In situ hybridization analysis of the expression of class I and II odorant receptors in the olfactory organ of the African spurred tortoise (Centrochelys sulcata)

doi:10.21203/rs.3.rs-3558622/v1

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In situ hybridization analysis of the expression of class I and II odorant receptors in the olfactory organ of the African spurred tortoise (Centrochelys sulcata)

https://doi.org/10.21203/rs.3.rs-3558622/v1

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In this study, in situ hybridization was used to determine the localization of odorant receptor (OR)-expressing cells in the olfactory organ of the African spurred tortoise (Centrochelys sulcata). The olfactory organ of turtles generally consists of an upper chamber epithelium (UCE) containing glands and a lower chamber epithelium (LCE) devoid of glands. The UCE is considered an air-nose that is receptive to airborne odorants, whereas the LCE is considered a water-nose that is receptive to waterborne odorants. The ORs are a family of olfactory receptors categorized into classes I and II based on their sequence similarities. Class I ORs are thought to be receptive to water-soluble odorants, and class II ORs to volatile odorants. However, where these receptors are expressed within the olfactory organ in turtles remains unclear. This study was performed to analyze the distribution of OR-expressing cells in the olfactory organ of C. sulcata. Class II ORs were revealed to be expressed in the UCE, implying that the UCE of C. sulcata functions as an air-nose. In contrast, class I ORs were expressed in both the UCE and LCE, implying that both of these epithelia function as a water-nose in the olfactory organ of C. sulcata. Furthermore, double-labeling experiments indicated that class I ORs are not co-expressed with the transcription factor B-cell lymphoma/leukemia 11b (Bcl11b), implying that the regulatory mechanism by which Bcl11b suppresses class I OR expression is conserved in C. sulcata. Further studies are needed to clarify the functional significance of the expression of class I ORs in the entire olfactory organ and to determine whether this is a trait shared among terrestrial turtles or found only in one species of tortoise that inhabits arid regions.

anatomy

nose

olfactory receptor

sense organ

turtle

The olfactory organ of turtles generally consists of an upper chamber epithelium (UCE) and a lower chamber epithelium (LCE). The UCE and LCE are situated dorsally and ventrally within the nasal cavity, respectively, and are separated by a non-sensory epithelium. Both the UCE and LCE are composed of olfactory receptor neurons (ORNs), supporting cells, and basal cells. The ORNs are further classified as either ciliated or microvillous according to the fine structure of their dendritic tips [1–3]. In many turtles, the UCE contains ciliated ORNs and the LCE contains microvillous ORNs. The UCE is regarded as the olfactory epithelium and the LCE as the vomeronasal organ. This is extrapolated from the fact that in mammals, ciliated ORNs are contained in the olfactory epithelium and microvillous ORNs are present in the vomeronasal organ. Furthermore, the UCE and LCE are distinguished by the presence or absence of associated glands, which the UCE contains but the LCE lacks. Based on the presence or absence of associated glands and their topographic arrangement within the nasal cavity, the UCE is regarded as an air-nose that is receptive to odorants in the air, and the LCE is regarded as a water-nose that is receptive to odorants in water [4, 5].

Turtles are classified into freshwater turtles, sea turtles, and terrestrial turtles according to their habitats. The African spurred tortoise Centrochelys sulcata is a terrestrial turtle that inhabits arid regions [6]. Like many turtles, its olfactory organ consists of both a UCE and an LCE. The UCE contains ciliated ORNs and associated glands, whereas the LCE contains microvillous ORNs and is devoid of associated glands (Fig. 1). The olfactory organ of C. sulcata is characterized by an LCE of smaller size than the UCE. Whereas the UCE lines the entire upper chamber, the LCE occupies only the anterior ventromedial wall of the nasal cavity, and the lower chamber is mostly lined by a nonsensory epithelium.

Odorant receptors (ORs) are a family of olfactory receptors that are expressed in the olfactory epithelium of many vertebrates. The ORs are seven-transmembrane receptors that are categorized as class I and II based on their sequence similarities [7]. In the olfactory organ of the African clawed frog Xenopus laevis, class I ORs are expressed in the middle chamber, whereas class II ORs are expressed in the principal chamber [8]. Because African clawed frogs perceive odorants within water in the middle chamber and odorants within air in the principal chamber, it has been suggested that class I ORs are receptive to water-soluble odorants and class II ORs to volatile odorants [9].

