Detection and localization of the thyroid hormone receptor beta mRNA in the immature olfactory receptor neurons of chum salmon

Thyroid hormone (TH) plays an important role in regulating multiple cellular and metabolic processes, including cell proliferation, cell death, and energy metabolism, in various organs and tissues of vertebrates. It is generally accepted that anadromous Pacific salmon (Oncorhynchus spp.) imprint odorants from their natal stream during their seaward migration, and they then use olfaction to discriminate their natal stream during the spawning migration. Both serum TH levels and the specific binding values of TH in the salmon olfactory epithelium were markedly increased during the seaward migration. However, thyroid hormone receptor (TR) expression in the olfactory epithelium has not been confirmed in vertebrates. We investigated gene expression of TR isoforms in chum salmon (O. keta) by both molecular biological and histochemical techniques. Expression of TRβ mRNA was detected in the olfactory epithelium by reverse transcriptase polymerase chain reaction (RT-PCR). Nucleotide sequencing demonstrated the existence of a remarkable homology between the RT-PCR product and part of the ligand-binding domain of other teleost TRβ isoforms. By in situ hybridization using a digoxygenin-labeled salmon olfactory TRβ cRNA probe, signals for salmon olfactory TRβ mRNA were observed preferentially in the perinuclear regions of immature olfactory receptor neurons (ORNs), as protein gene product 9.5 (PGP9.5)-immunopositive ORNs. Our results provide the first detection of TRβ gene expression in the olfactory epithelium, and suggested the possibility that TRβ may be involved in cell maturation and/or cell differentiation of the ORNs in Pacific salmon.


Introduction
Thyroid hormones (THs) are important regulators of multiple cellular and metabolic processes in various organs and tissues of vertebrates (Mullur et al., 2014). Thyroxine (T4) is the main product secreted by the thyroid follicular cells and is a precursor of the bioactive hormone 3, 3 0 , 5-triiodo-L-thyronine (T3), most of which is produced by outer ring deiodination of T4 in peripheral target tissues (Mullur et al., 2014). TH effects are mediated through thyroid hormone receptors (TRs).
TRs comprise part of a superfamily of nuclear receptors that are characterized by distinct regions or domains for DNA binding (C domain) and ligand-binding (E/F domain), separated by "hinge" regions (D domain) that may target the receptor to the nucleus (Evans, 1988;Lazar and Chin, 1990). TRs bind to thyroid hormoneresponsive elements in the promoter of their target genes to activate or repress transcription through interactions with other nuclear receptors such as retinoic acid receptor or retinoid X receptor (Rivas and Maranjo, 2007). TR isoforms, a and b, are differentially expressed in tissues and have distinct roles in TH signaling (Cheng et al., 2010). Although THs are required for neuronal proliferation, migration, synaptogenesis, and myelination during brain development (Howdeshell, 2002;Horn and Heuer, 2010), understanding of the function of THs in the peripheral olfactory system remains limited. In humans, TH deficiency may contribute to olfactory dysfunction in patients with hypothyroidism (McConnell et al., 1975). Propylthiouracil-treated hypothyroidism in rodents was associated with behavioral olfactory dysfunction Brosvic et al., 1996) and reduced proliferation of olfactory epithelial cells including olfactory receptor neurons (ORNs) (Mackay-Sim and Meisami, 1991, 1994). However, it is unknown which TRs mediate these effects of THs on the peripheral olfactory system of mammals or other vertebrates.
Anadromous Pacific salmon (Oncorhynchus spp.) imprint odorants from their natal streams during their seaward migration (Stabell, 1992), and they then use olfaction to identify their natal stream during their spawning migration (Wisby and Hasler, 1954;Nevitt and Dittman, 1998). Blood T4 levels of salmon gradually increase during the seaward migration and peak just before entering the sea (Dickhoff et al., 1982;Yamauchi et al., 1984). Our previous study indicated that T3-specific binding values markedly increased in the olfactory organs just before entering the sea during the seaward migration of masu salmon (O. masou) using in vitro autoradiography of 125 I -T3 in frozen sections (Kudo et al., 1994). In addition, the olfactory epithelia of T3 intraperitoneally implanted juvenile coho salmon (O. kisutch) indicated high DNA synthesis as detected using a 5-bromo-2 0 -deoxyuridine cell birth-dating technique, and the activation of olfactory cellular proliferation was reported in these tissues (Lema and Nevitt, 2004). Just as in mammals, teleost fishes have multiple TR subtypes (Nelson and Habibi, 2009). Although the number of TRs identified in a given species varies, all teleost TRs are orthologous to either the TR alpha or TR beta of mammals (Johnson and Lema, 2011). The teleost TR alpha and TR beta forms can have distinct patterns of tissue expression and transcriptional regulation (Chen et al., 2014;Johnson and Lema, 2011;Manchado et al., 2009;Lema et al., 2009), suggesting functional differentiation between receptor types. In salmonid fish, TR genes have been detected in various organs (brain, intestine, gill, liver, muscle, kidney, ovary, skin, etc.) of rainbow trout (O. mykiss) (Jones et al., 2002;Raine et al., 2005Raine et al., , 2011, coho salmon (Harada et al., 2008), and Atlantic salmon (Salmo salar) (Marchand et al., 2001). However, TR gene expression in the peripheral olfactory system has not been widely studied in vertebrates, including teleost. Therefore, a critical gap remains in understanding the mechanism whereby THs promote cell proliferation in the peripheral olfactory system.
The main goal of the present study was to examine gene expression of the TRs in the peripheral olfactory system of Pacific salmon and their involvement in regulating the proliferation of ORNs. For this purpose, we performed cloning of a partial TR cDNA from the olfactory organs of chum salmon (O. keta). In addition, to presume the function of TRs in the olfactory organs, we localized TR mRNAs within the olfactory epithelium using in situ hybridization, and compared the localization of TR transcripts to the distribution of proliferating cell nuclear antigen (PCNA) -a marker of newly proliferated cells -and protein gene product 9.5 (PGP9.5) -a commonly used indicator of neuronal cells.

