Journal of Oceanology and Limnology   2020, Vol. 38 issue(1): 294-305     PDF
Institute of Oceanology, Chinese Academy of Sciences

Article Information

SHANG Xiaomei, MA Aijun, WANG Xin'an, XIA Dandan, ZHUANG Jiao
Isolation, characterization and expression analysis of TRPV4 in half-smooth tongue sole Cynoglossus semilaevis
Journal of Oceanology and Limnology, 38(1): 294-305

Article History

Received Nov. 6, 2018
accepted in principle Feb. 26, 2019
accepted for publication Mar. 22, 2019
Isolation, characterization and expression analysis of TRPV4 in half-smooth tongue sole Cynoglossus semilaevis
SHANG Xiaomei1,2,3, MA Aijun1,2, WANG Xin'an1,2, XIA Dandan1,2, ZHUANG Jiao1,2     
1 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences; Shandong Key Laboratory of Marine Fisheries Biotechnology and Genetic Breeding; Qingdao Key Laboratory for Marine Fish Breeding and Biotechnology, Qingdao 266071, China;
2 Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology(Qingdao), Qingdao 266237, China;
3 College of Marine and Environmental Sciences, Tianjin University of Science and Technology, Tianjin 300457, China
Abstract: The transient receptor potential vanilloid 4 (TRPV4),another Ca2+ entry channel,belongs to the vanilloid subfamily and responds to a number of different physical and chemical stimuli and exists widely in mammals. However,our understanding of the TRPV4 in fish remains poor. Therefore,we studied the TRPV4 gene from Cynoglossus semilaevis,named CsTRPV4 that encodes a putative protein of 870 amino acids common in structure and characteristic of mammalian TRPV4,including the domains of ANK repeats,six TM,TRP domain,and CaMBD. The CsTRPV4 was expressed ubiquitously in examined tissues:higher expression in the heart,spleen,testis,and eye,but lower expression in kidney and liver. Surprisingly,the expression of CsTRPV4 in lateral line was significantly higher than in many other tissues as the CsTRPV4 was expressed significantly in the free neuromasts. In addition,CsTRPV4 in the free neuromast from the larval fish was significantly expressed in the hair cells of the free neuromasts. Therefore,the free neuromasts can act as a mechano-sensor to the mechanical stimulation in molecular level in C. semilaevis,which lays a foundation for further study of the functions of the free neuromasts.
Keywords: transient receptor potential vanilloid 4 (TRPV4)    Cynoglossus semilaevis    gene expression    free neuromasts    in situ hybridization    

Transient receptor potential vanilloid 4 (TRPV4) was identified in Caenorhabditis elegans in OSM-9 mutants (Colbert et al., 1997; Liedtke et al., 2003); it was the first number of the TRPV (vanilloid) subfamily described in mammals (Wissenbach et al., 2000).

The TRP superfamily is composed of seven protein subfamilies (Clapham, 2003; Pedersen et al., 2005; Eid and Cortright, 2009). The TRP superfamily is mainly involved in the mechanical and chemical senses. TRPM5 is a key protein for sensing bitter, sweet and umami (Zhang et al., 2007). TRPV1 and TRPV2 play a major role in the nociceptive perception of nociceptive stimuli (Caterina et al., 1999, 2000). TRPA1 is one of highly-expressed sensory neurons in hair cells of the inner ear. When the gene was mutated or knocked out in mice, it would cause some mechanical sensitive behavior defects just like the head contraction and the nasal tip contact response (Kwan et al., 2009; Rui and Xu, 2010). The TRPV subfamily is involved in mechano- and osmotransduction (O'Neil and Heller, 2005; Liedtke, 2007). In particular, TRPV4 is a well-recognized mechanoreceptor, apparently playing important roles in sensing physical and chemical stimuli, including warm temperature, hypotonicity, mechanical stimuli, and endogenous lipids (Garcia-Elias et al., 2014).

