Kimiko Hagino-Yamagishi

Expression of vomeronasal receptor genes in Xenopus laevis.

In the course of evolution, the vomeronasal organ (VNO) first appeared in amphibians. To understand the relationship between the VNO and the vomeronasal receptors, we isolated and analyzed the expression of the vomeronasal receptor genes of Xenopus laevis. We identified genes of the Xenopus V2R receptor family, which are predominantly expressed throughout the sensory epithelium of the VNO. The G‐protein Go, which is coexpressed with V2Rs in the rodent VNO, was also extensively expressed throughout the vomeronasal sensory epithelium. These results strongly suggest that the V2Rs and Go are coexpressed in the vomeronasal receptor cells. The predominant expression of the Xenopus V2R families and the coexpression of the V2Rs and Go imply that V2Rs play important roles in the sensory transduction of Xenopus VNO. We found that these receptors were expressed not only in the VNO, but also in the posterolateral epithelial area of the principal cavity (PLPC). Electron microscopic study revealed that the epithelium of the PLPC is more like that of the VNO than that of the principal and the middle cavity. These results suggest that in adult Xenopus the V2Rs analyzed so far are predominantly expressed in the vomeronasal and vomeronasal‐like epithelium. The analysis of V2R expression in Xenopus larvae demonstrates that V2Rs are predominantly expressed in the VNO even before metamorphosis. J. Comp. Neurol. 472:246–256, 2004.

Xenopus V1R Vomeronasal Receptor Family Is Expressed in the Main Olfactory System

To date, over 100 vomeronasal receptor type 1 (V1R) genes have been identified in rodents. V1R is specifically expressed in the rodent vomeronasal organ (VNO) and is thought to be responsible for pheromone reception. Recently, 21 putatively functional V1R genes were identified in the genome database of the amphibian Xenopus tropicalis. Amphibians are the first vertebrates to possess a VNO. In order to determine at which point during evolution the vertebrate V1R genes began to function in the vomeronasal system, we analyzed the expression of all putatively functional V1R genes in Xenopus olfactory organs. We found that V1R expression was not detected in the VNO but was specifically detected in the main olfactory epithelium (MOE). We also observed that V1R-expressing cells in the MOE coexpressed Gi2, thus suggesting that the V1R-Gi2-mediated signal transduction pathway, which is considered to play an important role in pheromone reception in the rodent VNO, exists in the amphibian MOE. These results suggest that V1R-mediated signal transduction pathway functions in Xenopus main olfactory system.

Predominant expression of Brn-2 in the postmitotic neurons of the developing mouse neocortex

The expression of Brn-2, a central nervous systems (CNS)-specific POU domain transcription factor, in the developing mouse neocortex was examined with an anti-Brn-2 antibody. Brn-2 protein was first detected in CNS on embryonic day (E) 11.5, and remained strong until E15.5. From E11.5 to postnatal day (P) 0, a high level of Brn-2 expression was observed in the subventricular zone, the intermediate zone, and the outer layer of the neocortex, but not in the ventricular zone. In the double-staining experiments, most of the Brn-2 positive cells were also positive for NCAM-H, an adhesion molecule specific to post-mitotic neurons. Furthermore, BrdU-labeling experiments demonstrated the presence of Brn-2 protein exclusively in postmitotic cells. These results indicated that, in the developing neocortex, Brn-2 expression is up-regulated after the final cell division. Therefore, this transcription factor may be involved in the migration and/or maturation process of the immature neuronal cells.

Lineage-Specific Expansion of Vomeronasal Type 2 Receptor-Like (OlfC) Genes in Cichlids May Contribute to Diversification of Amino Acid Detection Systems