Animals
TR mRNA expression in the chum salmon olfactory epithelia were examined at feeding immature salmon. Fish were collected from the Northwest Pacific Ocean (43 N, 155 E) using a drift gill net deployed from the training ship "Oshoryo- 2092 AE 95.12 g, age: 3e5 years, n ¼ 10]. Fish were immobilized in carbonated water, and then the olfactory organs (i.e., olfactory rosettes) were dissected. The experiments were performed according to "National University Corporation Hokkaido University Provisions on Animal Experiments" which "Hokkaido University Animal Care and Use Committee" approved.

RNA preparation
Dissected tissues were immediately immersed in RNAlater (Ambion, Austin, TX, USA) and stored at À30 C until RNA extraction. Total RNA was prepared from the olfactory rosette using a High Pure RNA Tissue Kit (Roche, Indianapolis, IN, USA) in accordance with the manufacturer's instructions. Poly (A) þ RNA was isolated using oligo (dT)-latex beads (Oligotex-dT30<Super>; Takara, Otsu, Japan) from total RNA.

Oligonucleotides
Oligonucleotides used as PCR primers are shown in Table 1. The degenerate fish TR primer pair (Fish TR-F and Fish TR-R) was designed from consensus sequences of TR found in several teleostean fish species (e.g., Kawakami et al., 2003Kawakami et al., , 2007Kawakami et al., , 2008. b-actin primers (b-actin-F and b-actin-R) were synthesized based on sequences of rainbow trout b-actin cDNA (GenBank accession no. AF254414) (Celius et al., 2000).

Cloning and sequencing of salmon TR cDNA
In brief, poly (A) þ RNA from the olfactory rosettes of immature chum salmon were reverse transcribed using a Random Primer pd (N) 6 and PrimeScript Reverse Transcriptase (Takara, Tokyo, Japan) in accordance with the manufacturer's instructions.
The PCR conditions were: the first cycle, denaturation at 94 C for 3 min, annealing at 55 C for 5 min, and extension at 72 C for 5 min; 35 cycles of incubation, 30 sec at 94 C, 30 sec at 56 C, and 1 min at 72 C; and finally extension at 72 C for 5 min for 3 0 A overhangs using the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). The amplicons were T-A ligated to pCRII-TOPO vector (TOPO TA Cloning Kit; Invitrogen, San Diego, CA) and sequenced. Table 1. Primer sets used for RT-PCR for sequence and expression analysis of chum salmon TRs and b-actin.

Name
Primer sequence DNA sequencing was conducted using a PRISM 3730 DNA analyzer and BigDye Terminator v3.1 (Applied Biosystems). Nucleotide sequence alignment was conducted using a nucleotide basic local alignment tool (blastn) offered by NCBI. The partial cDNA encoding chum salmon trb was deposited in GenBank as accession no. AB669091.

In situ hybridization
After the plasmid vector was linearized, digoxigenin (DIG)-labeled antisense and sense cRNA probes were prepared using T7 and SP6 RNA polymerases with a DIG RNA Labeling Kit (Boehringer Mannheim, Mannheim, Germany) in accordance with the manufacturer's instructions. Olfactory rosette tissues were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) at 4 C for 72 h, and then embedded in Histosec. Then, approximately 5-mm thick sections were prepared.
Serial paraffin sections were digested with 20 mg/mL proteinase K in 10 mmol/L Tris-HCl, 1 mmol/L EDTA (pH 8.0) for 20 min at 37 C, and then treated with 0.2 mol/L HCl for 10 min and 0.25% acetic anhydrate in 0.1 mol/L triethanolamine-HCl (pH 8.0) for 10 min. Hybridization and immunohistochemical detection for hybridized signals were performed using the previously reported method (Kudo et al., 1999). To assess the specificity of the in situ hybridization signals, two negative control procedures were performed. First, RNA was digested in randomly chosen sections with 20 mg/mL RNase A for 30 min at 37 C prior to in situ hybridization. Second, digoxygenin-labeled sense RNA probes were hybridized in parallel to antisense RNA probes in all cases.

