":false,"title":"Zoological Letters"},"publishedOn":"2026-02-07 15:57:17","publishedOnDateReadable":"February 7th, 2026"},"versionCreatedAt":"2025-12-01 15:27:12","video":"","vorDoi":"10.1186/s40851-026-00260-z","vorDoiUrl":"https://doi.org/10.1186/s40851-026-00260-z","workflowStages":[]},"version":"v1","identity":"rs-8198358","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8198358","identity":"rs-8198358","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}