Although animal TRPV4s have been studied extensively, information concerning fish TRPV4 is scarce. At present, studies of TRPV4 in bony fish have been conducted in three species, zebrafish (Danio rerio) (Mangos et al., 2007), Nile tilapia (Oreochromis niloticus) (Watanabe et al., 2012), and European sea bass (Dicentrarchus labrax) (Bossus et al., 2011).

The half-smooth tongue sole (Cynoglossus semilaevis) is a rare commercial marine fish species in China (Sha et al., 2011). It lives in sandy or muddy seabed areas (Ma et al., 2006). C. semilaevis sensory system is well developed (Ma et al., 2007a, b ). There is no information about the mechanical and chemical sensor genes in this type of fish. We investigated the pattern of TRPV4 expression during the adult life of half-smooth tongue sole. Our analysis may offer new insight into the sensory functions of TRPV4.


Half-smooth tongue sole specimens were sampled from healthy two-year-old fish (size: 48±1.20 cm; weight: 750±2.265 g) and larvae at one-day posthatching (dph) (size: 1.5±1.125 cm; weight: 0.1±0.050 g) provided by the Mingbo Aquaculture Company (Yantai, China).

2.2 Tissues sampling and RNA extraction

All experiments were carried out with the approval of the UK Home Office Regulatory Requirements and the local Ethics Committee. Fish were anesthetized by speed-freezing and were decapitated after blood sampling.

Three two-year-old half-smooth tongue sole specimens were fasted prior to tissue collection. Scissors were first used to separate the abdomen, and forceps were used to remove organs and tissues. Six sensory tissues (canal neuromasts, free neuromasts, pharynx, olfactory sac, eye, and skin) and nine normal tissues (brain, gill, heart, liver, intestine, spleen, kidney, testis, and muscle) were sampled, stored at -80℃ until RNA extraction.

RNA was extracted using RNAprep pure Tissue Kit (TIANGEN). First-strand cDNA was synthesized directly from the extracted RNA using oligo (dT) 18 primer with Reverse Transcription Reagents (Takara), according to the manufacturer's instructions. The resulting cDNA was used as the template for quantitative reverse transcription polymerase chain reaction (qRT-PCR).

2.3 Full-length cDNA isolation of Cynoglossus semilaevis (CsTRPV4)

One of CsTRPV4 cDNA fragment was amplified from the adult liver using the primer pair F and R and ExTaq DNA polymerase (TaKaRa Code No. RR001A, Japan). A pair of degenerate primers F and R was designed based on the conserved amino acid sequence of the TRPV4 proteins (Table 1). PCR was carried out as follows: 95℃ for 5 min; 35 cycles of 95℃ for 30 s, 58℃ for 60 s and 72℃ for 30 s; plus a final extension step at 72℃ for 7 min. To obtain the 5′ and 3′ cDNA ends of CsTRPV4, 5′- and 3′- rapid amplification of cDNA ends (RACE) was performed using the SMART RACE cDNA Amplification Kit (Clontech), according to the manufacturer's instructions. Two specific primers (RACE5′ and RACE3′), based on the sequence of the initial cDNA fragment, were used for 3′ and 5′ RACE (Table 1). The cDNA fragments were purified using a TIANgel Midi Purification Kit (TIANGEN), ligated into vector pMD18-T (TaKaRa, Dalian), and then propagated in E. coliTop10 cells. Ten recombinant plasmids for the 3′ and 5′ RACE products were sequenced (Sangon, Shanghai, China). Fusion of the 3′ and 5′ RACE fragments generated the full-length cDNA of CsTRPV4. Analysis of the whole sequence used the DNAMAN software. We downloaded representative sequences from NCBI (Table 2) and employed DNAMAN to align them and to construct a phylogenetic neighbor-joining tree. Bootstrap resampling was repeated 1 000 times to construct cluster stability, and bootstrap values > 50% were shown near nodes.