Previous studies have shown that turtles express ORs in both the UCE and LCE [3, 10–12]. We recently examined the distribution of OR-expressing cells in the olfactory organ of the green sea turtle Chelonia mydas and demonstrated the expression of class I ORs in the LCE and class II ORs in both the UCE and the LCE [13]. No relationship has been established between the olfactory receptor expression and the ecology of turtles. However, the expression of class II ORs, which have been suggested to be receptive to volatile odorants, throughout the olfactory organ has been assumed to be correlated with the ecology of sea turtles, which spend most of their lives underwater and do not come ashore except when laying eggs. In contrast, where class I and II ORs are expressed within the olfactory organ of terrestrial turtles remains unknown. Therefore, we examined the distribution of OR-expressing cells in the olfactory organ of C. sulcata to identify any differences from the distribution in sea turtles.

B-cell lymphoma/leukemia 11B (Bcl11b), a transcription factor involved in the regulation of OR gene expression, is expressed throughout the olfactory epithelium of mice except the dorsomedial portion [14]. It represses the expression of class I ORs, allowing class II ORs to be expressed in all but the dorsomedial portion of the mouse olfactory epithelium [15]. In the African clawed frog X. laevis, Bcl11b is expressed in the principal chamber where class II ORs are expressed, but not in the middle chamber where class I ORs are expressed [15]. Because Bcl11b is expressed in the UCE in the olfactory organ of turtles [16], we further examined whether the Bcl11b-mediated regulation of OR gene expression found in mammals and amphibians is conserved in the olfactory organ of C. sulcata.

Animals

All animal handling and tissue harvesting procedures were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the Guide for the Care and Use of Experimental Animals at Iwate University. Eggs and hatchlings of C. sulcata were provided by the Ishikawa Zoo. Embryos (n = 5) and hatchlings (n = 4) were decapitated under anesthesia induced by intraperitoneal injection of sodium pentobarbital.

Identification and classification of C. sulcata ORs

The olfactory organ was dissected from an embryo and homogenized with ISOGEN (Nippon Gene, Tokyo, Japan) to extract total RNA, which was digested with DNase I (Takara Bio, Shiga, Japan) to remove contaminating genomic DNA. RNA sequencing (RNA-seq) was performed on the olfactory organ as previously described [17]. Briefly, total RNA extracted from the olfactory organ of C. sulcata was subjected to RNA-seq using NovaSeq 6000 sequencers (Illumina, San Diego, CA, USA) after constructing a sequence library using the TruSeq RNA kit (Illumina). Construction of the library and sequencing were performed by Macrogen Japan (Tokyo, Japan) (https://macrogen-japan.co.jp/). The sequence reads were deposited in the DNA Data Bank of Japan Sequence Read Archive (accession number: DRAXXXXX). De novo transcriptome assembly was performed using Trinity PE [18] after quality checking and filtering. The curated contig sequences were then subjected to exploration of the ORs of C. sulcata. ORs were identified from the contigs using previously identified intact ORs [19, 20] as queries. The series of identification processes was performed using the FATE program (https://github.com/Hikoyu/FATE). Briefly, previously identified intact ORs [19, 20] were used as a query for performing a tBLASTn search, and this was followed by coding region prediction using GeneWise v2.4.1 [21]. Next, the output genes were confirmed as a homolog of known ORs by a BLASTX search against the Uniref50 database.

To classify the OR-like genes of C. sulcata, we first aligned them with previously classified ORs [19, 20] using MAFFT with the --linsi option (v7.505; [22]), and a phylogenetic tree was constructed to display the orthology with known OR subfamilies using RAxML-NG v1.0.1 [23] using the JTT + G4 model (which was selected by ModelTest-NG [24, 25] based on the corrected Akaike information criterion score). Finally, eight class I ORs and 61 class II ORs were identified as intact ORs. Of the identified genes, five class I ORs (CSU02, CSU03, CSU04, CSU05, and CSU06) and ten class II ORs (CSU08, CSU09, CSU10, CSU11, CSU13, CSU14, CSU15, CSU16, CSU17, and CSU18) were chosen as representatives of C. sulcata ORs (Fig. 2). A gene tree of all 69 African spurred tortoise ORs was also constructed using the method described above, along with 690 intact ORs of Carettochelys insculpta identified in our previous study [26].

