RESULTS AND DISCUSSION

Trpc2 expression has so far not been reported in any anuran species, so we used RT-PCR to test whether the trpc2 transcript is present in the olfactory organ of X. laevis. The X. laevis genome sequence is not available, and the trpc2 gene sequence was not known. Therefore, we designed degenerate primers based on the trpc2 sequence of Xenopus tropicalis, a species closely related to X. laevis. The primers were designed to target a highly conserved region among different vertebrate species (Fig. 1A). We then performed RT-PCR on the olfactory organ (MOE and VNO) of larval X. laevis

and a 1402 bp fragment was isolated and sequenced (Fig. 1B). In BLAST searches, the obtained sequence (accession no. HG326501, European Nucleotide Archive) showed the X. tropicalis gene as the closest ortholog (90% nucleotide identity). A multi-species alignment (see supplementary material Fig. S1) showed a high degree of similarity between the X. laevis trpc2 fragment and the sequence of diverse vertebrate species (identity: Plethodon shermani 78%, Danio rerio 72%, Mus musculus 72%, Macropus eugenii 72%). Next we analyzed the tissue specificity of the trpc2 gene expression by performing RT-PCR with a second set of primers specific for the X. laevis sequence (see Materials and methods). Amplified products of the expected size were reproducibly found in the larval MOE and the VNO, whereas no signals were detected from the olfactory bulb and other organs, such as the brain, heart and eye (Fig. 1C). In a next step, the expression of trpc2 was examined by in situ hybridization of larval X. laevis tissue sections encompassing both MOE and VNO. Numerous trpc2-positive cells were observed in the epithelia of both the MOE and the VNO (Fig. 2A). This bimodal expression in the two main olfactory organs is different from the situation in all other tetrapods examined so far. Trpc2 expression in salamander (Plethodon shermani), the only non-mammalian tetrapod examined, is limited to the VNO (Kiemnec-Tyburczy et al., 2012), as in all mammals investigated so far (Liberles, 2014).

Xenopus laevis is also peculiar in that some v2rs are expressed in the MOE, whereas other v2rs are expressed in the VNO (Gliem et al., 2013; Syed et al., 2013). Thus, the TRPC2 distribution we report here parallels the distribution of V2Rs. In a terrestrial salamander, v2r expression is confined to the VNO, in other words, it again parallels the trpc2 expression, which is also restricted to the VNO in this species (Kiemnec-Tyburczy et al., 2012). Such co-localization supports the hypothesis that TRPC2 is involved in V2R signal transduction.

To obtain a more stringent criterion of co-localization, we examined the relative height (in basal-to-apical direction) of cells expressing trpc2. This parameter shows a distinct, non-random distribution for cells expressing v2rs in the MOE of Xenopus laevis (Syed et al., 2013) (see also Fig. 2B). Evaluation of more than 300 trpc2-expressing cells showed that the trpc2 gene is expressed in a distinct zone of the MOE that closely resembles the expression zone of v2r genes, particularly v2r-C (Fig. 2C,D), determined in earlier work of our group (Syed et al., 2013). In fact, the epithelial distribution of trpc2 and v2r-C is almost identical, as judged by peak position, half-width, median value and skewness (Fig. 2D). Similar to v2rs (Syed et al., 2013), the medial-to-lateral distribution of trpc2-positive cells within the MOE was uniform with no tendency for lateralization (not shown). Together, these results lead to the hypothesis that in Xenopus, TRPC2 and V2Rs might be present in the same subpopulation of cells.

Recent work of our group showed that a large subpopulation of amino acid odor-sensitive microvillous ORNs of the Xenopus MOE has a phospholipase C- and diacylglycerol-mediated transduction pathway that may couple to TRPC2 (Gliem et al., 2013; Sansone et al., 2014). Microvillous ORNs in the single sensory surface of fishes are also known to be sensitive to amino acid odors and to express v2rs and trpc2 (Sato et al., 2005). In fact, two fish V2Rs, OlfCa1 and OlfCc1, have been shown to be sensitive to amino acid odors (DeMaria et al., 2013; and references therein). Together, these data suggest that in Xenopus, TRPC2 could be involved in mediating the amino acid response of V2Rs, similar to the situation in fishes. Further investigations will be necessary to substantiate this hypothesis.

Our results strengthen the general concept that sensory neurons expressing v2rs and trpc2 may be connected to the detection of non-volatile odors. In fully terrestrial vertebrates (Liberles, 2014), including a terrestrial salamander (Kiemnec-Tyburczy et al., 2012), vomeronasal receptors and trpc2 are solely expressed in the sensory neurons of the VNO, mainly specialized for the detection of large non-volatile molecules. In contrast, in teleost fishes, vomeronasal receptors and trpc2 are expressed in the single sensory epithelium (Sato et al., 2005; Hamdani and Døving, 2007). In the fully aquatic larvae of X. laevis, vomeronasal receptors (Gliem et al., 2013; Syed et al., 2013) and trpc2 (present study) are expressed in both the MOE and VNO. It will be interesting to see whether the correlation of sensory neurons expressing vomeronasal receptors and trpc2 for non-volatile odors holds up in adult X. laevis, in which the larval MOE has metamorphosed into an air nose and a new adult water nose has emerged.

Certainly the results of the present study add to growing evidence that the olfactory regionalization in X. laevis, and very likely also in other aquatic amphibians, is still incomplete. They possess an anatomically segregated vomeronasal system, but their main olfactory system is still very similar to that of teleost fishes, including cellular and genetic components that are already confined to the VNO in fully terrestrial vertebrates. This intermediate segregation of the Xenopus olfactory system results in an excellent model system to study the molecular driving forces governing the evolution of the vertebrate olfactory system.