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
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 TRPV4.2 MATERIAL AND METHOD 2.1 Animal
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.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.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.
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.
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.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).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).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.5 CONCLUSION
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 CsTRPV4.6 DATA AVAILABILITY STATEMENT
The datasets generated during and/or analyzed during this study are available from the corresponding author on reasonable request.
Amato V, Viña E, Calavia M G, Guerrera M C, Laurà R, Navarro M, De Carlos F, Cobo J, Germanà A, Vega J A. 2011. Trpv4 in the sensory organs of adult zebrafish. Microscopy Research & Technique, 75(1): 89-96.
Bossus M, Charmantier G, Lorin-Nebel C. 2011. Transient receptor potential vanilloid 4 in the European sea bass Dicentrarchuslabrax: a candidate protein for osmosensing. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 160(1): 43-51.
Carton A G, Montgomery J C. 2002. Responses of lateral line receptors to water flow in the Antarcticnotothenioid, Trematomusbernacchii. Polar Biology, 25(10): 789-793. DOI:10.1007/s00300-002-0416-5
Caterina M J, Leffler A, Malmberg A B, Martin W J, Trafton J, Petersen-Zeitz K R, Koltzenburg M, Basbaum A I, Julius D. 2000. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science, 288(5464): 306-313. DOI:10.1126/science.288.5464.306
Caterina M J, Rosen T A, Tominaga M, Brake A J, Julius D. 1999. A capsaicin-receptor homologue with a high threshold for noxious heat. Nature, 398(6726): 436-441. DOI:10.1038/18906
Clapham D E. 2003. TRP channels as cellular sensors. Nature, 426(6966): 517-524. DOI:10.1038/nature02196
Colbert H A, Smith T L, Bargmann C I. 1997. OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. The Journal of Neuroscience, 17(21): 8 259-8 269. DOI:10.1523/JNEUROSCI.17-21-08259.1997
Cuajungco M P, Grimm C, Oshima K, D'hoedt D, Nilius B, Mensenkamp AR, Bindels RJ M, Plomann M, Heller S. 2006. PACSINs bind to the TRPV4 cation channel PACSIN 3 modulates the subcellular localization of TRPV4. Journal of Biological Chemistry, 281(27): 18 753-18 762. DOI:10.1074/jbc.M602452200
D'Hoedt D, Owsianik G, Prenen J, Cuajungco MP, Grimm C, Heller S, Voets T, Nilius B. 2008. Stimulus-specific modulation of the cation channel TRPV4 by PACSIN 3. Journal of Biological Chemistry, 283(10): 6 272-6 280. DOI:10.1074/jbc.M706386200
Dambly-Chaudière C, Sapède D, Soubiran F, Decorde K, Gompel N, Ghysen A. 2003. The lateral line of zebrafish: a model system for the analysis of morphogenesis and neural development in vertebrates. Biology of the Cell, 95(9): 579-587. DOI:10.1016/j.biolcel.2003.10.005
Dijkgraaf S. 1963. The functioning and significance of the lateral-line organs. Biological Reviews, 38(1): 51-105. DOI:10.1111/j.1469-185X.1963.tb00654.x
Eid S R, Cortright D N. 2009. Transient receptor potential channels on sensory nerves. In: Canning B, Spina D eds. Sensory Nerves. Springer, Berlin, Heidelberg.
Engelmann J, Hanke W, Mogdans J, Bleckmann H. 2000. Hydrodynamic stimuli and the fish lateral line. Nature, 408(6808): 51-52. DOI:10.1038/35040706
Everaerts W, Nilius B, Owsianik G. 2010. The vanilloid transient receptor potential channel TRPV4: From structure to disease. Progress in Biophysics and Molecular Biology, 103(1): 2-17.