Immunohistochemistry
Sections semi-adjacent to those used for the above in situ hybridization were prepared. After deparaffinization and rehydration, the sections were blocked with 0.3% H 2 O 2 in absolute methanol for 20 min to deactivate endogenous peroxidases.
After rinsing with phosphate-buffered saline (PBS), the sections were incubated with antigen-retrieval solution (L.A.B. Solution; Polysciences, Warrington, PA) for 10 min at room temperature, and then rinsed with PBS. Antigen-retrieved sections were incubated with 10% bovine serum albumin in PBS for 60 min at room temperature followed by incubation in a humid chamber with one of the following: rabbit anti-Nile tilapia (Oreochromis niloticus) PGP9.5 recombinant polyclonal antibody (Mochida et al., 2002) at a dilution of 1:1000 in PBS, or mouse anti-rat PCNA monoclonal antibody (PC10; Santa Cruz Biotechnology) at a dilution of 1:50 in PBS for 18 h at 4 C. After rinsing in PBS, sections were reacted using the indirect immunoperoxidase method (Histofine Simple Stain MAX-PO Kit, Nichirei, Tokyo, Japan).
PGP9.5 and PCNA localized the immature ORNs and the proliferating cells, respectively. The peroxidase complex was visualized by treatment with a freshly prepared diaminobenzidine tetrahydrochloride (0.1 mg/mL) solution with 0.01% H 2 O 2 for 5 min at room temperature. The specificities of the above immunoreactivities were confirmed by replacing the primary antibody with a pre-immuno serum for PGP9.5, or a pre-absorption PCNA antibody with antigenic peptide for it. Sections semi-adjacent to those used for the above immunohistochemical analyses were counterstained with Delafield's hematoxylin and eosin staining.

Characterization of a partial salmon TR gene in the olfactory organ
The amplified cDNA fragments of the TR and b-actin mRNAs of the expected sizes (422 and 385 bp, respectively) were detected in the olfactory organ of immature chum salmon using RT-PCR ( Fig. 1; Supplemental data 1). The nucleotide sequence of the partial TR amplicon (AB669091) exhibited high homology (99% and 96%) with the corresponding region of and TRb2 (AB303987 and AF302252) previously examined in coho salmon and Atlantic salmon liver (Harada et al., 2008;Marchand et al., 2001), respectively. This amplicon included the sequence of the ligandbinding domain (E/F domain) in salmonid TR (Marchand et al., 2001). For this reason, the deduced amino acid sequence was identical to salmon TRb. Sequencing resulted in identification of only a single TRb cDNA from the salmon olfactory rosette; TRa in this tissue was not able to detect at the present study.

Expression of TRb mRNA in the olfactory epithelium
Signals of TRb mRNA using a labeled cRNA antisense probe were seen preferentially in the perinuclear regions of the olfactory epithelial cells in the lower twothirds of the middle layer and the upper area of the lower layer in the olfactory epithelium of immature chum salmon ( Fig. 2A and B). In the RNase A pretreated control sections, signals were abolished in all cases (not shown). Hybridization with a labeled sense control probe identified no detectable hybridized signals in adjacent sections to Fig. 2A and B of the olfactory epithelium ( Fig. 2C and D). No clear differences in the localization or signal intensity for TRb were observed between males and females in in situ hybridization examinations. Analysis of semi-serial sections of the olfactory epithelium showed signals of TRb mRNA and immunoreactivities for PGP9.5 and PCNA in immature chum salmon (Fig. 3). Immunoreactivity for PCNA was mainly observed in the globose basal cells and part of horizontal basal cells (Fig. 3C). Immunoreactivities for PGP9.5 were observed in the perinuclear regions of the immature ORNs in the lower two-thirds of the middle layer and the upper area of the lower layer, and the dendrites of these neurons (Fig. 3D). Both signals of TRb mRNA and immunoreactivity for PGP9.5 overlapped in the perinuclear regions of immature ORNs (Fig. 3B and D). Horizontal basal cells did not show either positive reaction for TRb mRNA and PGP9.5 near the basal lamina. The above immunoreactivities were not detected with the pre-immuno antibody or the preabsorbed antibody with antigenic peptide.