Probe preparation

Complementary DNA was synthesized from the total RNA with a ReverTra Ace kit (Toyobo, Osaka, Japan) using oligo dT primers according to the manufacturer’s protocol. DNA fragments of ORs were amplified from the olfactory organ complementary DNA using ExTaq (Takara Bio) with the primer pairs listed in Table 1. All DNA fragments were inserted into a pCRII TOPO vector using a TOPO TA Cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) and confirmed by sequencing using a BigDye Terminator v3.1 Cycle Sequencing Kit and a 3730xl DNA analyzer (Thermo Fisher Scientific). Digoxigenin (DIG)-labeled RNA probes were synthesized from linearized plasmids using a SP6/T7 DIG RNA labeling kit (Sigma-Aldrich, St. Louis, MO, USA), treated with DNase and ethylenediaminetetraacetic acid, precipitated with ethanol, and dissolved in water. The probes were aliquoted and stored at − 80°C until use. Probes against partial sequences of olfactory marker protein (OMP) (XM_037884963) and Gαolf (XM_037892876), the alpha subunit of G-protein coupled to ORs, of the green sea turtle were also used in this study. These probes were prepared using the following primers: OMP forward primer (5′-AGCTTGAGTTCCCCTTCGTC-3′), OMP reverse primer (5′-CTACGGAGCACTTGAGGTGG-3′), Gαolf forward primer (5′-CAGAAGCGGGAGTACAAGCA-3′), and Gαolf reverse primer (5′-TAGCTGCTACAGGCCACAAC-3′) [13].

Tissue preparation

Olfactory organs were dissected under a stereomicroscope, fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) (pH 7.4), and decalcified in 10% ethylenediaminetetraacetic acid in 0.1 M PB. After cryoprotection in 10%, 20%, and 30% sucrose in 0.1 M PB, the tissues were embedded in O.C.T. Compound (Sakura Finetek, Torrance, CA, USA), sectioned at 20-µm thickness, and thaw mounted onto MAS-coated glass slides (Matsunami, Osaka, Japan). The sections were air-dried and stained with hematoxylin–eosin for general histological examination or processed for in situ hybridization.

In situ hybridization

In situ hybridization was performed as described previously [17]. Briefly, the sections were fixed in 4% PFA for 10 min at room temperature, treated with 10 µg /mL proteinase K for 15 min at 37ºC, and washed with phosphate-buffered saline containing 0.2% glycine. The sections were then fixed again with 4% PFA and incubated in 0.25% acetic anhydrase in 0.1 M triethanolamine (pH 8.0) for 15 min. After washing in 4× saline sodium citrate (SSC), the sections were incubated in 50% formamide in 2× SSC for 30 min. Hybridization was performed in the hybridization buffer ISHR7 (Nippon Gene) overnight at 55°C. Post-hybridization washing was performed in 50% formamide in 2× SSC for 1 h and 0.1× SSC for 2 h at 55°C. Signals were detected using anti-DIG antibody coupled to alkaline phosphatase and 4-nitroblue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl-phoshate (BCIP) stock solution (Sigma-Aldrich). Because the results obtained from hatchlings were identical to those obtained from embryos, all photomicrographs shown in this paper are of embryos. Negative control experiments using sense probes yielded no specific signals.

Combined in situ hybridization and immunohistochemistry

Combined in situ hybridization for ORs and immunohistochemistry for Ctip2 (Bcl11b) were carried out according to a previous study [27] with partial modification. First, sections were hybridized with DIG-labeled OR probes, and signals for the OR probes were stained dark purple with alkaline phosphatase-labeled anti-DIG antibody and NBT/BCIP stock solution as described above. After inactivation of endogenous peroxidase with 0.3% hydrogen peroxide in methanol, the sections were incubated overnight at 4ºC with an anti-rat Ctip2 antibody (ab18465; Abcam, Cambridge, UK), a rat monoclonal antibody to a recombinant protein corresponding to 1–150 amino acids of human Ctip2 (Bcl11b) that was used to detect Ctip2 (Bcl11b) in turtle tissues in a previous study [28], and then incubated with biotinylated donkey anti-rat antibody for 2 h at room temperature. After incubation with streptavidin-conjugated horseradish peroxidase, a TSA Biotin System (NEL700A001KT; PerkinElmer, Waltham, MA, USA) was used for amplification of immunosignals. Following washing, immunosignals for Bcl11b were stained brown with a VECTASTAIN Elite ABC kit (Vector Laboratories, Newark, CA, USA) and diaminobenzidine substrate. Images of signals in the same field of view were taken sequentially after each color reaction.

RNA-seq and tBLASTn searches identified 69 ORs expressed in the olfactory organ of C. sulcata. To classify these genes into class I or II, a phylogenetic tree was constructed using previously classified ORs [19, 20], and the genes were classified according to monophyly with the existing subfamilies. Consistent with the previous studies, the ORs were extensively duplicated in a species-specific manner. The 69 ORs of C. sulcata were classified into eight class I and 61 class II ORs (Additional Files 1, 2). Figure 2 shows a gene tree of the 69 ORs with 690 ORs of the pig-nosed turtle identified in our previous study [26]. Several ORs were chosen from each of the class I and II ORs of the phylogenetic tree to design the probes for in situ hybridization (Fig. 2, Table 1).