Fish use olfaction to sense a variety of nonvolatile chemical signals in water. However, the evolutionary importance of olfaction in species-rich cichlids is controversial. Here, we determined an almost complete sequence of the vomeronasal type 2 receptor-like (OlfC: putative amino acids receptor in teleosts) gene cluster using the bacterial artificial chromosome library of the Lake Victoria cichlid, Haplochromis chilotes. In the cluster region, we found 61 intact OlfC genes, which is the largest number of OlfC genes identified among the seven teleost fish investigated to date. Data mining of the Oreochromis niloticus (Nile tilapia) draft genome sequence, and genomic Southern hybridization analysis revealed that the ancestor of all modern cichlids had already developed almost the same OlfC gene repertoire, which was accomplished by lineage-specific gene expansions. Furthermore, comparison of receptor sequences showed that recently duplicated paralogs are more variable than orthologs of different species at particular sites that were predicted to be involved in amino acid selectivity. Thus, the increase of paralogs through gene expansion may lead to functional diversification in detection of amino acids. This study implies that cichlids have developed a potent capacity to detect a variety of amino acids (and their derivatives) through OlfCs, which may have contributed to the extraordinary diversity of their feeding habitats.

Characterization of V1R receptor (ora) genes in Lake Victoria cichlids.

Although olfaction could play a crucial role in underwater habitats by allowing fish to sense a variety of nonvolatile chemical signals, the importance of olfaction in species-rich cichlids is still controversial. In particular, examining whether cichlids rely on olfaction for reproduction is of primary interest to understand the mechanisms of speciation. In the present study, we explored the V1R (also known as ora) genes, which are believed to encode reproductive pheromone receptors in fish, in the genomes of Lake Victoria cichlids. By screening a bacterial artificial chromosome library, we identified all six intact V1R genes (V1R1 to V1R6) that have been reported in other teleost fish. Furthermore, RT-PCR and in situ hybridization analyses showed that all of the V1R genes were expressed in the olfactory epithelium, indicating that these receptors are functional in cichlids. These observations indicate that cichlids use V1R-mediated olfaction in some ways for their social behaviors.

Multiple Episodic Evolution Events in V1R Receptor Genes of East-African Cichlids

Fish use olfaction to detect a variety of nonvolatile chemical signals, and thus, this sense is key to survival and communication. However, the contribution of the olfactory sense to social—especially reproductive—interactions in cichlids is still controversial. To obtain insights into this issue, we investigated the genes encoding V1Rs—possible candidates for reproductive pheromone receptors—among East-African cichlids. Interestingly, we found an excess of nonsynonymous over synonymous substitutions in four of six V1R genes in multiple cichlid lineages. First, we found that highly dimorphic V1R2 allele groups were shared among the cichlids inhabiting all East-African Great Lakes emerged through the episodic accumulation of the nonsynonymous substitutions prior to the radiation of the Lake Tanganyika species flock. We further detected such episodic events in V1R1 of the tribe Tropheini, and in V1R3 and V1R6 of the tribe Trematocarini. The excess of nonsynonymous substitutions in these examples were indicated as dN/dS > 1, which were all statistically significant by Fisher’s exact test. Furthermore, we speculate that the amino acid changes in these episodic events are likely functional switch because they occurred in the putative ligand-binding pocket. Our finding of the occurrence of multiple episodic events and the unexpected gene diversity in one unique gene family is suggestive of the contribution of the V1R to the species diversification and the social interaction in cichlids.

Involvement of Gαolf-expressing neurons in the vomeronasal system of Bufo japonicus

Most terrestrial vertebrates possess anatomically distinct olfactory organs: the olfactory epithelium (OE) and the vomeronasal organ (VNO). In rodents, olfactory receptors coupled to Gαolf are expressed in the OE, whereas vomeronasal receptors type 1 (V1R) and vomeronasal receptors type 2 (V2R), coupled to Gαi2 and Gαo, respectively, are expressed in the VNO. These receptors and G proteins are thought to play important roles in olfactory perception. However, we previously reported that only V2R and Gαo expression is detected in the Xenopus laevis VNO. As X. laevis spends its entire life in water, we considered that expression of limited types of chemosensory machinery in the VNO might be due to adaptation of the VNO to aquatic life. Thus, we analyzed the expression of G proteins in the VNO and the accessory olfactory bulb (AOB) of the adult Japanese toad, Bufo japonicus, because this species is well adapted to a terrestrial life. By using immunohistochemical analysis in combination with in situ hybridization and DiI labeling, we found that B. japonicus Gαolf and Gαo were expressed in the apical and middle‐to‐basal layer of the vomeronasal neuroepithelium, and that the axons of these Gαolf‐ and Gαo‐expressing vomeronasal neurons projected to the rostral and caudal accessory olfactory bulb, respectively. These results strongly suggest that both the Gαolf‐ and Gαo‐mediated signal transduction pathways function in the B. japonicus VNO. The expression of Gαolf in the B. japonicus VNO may correlate with the detection of airborne chemical cues and with a terrestrial life. J. Comp. Neurol. 519:3189–3201, 2011.