Table 1 Primers used for amplification of full-length CsTRPV4 gene, for qRT-PCR, and for semi- qRT-PCR
Table 2 Amino acid identity comparison of CsTRPV4 with other Trpv4 homologs
2.4 CsTRPV4 expression analysis

To analyze the expression pattern of CsTRPV4 in different tissues, qRT-PCR was performed using the first-strand cDNAs as templates. The primer pair F1 and R1 were designed from the full-length cDNA of CsTRPV4 as detection primer. The qRT-PCR reactions were performed using an ABI 7500 real-time PCR system (Applied Biosystems). SYBR Premix ExTaq (TaKaRa, Dalian) was used with a primer concentration of 200 nmol/L, in a 20-μL reaction mixture containing 10 μL SYBR Premix, 0.4 μL ROX Reference Dye II, 1 μL cDNA, 7.8 μL nuclease-free water, and 0.4 μL of forward and reverse primers. The expression of β-actin was used as endogenous control. Reaction conditions were 95℃ for 30 s, followed by 40 cycles of 95℃ for 5 s, and 60℃ for 34 s. Triplicate reactions of each sample were performed. Relative gene expression levels were calculated using the comparative Ct method (2-ΔΔCt method) based on Ct values for CsTRPV4 and β-actin (Livak and Schmittgen, 2001). All values are expressed as means and standard deviations (mean±SD). One-way analysis of variance (ANOVA) followed by a Dunnett's two-tailed posthoc test was used to determine differences among different groups. All the primer sequences used in this paper are listed in Table 1.

2.5 In situ hybridization histochemistry

The vector pGEM-T was digested with Nco I, and the lye-specific antisense probe was synthesized using Sp6 RNA polymerase. A sense probe was synthesized using T7 RNA polymerase and the vector pGEM-T digested with Sa II. The abocular skin of the head of two years old half-smooth tongue sole was cut into 6 or 8 pieces. These sections, as well as 1 dph larvae, were fixed in freshly prepared 4% paraformaldehyde in 100 mmol/L phosphate buffered saline (PBS; pH 7.4) at 4℃ for 12 h. The samples were dehydrated, embedded in paraffin, and sectioned at 6 μm. Whole mount in situ hybridization of 1 dph larvae was performed as described previously (Thisse and Thisse, 2008).

3 RESULT 3.1 Sequence analysis

The primer pairs F and R were used to amplify a 2 364-bp fragment with significant similarity to the TRPV4 sequence published in GenBank. This sequence was then used to design gene specific primers for 5′- and 3′-RACE, in order to obtain the full cDNA. The assembled CsTRPV4 cDNA (GenBank accession number KU248476) was 3 104 bp. The full-length of CsTRPV4 had an open reading frame of 2 613 bp, encoding a protein of 870 amino acids. CsTRPV4 contained a 5′ untranslated region (UTR) of 237 bp, and a 3′-UTR of 254 bp. The 3′-UTR contains a stop codon (TGA) and a poly (A)+ tail; however, the typical AATAAA poly-adenylation signal was not found in upstream of the poly(A)+ tail (Fig. 1). The predicted molecular mass and pI of mature CsTRPV4 were 98.75 kDa and 8.07, respectively.

Fig.1 Full cDNA sequence and predicted the amino acid sequence of C. semilaevis TRPV4 The bold underlined letters indicate start codon (ATG) and the stop codon (TGA). The blue underlined letters indicate ankyrin repeats. The letters in box represent the transmembrane domains. The red underlined letters represent the pore loop.
3.2 Phylogenetic analysis of CsTRPV4

Alignment of the CsTRPV4 with homologs of other species revealed high homology (Fig. 2). Homology between half-smooth tongue sole and bicolor damselfish, tilapia, zebrafish, tropical clawed frog, rat, and human was 90.1%, 84.8%, 77.1%, 74.1%, 72.4%, and 72.9%, respectively (Table 2). CsTRPV4 showed the highest similarity to the damselfish TRPV4, deferring mostly at the N-terminal region and C-terminus (Fig. 2). CsTRPV4 had conserved structural and functional domains as other TRPs, such as ankyrin repeat (ANK), proline-rich domain (PRD), transmembrane (TM), pore loop (PL), TRP domain, and calmodulin-binding domains (CaMBD) (Figs. 1 and 2).CsTRPV4 also contained the NLS motif (QKRRRKKL), which had a low level of conservation, relative to other TRPV4s (Fig. 2). The PL sequence between the TM5 and TM6 is important for the ion transporting domain. The predicted CsTRPV4 protein has the PL signature sequence of TRPV4 (L693D694L695F696K697L698T699I700G701M702G703E704), which has high similarity to that of other species. The typical PL sites D694, L698, and M702 were conserved in CsTRPV4. There was only one mutation at the end of the PL sequence: D704 to E704, which also found in bicolor damselfish.