In situ hybridization revealed that OMP, encoding a marker for ORNs [29], was expressed by cells ubiquitously distributed in the middle to basal layers of both the UCE and the LCE (Fig. 3). In both the UCE and the LCE, the Gαolf probe showed broad labeling extending throughout the sensory epithelia defined by OMP expression (Fig. 3). These observations imply the expression of ORs in most ORNs of the UCE and LCE.

All ten class II ORs examined, including CSU08 (Fig. 4), CSU09, CSU10, CSU11, CSU13, CSU14, CSU15, CSU16, CSU17, and CSU18 (Fig. 5, Supplementary Fig. S1), were expressed in the UCE, with little expression in the LCE. In contrast, all five class I ORs examined, including CSU02 (Fig. 4), CSU03, CSU04, CSU05, and CSU06 (Fig. 6), were expressed both in the UCE and LCE.

In the turtle olfactory organ, Bcl11b is expressed in the UCE but not in the LCE [16]. If the Bcl11b-mediated regulation of OR gene expression seen in mammals and amphibians is conserved in reptiles, then class I ORs should be expressed only in the LCE in the turtle olfactory organ. Nevertheless, class I ORs were found to also be expressed in the UCE in C. sulcata. Thus, we next examined whether the UCE of C. sulcata, which expresses class I ORs, contains ORNs that do not express Bcl11b or whether the expression of class I ORs in C. sulcata is not regulated by Bcl11b.

As described earlier, OR in situ hybridization and double staining with Bcl11b antibody was performed among the class I OR-expressing cells and class II OR-expressing cells distributed in the UCE of C. sulcata. A positive Bcl11b antibody reaction was observed in the nuclei of class II OR-expressing cells (Fig. 7) but not in the nuclei of class I OR-expressing cells (Fig. 8).

The number of ORs identified in this study (eight class I ORs and 61 class II ORs) is roughly consistent with the number of OR genes obtained by polymerase chain reaction (PCR) cloning from turtle genomic DNA [30], less than the number of turtle OR genes identified by genome sequencing [31–33], and less than the number of pig-nosed turtle OR genes (375 class I ORs and 315 class II ORs) that we recently reported [26]. This is probably because the OR genes expressed in the olfactory organ of C. sulcata were identified by RNA-seq, and it is assumed that the OR genes revealed in this study are only a portion of all OR genes.

The sequences of the reverse-transcription PCR products amplified by five pairs of primers (CSU02, CSU03, CSU05, CSU09, and CSU17) were not identical to those identified by RNA-seq, although they showed > 80% similarity with the corresponding genes (Additional File 3). The slight inconsistency in OR sequences between the RNA-seq and reverse-transcription PCR products may have been due to low expression and consequent improper assembly of the RNA-seq reads.

In the present study, in situ hybridization analysis revealed that class I ORs of C. sulcata were expressed in both the UCE and the LCE, whereas class II ORs were expressed solely in the UCE. The expression of class II ORs, which have been suggested to be receptive to volatile odorants, in the UCE suggests that airborne odorants are received in the UCE of C. sulcata. Meanwhile, the expression of class I ORs, which have been suggested to detect water-soluble odorants, in the entire olfactory organ of C. sulcata suggests that waterborne odorants are received not only in the LCE but also in the UCE. Based on the findings of previous studies, the UCE with associated glands is regarded as an air-nose that is receptive to airborne odorants, and the LCE, lacking associated glands, is regarded as a water-nose that is receptive to waterborne odorants [4, 5]. This assumption is supported by the observations that the LCE is larger than the UCE in highly aquatic turtles such as soft-shelled turtles [34] and the LCE is smaller than the UCE in terrestrial turtles such as C. sulcata. However, the present study demonstrated that in the olfactory organ of C. sulcata, class I ORs are expressed in both the UCE and LCE and class II ORs are expressed in the UCE. By contrast, in the olfactory organ of the green sea turtle C. mydas, class I ORs are expressed in the LCE and class II ORs are expressed in both the UCE and LCE, suggesting that odorants within air are received not only in the UCE but also in the LCE [13]. According to these findings, the anatomy-based assumption that the UCE is an air-nose and the LCE is a water-nose may be an oversimplification that does not necessarily apply to all turtles.