Characterization of two strains of avian sarcoma virus isolated from avian lymphatic leukosis virus-induced sarcomas.

Two replication-defective avian sarcoma viruses, S1 and S2, which were independently isolated from tumors of chickens inoculated with avian lymphatic leukosis virus (LLV) were characterized. The genomes of S1 and S2 contain src-related sequences and are, respectively, about 3.9 and 4.5 kilobases long. pp60src-related proteins with molecular weights of 62,000 (p62) were detected in cells infected with these viruses, and protein kinase activity was found to be associated with these proteins. No other viral proteins, such as gag, pol, and env gene products, were detected. These results suggested that the c-src sequence in normal chicken cells was incorporated into LLV genomes by recombination at the expense of most of the viral genes to generate highly defective new sarcoma viruses.

Expression of G proteins in the olfactory receptor neurons of the newt Cynops pyrrhogaster: Their unique projection into the olfactory bulbs

We analyzed the expression of G protein α subunits and the axonal projection into the brain in the olfactory system of the semiaquatic newt Cynops pyrrhogaster by immunostaining with antibodies against Gαolf and Gαo, by in situ hybridization using probes for Gαolf, Gαo, and Gαi2, and by neuronal tracing with DiI and DiA. The main olfactory epithelium (OE) consists of two parts, the ventral OE and dorsal OE. In the ventral OE, the Gαolf‐ and Gαo‐expressing neurons are located in the apical and basal zone of the OE, respectively. This zonal expression was similar to that of the OE in the middle cavity of the fully aquatic toad Xenopus laevis. However, the Gαolf‐ and Gαo‐expressing neurons in the newt ventral OE project their axons toward the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively, whereas in Xenopus, the axons of both neurons project solely toward the MOB. In the dorsal OE of the newt, as in the principal cavity of Xenopus, the majority of the neurons express Gαolf and extend their axons into the MOB. In the vomeronasal organ (VNO), the neurons mostly express Gαo. These neurons and quite a few Gαolf‐expressing neurons project their axons toward the AOB. This feature is similar to that in the terrestrial toad Bufo japonicus and is different from that in Xenopus, in which VNO neurons express solely Gαo, although their axons invariably project toward the AOB. We discuss the findings in the light of diversification and evolution of the vertebrate olfactory system. J. Comp. Neurol. 522:3501–3519, 2014.

Transcriptional regulatory region of Brn-2 required for its expression in developing olfactory epithelial cells

Brain-2 is a class III POU transcription factor expressed in developing nervous system. In this study, we have examined the transcriptional regulatory region of Brn-2. Expression of Brn-2 is activated when P19 embryonal carcinoma cells are induced to differentiate into neural cells with retinoic acid (RA). In P19 cells, the 0.5 kb upstream region of Brn-2 was sufficient for the transcriptional activation during RA-induced differentiation. Deletion analysis of the 0.5 kb region located a proximal enhancer (between -422 and -379 with respect to the translational initiation codon), which was essential for the activation. By gel shift assay and methylation interference assay, a specific binding factor was detected that recognized a core sequence GAGCCAAT found within the proximal enhancer. To examine whether the 0.5 kb upstream region can function in embryos, transgenic mice were generated that contained LacZ gene driven by the 0.5 kb upstream region. In these transgenic mice, LacZ was expressed in developing olfactory epithelial cells between embryonic day 12.5 and 14.5. Immunostaining with an anti-Brain-2 antibody demonstrated the expression of Brain-2 in the olfactory epithelium (most likely olfactory receptor neurons) at similar developmental stages. These results suggest that the 0.5 kb upstream region of Brn-2 is sufficient for the expression in the developing olfactory cells and that the DNA binding factor recognizing the proximal enhancer may be involved in the olfactory cell specific expression.