Fig.2 Multiple alignments of C. semilaevis TRPV4 with other TRPV4s homologs by the DNAMAN program The GenBank accession numbers for the sequences used in this alignment are provided in Table 2. Identical residues are shaded in dark gray; dots (.) indicate gaps inserted for maximizing similarity. Numbers shown on the right are the amino acid positions for each sequence. PRD, TM, PL, CaMBD indicate prolinerich domain, transmembrane, pore loop, and calmodulin-binding domains, respectively.

A phylogenetic tree was constructed using the predicted amino acids of CsTRPV4 to identify its evolutionary position (Fig. 3). The GenBank accession numbers of TRPV4 amino acid sequences used in this study are shown in Table 2. As being shown, TRPV4s are divided into two groups, one of them only contains C. elegans; the other is the vertebrate group (Fig. 3). The vertebrate group is divided into two main groups: bony fish are clustered in one branch, while amphibians, reptiles, and mammals formed the second main branch. TRPV4 from half-smooth tongue sole and bicolor damselfish clustered together with tilapia but were located as a different clade from zebrafish and rainbow trout.

Fig.3 Phylogenetic tree of TRPV4 constructed by the neighbor-joining method use the DNAMAN program The GenBank accession numbers are provided in Table 2. Bootstrap majority consensus values on 1 000 replicates are shown at each branch point.

In CsTRPV4, the residues Ser, Thr, and Tyr, are susceptible to phosphorylation and dephosphorylation. Analysis of protein phosphorylation plays a major role in elucidating its function. We predicted phosphorylation sites of CsTRPV4 using NetPhos3.1. CsTRPV4 contained 47 Ser, 32 Thr, and 12 Tyr residues (Fig. 4), which is consistent with previous studies in other TRPV4s.

Fig.4 Possible phosphorylation sites of CsTRPV4 identified using NetPhos 3.1
3.3 Tissue expression analysis of CsTRPV4

RT-PCR was performed using total RNA extracted from adult half-smooth tongue sole. The first part of CsTRPV4 transcript was amplified from kidney, a tissue well known to express TRPV4 (Strotmann et al., 2000; Liedtke et al., 2000). CsTRPV4 was also expressed at high levels in the heart, spleen, testis, and eye, but it had low expression levels in the kidney (Fig. 5). The expression of CsTRPV4 in the lateral line was significantly higher than in the kidney and liver but similar to the expression in the testis (Fig. 5). We next examined CsTRPV4 expression in six sense organs of half-smooth tongue sole. CsTRPV4 was expressed in all sense organs and was expressed at high levels in the eye and in free neuromasts (Fig. 6).

Fig.5 Expression pattern of CsTRPV4 in different body tissues Expression analysis was performed by qRT-PCR, using β-actin as an internal control. 2-ΔΔCt values were calculated to normalize data. Results represent three independent experiments and are expressed as mean ± standard deviation. Different letters denote significant differences (P < 0.05).
Fig.6 Expression pattern of CsTRPV4 in different sensory tissues Expression analysis was performed by qRT-PCR, using β-actin as an internal control. 2-ΔΔCt values were calculated to normalize data. Results represent three independent experiments and are expressed as mean ± standard deviation. Different letters denote significant differences (P < 0.05)
3.4 Location of CsTRPV4 in free neuromast

Whole mount in situ hybridization revealed that the CsTRPV4 mRNA was detected in free neuromasts at 1 dph (Fig. 7). The in situ hybridization show that CsTRPV4 was expressed in the free neuromast of adult fish, especially in the hair cells (Fig. 8).