Discussion
Transcripts encoding a TRb were identified for the first time in the peripheral olfactory system of a vertebrate. These results indicated that the olfactory organ was one of target tissues for THs in chum salmon. RT-PCR analysis in the present study revealed the presence of TRb mRNA in the olfactory organs of chum salmon, but the expression of TRa mRNA was not detected. Previous studies indicated that TRb was highly expressed in the brain and pituitary of teleostean fish, Japanese conger eel (Conger myriaster) (Kawakami et al., 2003), and Japanese eel (Anguilla japonica) (Kawakami et al., 2007). The present partial TR sequence from chum salmon olfactory organs exhibited high homology to coho salmon TRb isoform (Harada et al., 2008), and was identified as an ortholog of fish TRb. The TRa and b subtypes are the products of two distinct genes, whereas the two TRb isoforms (TRb1and TRb2) are splice variants from the same gene. In mammals, TRb1 is widely expressed and is particularly abundant in the liver and kidney (Lazar and Chin, 1990). Conversely, in mammals TRb2 is most highly expressed in the pituitary and hypothalamus (Hodin et al., 1989;Cook et al., 1992). However, the present partial TRb amplicon was not able to distinguish TRb1 or TRb2 isoforms. Because, the deduced amino acid sequence of this amplicon was a region in which TRb1 and TRb2 hold the same amino acid sequence in coho salmon (Harada et al., 2008).
Histological examination revealed that a similar pattern of localization of TRb mRNA and PGP9.5-immunoreactivity to in the lower two-thirds of the middle layer and the upper area of the lower layer of the olfactory epithelium. In mammals, PGP9.5 is expressed in ORNs over a broad range of developmental stages, including immature ORNs (Taniguchi et al., 1993). In teleosts, Nile tilapia ORNs expressed a ubiquitin C-terminal hydrolase (EC 3.4.19.12) homologous to PGP9.5 (Mochida et al., 2002). Moreover, our previous immunohistochemical study revealed that the immunoreactivities for PGP9.5 were located in the cytosol of immature ORNs of lacustrine sockeye salmon (O. nerka) (Kudo et al., 2009). PCNA is an intranuclear polypeptide synthesized in the late G1 and S phase (Mathews et al., 1984). The localizations of TRb-expressing cells and PCNA-immunoreactive basal cells differed in the olfactory epithelia. These findings strongly suggested that TRb is expressed in immature ORNs in salmon olfactory epithelia (Fig. 4). The differentiation period from basal cell to mature ORN was reported to be about 1 week in mouse and salmon using in vivo 3 H-thymidine (Graziadei and Monti-Graziadei, 1979) and 5-bromo-2 0deoxyuridine (Yanagi et al., 2004) labeling experiments, respectively. Based on the detection of TRb only in the salmon olfactory rosette and the localization pattern of TRb mRNA within the epithelium, it is possible that TRb may mediate the effects of THs on cell maturation and/or cell differentiation of ORNs during this period in the olfactory organs of Pacific salmon. In fact, a previous report indicated that THs have an essential role in the maturation of ORNs in developing rats (Paternostro and Meisami, 1996). In mammalian brains, TH controls the expression of many genes encoding proteins with roles in cell differentiation, such as cytoskeletal proteins, neurotrophins, neurotrophin receptors, cell cycle regulators, and extracellular matrix proteins (Bernal, 2015). Presumably also in salmon ORNs, THs may regulate the transcription of these proteins via TRs as well as mammalian neurons, but direct support for this assumption was not available.
Filial imprinting is caused by visual stimuli, and it has been demonstrated that precocial chicks visually imprint on the first conspicuous moving object, and the visual stimulus then elevated THs in the chick's brain thereby initiating the imprinting phase (Yamaguchi et al., 2012). The relationship between the blood T4 PCNA: proliferating cell nuclear antigen, PGP9.5: protein gene product 9.5. concentration peak and the onset of seaward migration (i.e., olfactory imprinting period) has been reported in juvenile chum salmon (Ojima and Iwata, 2007) and in parr-smolt transformation of coho salmon (Dittman and Quinn, 1996). More recently, thyrotropin-releasing hormone gene expression in hatchery-reared juvenile chum salmon brain increased immediately after release from a hatchery into the natal stream, and the expression of the essential NR1 subunit of the N-methyl-D-aspartate receptor increased during downstream migration (Ueda et al., 2016). In addition, oral T4 administration experiments in the same stage fish enhanced NR1 gene activation (Ueda et al., 2016). These results suggested that olfactory memory formation during juvenile seaward migration of chum salmon was controlled by THs in the central nervous system. THs that increase during downstream migration may also act on the peripheral olfactory system, and may be increasing the sensitivity of olfaction for their natal stream odorants.
The present study is the first to describe the detection and localization of TRb mRNA in the vertebrate olfactory epithelium. These findings suggested the possibility that TRb may be involved in cell maturation and/or cell differentiation of ORNs in Pacific salmon.

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