The expression of class I ORs, which have been suggested to be receptive to water-soluble odorants, in the entire olfactory organ of C. sulcata may be related to the habit of terrestrial turtles to suck water through the nose for ingestion. Terrestrial turtles use their nose to drink water from shallow puddles, including those that are too shallow to drink from using their mouth. However, the influx of water into the nasal cavity through the external nostrils is not restricted to terrestrial turtles; it is also observed in freshwater turtles and sea turtles. Thus, the functional significance of the expression of class I ORs in the entire olfactory organ remains elusive. Additionally, further investigation is needed to determine whether the distribution of class I OR-expressing cells throughout the olfactory organ is common to terrestrial turtles or is found only in one species of turtles living in arid regions.

How the diversity in the expression of ORs among turtle species arises remains unknown. One possibility is that although ORNs contained in the UCE and LCE are potentially capable of expressing both class I and II ORs, class I ORs are expressed solely in the LCE of C. sulcata as a result of suppression of class II OR expression in the LCE, whereas class II ORs are expressed solely in the UCE of the green sea turtle as a result of suppression of class I OR expression in the UCE. The mechanism by which ORNs select one among many OR genes in the genome for expression remains unclear [35]. This study showed that Bcl11b is co-expressed with class II ORs but not with class I ORs in the UCE of C. sulcata. These results suggest that the mechanism by which Bcl11b regulates (suppresses) the expression of class I ORs is conserved in the olfactory organ of C. sulcata, as in other turtles. Additionally, in the olfactory organ of C. sulcata, expression of class II ORs was restricted to the UCE and was not found in the LCE in the present study. Unlike class I ORs, mechanisms regulating the expression of class II OR genes are currently unknown and are the subject of future research.

In this study, transcriptome analysis identified 69 OR genes, including eight class I and 61 class II ORs, expressed in the olfactory organ of the terrestrial turtle C. sulcata. By analyzing the expression sites of ORs in the olfactory organ of C. sulcata, we found that class I ORs are expressed in both the UCE and LCE, whereas class II ORs are expressed solely in the UCE. These observations are in marked contrast to what we reported previously in the olfactory organ of the sea turtle C. mydas, suggesting that differences in the habitats of turtle may be correlated with the expression site of ORs in the olfactory organ.

LCE

lower chamber epithelium

ORs

odorant receptors

ORNs

olfactory receptor neurons

UCE

upper chamber epithelium.

Ethics approval and consent to participate

This study was approved by the Animal Ethics Committee of Iwate University (Approval no. A201720; A202143-2).

Consent for publication

Not applicable

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Funding

This research did not receive any specific grant from funding, agencies in the public, commercial, or not-for-profit sectors.

Authors' contributions

NN designed the project. SN and ZZ performed investigation. MN and NN wrote the manuscript and prepared figures. HN provided the experimental materials. TY and YY provided overall supervision. All authors read and agreed to the final version of the manuscript.

Acknowledgements

The authors thank Ishikawa Zoo (Ishikawa, Japan) for providing eggs and hatchlings of African spurred tortoise Centrochelys sulcata, Single-cell Genome Information Analysis Core (SignAC) at WPI-ASHBi (Kyoto University, Japan) for providing the computing resource, and Haruka Sasaki (Iwate University, Japan) for technical assistance with in situ hybridization.

Authors' information (optional)

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Table 1 is available in the Supplementary Files section.

Additionalfile1.txt
Additional file 1 Nucleotide sequences of C. sulcata class I ORs. Eight class I ORs were identified by RNA-seq.
Additionalfile2.txt
Additional file 2 Nucleotide sequences of C. sulcata class II ORs. Sixty-one class II ORs were identified by RNA-seq.
Additionalfile3.txt
Additional file 3 The DNA fragment sequences amplified by primer pairs CSU02, CSU03, CSU05, CSU09, and CSU17, and multiple sequence alignments with their close relatives.
SupplementaryFigureS1231025.jpg
Additional file 4 Supplementary Fig. S1. Class II ORs are expressed solely in the UCE of C. sulcata. In situ hybridization analyses with probes specific to CSU14-CSU18 (class II ORs). Transverse sections through the rostral, middle, and caudal regions of the olfactory organ. Dorsal is up. Labeled cells are shown as blue circles or asterisks. Scale bars: 1 mm.
Table1231023.xlsx
Table 1 Primers for African spurred tortoise Centrochelys sulcata odorant receptors

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In situ hybridization analysis of the expression of class I and II odorant receptors in the olfactory organ of the African spurred tortoise (Centrochelys sulcata)

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Abstract

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Background

Materials and methods

Animals

Probe preparation

Tissue preparation

In situ hybridization

Combined in situ hybridization and immunohistochemistry

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Table 1

Supplementary Files

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Version 1