Fig.7 In situ hybridization of CsTRPV4 in free neuromast of half-smooth tongue sole a and c. the free neuromast with antisense RNA probe; b and d. the free neuromast with sense RNA probe; Scale bars represent 100 μm. sk: skin; fn: free neuromast; hc: hair cells.
Fig.8 Whole mount in situ hybridization of free neuromast in half-smooth tongue sole a. 1 dph larvae with antisense RNA probe; b. high magnification of the place of the black box. The black arrow indicates free neuromasts. Scale bars represent 100 μm. fn: free neuromast; Ffn: free neuromasts of the fish front; Bfn: free neuromast of fish back.
4 DISCUSSION 4.1 Structure analysis

TRPV4 channel, a nonselective cation channel, can integrate different stimuli and confers many distinct cellular functions in various cell types throughout the body, involving in the pathogenesis of several diseases (Everaerts et al., 2010). In this study, we cloned the full-length CsTRPV4 and investigated the expression of CsTRPV4 in various organs. Importantly, we showed the expression of CsTRPV4 in free neuromast of the lateral line system, which is as a sensory organ in fish. Finally, we demonstrated the location of CsTRPV4 in free neuromast that belongs to the lateral line system as a kind of sensory organs in fish.

The CsTRPV4 was similar in length and shared a high degree of sequence identity with other TRPV4s of teleost fish, and therefore that it is possibly involved in critical cellular and/or physiological functions. The putative protein of CsTRPV4 contained the conserved domain of ankyrin (ANK) repeats, six transmembranes (TM), TRP domain, and CaMBD. The NLS(QKRRRKKL) belonged to the typical monopartite type that is a short peptide composed of 4–8 amino acids, with a positive charge in K and R residues, usually also contains P (see Fig. 2). Therefore, the conserved NLS may play a different role with other organisms by a variety of activation methods determining the behavior of the protein in the nucleus (Zhang et al., 2002).

Inmammalian, TRPV4s generally have six ANK repeats, which were more than CsTRPV4. CsTRPV4 contained four ANK repeats, as so do other fish, such as zebrafish (Mangos et al., 2007). The ANK repeats might be involved in self-association of N-termini to form a tetrameric structure that is necessary for the oligomerization of TRPV4 (Gaudet, 2008). The lack of TRPV4 oligomerization can reduce its accumulation in the endoplasmic reticulum impairing its mechanosensitivity (Liedtke et al., 2000; Suzuki and Mizuno, 2012). This ANK domain is responsible for a correct trafficking of TRPV4 to the plasma membrane and might anchor the channel to the cytoskeleton or constitute a mechanical link for gating in sensory cells.

Compared to other known species, the PRD of CsTRPV4 was not conserved. The mutation of PRD, which forms an interaction site with PACSIN 3 (a cytoskeleton protein involved in synaptic vesicular membrane trafficking and endocytosis) (Cuajungco et al., 2006), can result in low sensitivity to heat and cell swelling (D'Hoedt et al., 2008).

The PL of CsTRPV4 had higher similarity to teleost fish. The three critical residues D694, L698, and M702 were the same as in other organisms. D694 is important for Ca2+ sensitivity of the TRPV4 pore. In contrast, neutralizing the only positively charged residue in the putative pore region, L698, has no noticeable effects on the properties of the TRPV4 channel pore. M702 is located at the center of a putative selectivity filter when it changes the whole cell current amplitude and impairs Ca2+ permeation are reduced. The end amino acid of PL in CsTRPV4, D704 changed to E704, which might reduce the permeability for Ca2+ and Mg2+, and decrease the affinity of the channel to the voltage-dependent pore blocker ruthenium red (Voets et al., 2002). In a previous report, a mutant of Y555A in the third TM domain of TRPV4 in humans is not activated by heat or by 4α-PDD but activated by cell swelling or by arachidonic acid (Vriens et al., 2004). Thus, if a site in the conserved domain changes, the function of the protein could be affected. Our data indicate that the protein sequence of CsTRPV4 is more much conservative than other TRPV4, and CsTRPV4 can be also affected by osmotic cell swelling, heat, phorbol ester compounds, and 5, 6-epoxyeicosatrienoic acid.

4.2 Expression

The CsTRPV4 expression in normal tissues was similar to that of other species, showing a broad expression spectrum but at various expression levels. The highest expression of CsTRPV4 was found in the heart, followed by spleen, eye, and testis. This expression pattern is different from that of mammals. In mammals, the highest expression of CsTRPV4 was found in the kidney, followed by liver and heart. CsTRPV4 was revealed the highest in the heart, which might be related to its many functions. The disease of cardiac fibrosis was closely related to TRPV4 in mammalians.

We also investigated the expression of CsTRPV4 in six sensory organs of adult half-smooth tongue sole. The expression of CsTRPV4 was the highest in the eyes and lowest in the skin. In zebrafish, the TRPV4 was expressed at high levels in the skin, which plays a main role in sensing hyposmolarity (Galindo-Villegas et al., 2015). The TRPV4 was also detected in the eye, mainly localized in the retina, and maybe play specific roles in visual processing (Amato et al., 2012). A recent report showed that TRPV4 is closely related to the flow of Ca2+ and to retinal ganglion cell apoptosis in mouse (Ryskamp et al., 2014). In halfsmooth tongue sole, the CsTRPV4 had low expression in the skin and high expression in the eye, which might indicate the sensing of osmotic pressure in the eye, not in the skin. Now, TRPV4 has proved to have a positive correlation with ophthalmic diseases.

Importantly, compared to other sensory organs, CsTRPV4 was expressed at a higher level in free neuromasts than that of the later line. The section in situ hybridization results show that CsTRPV4 was detected in the mantle and sensory cells of free neuromasts in adult fish. Whole mount in situ hybridization revealed that CsTRPV4 was also expressed in free neuromasts of 1 dph larvae. The expression of TRPV4 in hair cells of neuromasts in zebrafish embryos was previously reported by Mangos et al. (2007). At 40 hpf in zebrafish, TRPV4 expression can be detected in sensory structures, specifically in free neuromasts (Dambly-Chaudiere et al., 2003). TRPV4 was detected in several sensory organs, including neuromasts or olfactory pits in adult zebrafish (Amato et al., 2012). In our previous study, 3 dph larvae began ingesting rotifers, but the retinomotor responses were not visible; the retinomotor responses were visible at 15 dph (Ma et al., 2007b). At 1 dph larvae, the canal neuromasts of half-smooth tongue sole had not formed, so presumably free neuromasts, like the bluefin tuna larvae hatch (Kawamura et al., 2003), played an important role in detecting stimuli to promote feeding. So the expression of TRPV4 in free neuromasts in half-smooth tongue sole may provide sensitivity to mechanical stimulation playing a central role in mechanical feeling. Previous studies have found that free neuromasts could present more on the fish of living in the still water or little movement (Dijkgraaf, 1963), which innervated by the anterolateral nerve playing a role of mechanical sense to percept of flow velocity (Koyama et al., 1990; Marshall, 1996; Engelmnan et al., 2000; Carton and Montgomery, 2002). Present results added new data to the mechanosensitive of free neuromasts.


In summary, this is the first time that sensory gene TRPV4 is isolated from flatfish. The gene CsTRPV4 was widely expressed in various tissues of halfsmooth tongue sole indicating that it played a very important role in the fish and the expression of CsTRPV4 in all sensory organs may perform sensory functions. Future research on the TRPV4 of flatfish, including isolation and characterization of TRPV4 from more kinds of flatfish to discover the commonalities and differences of the expression may help us knowing the potential function of the gene. Gene interference or silencing will help us further study the sensory function of CsTRPV4.


The datasets generated during and/or analyzed during this study are available from the corresponding author on reasonable request.

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