Galindo-Villegas J, Montalban-Arques A, Liarte S, De Oliveira S, Pardo-Pastor C, Rubio-Moscardo F, Meseguer J, Valverde M A, Mulero V. 2016. Correction: cutting edge: trpv4-mediated detection of hyposmotic stress by skin keratinocytes activates developmental immunity. Journal of Immunology, 196(8): 3 494. DOI:10.4049/jimmunol.1600243
Garcia-Elias A, Mrkonjić S, Jung C, Pardo-Pastor C, Vicente R, Valverde M A. 2014. The trpv4 channel. In: Nilius B, Flockerzi V eds. Mammalian Transient Receptor Potential (TRP) Cation Channels.Springer, Berlin, Heidelberg.
Gaudet R. 2008. A primer on ankyrin repeat function in TRP channels and beyond. Molecular BioSystems, 4(5): 372-379. DOI:10.1039/b801481g
Kawamura G, Masuma S, Tezuka N, Koiso M, Jinbo T, Namba K. 2003. Morphogenesis of sense organs in the bluefin tuna Thunnus orientalis: The big fish bang. In: Proceedings of the 26th Annual Larval Fish Conference. Norwegian Institute of Marine Research, Bergen, Norway. p.123-135.
Koyama H, Kishida R, Goris R C, Kusunoki T. 1990. Organization of the primary projections of the lateral line nerves in the lamprey Lampetra japonica. The Journal of Comparative Neurology, 295(2): 277-289. DOI:10.1002/cne.902950210
Kwan K Y, Glazer J M, Corey D P, Rice F L, Stucky C L. 2009. TRPA1 modulates mechanotransduction in cutaneous sensory neurons. Journal of Neuroscience, 29(15): 4 808-4 819. DOI:10.1523/JNEUROSCI.5380-08.2009
Liedtke W, Choe Y, Martí-Renom M A, BellAM, Denis C S, Šali A, Hudspeth A J, Friedman J M, Heller S. 2000. Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. Cell, 103(3): 525-535. DOI:10.1016/S0092-8674(00)00143-4
Liedtke W, Tobin D M, Bargmann C I, Friedman J M. 2003. Mammalian TRPV4 (VR-OAC) directs behavioral responses to osmotic and mechanical stimuli in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America, 100(Suppl. 2): 14 531-14 536.
Liedtke W. 2007. Role of TRPV ion channels in sensory transduction of osmotic stimuli in mammals. Experimental Physiology, 92(3): 507-512. DOI:10.1113/expphysiol.2006.035642
Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method. Methods, 25(4): 402-408. DOI:10.1006/meth.2001.1262
Ma A J, Liu X Z, Xu Y J, Liang Y, Zhuang Z M. 2006. Feeding rhythm and growth of the tongue sole, Cynoglossus semilaevis Günther, during its early life stages. Aquaculture Research, 37(6): 586-593. DOI:10.1111/j.1365-2109.2006.01466.x
Ma A J, Wang X A, Zhuang Z M, Zhang X M, Zhang L J. 2007b. Structure of retina and visual characteristics of the half-smooth tongue-sole Cynoglossus semilaevis Günter. Acta Zoologica Sinica, 53(2): 354-363. (in Chinese with English abstract)
Ma A J, Wang X A, Zhuang Z M. 2007a. Lateral-line sense organs and dermal surface structures of the tongue sole Cynoglossus semilaevis. Acta Zoologica Sinica, 53(6): 1 113-1 120. (in Chinese with English abstract)
Mangos S, Liu Y, Drummond I A. 2007. Dynamic expression of the osmosensory channel trpv4 in multiple developing organs in zebrafish. Gene Expression Patterns, 7(4): 480-484. DOI:10.1016/j.modgep.2006.10.011
Marshall N J. 1996. Vision and sensory physiology the lateral line systems of three deep-sea fish. Journal of Fish Biology, 49(SA): 239-258. DOI:10.1111/j.1095-8649.1996.tb06079.x
O'Neil R G, Heller S. 2005. The mechanosensitive nature of TRPV channels. Pflügers Archiv., 451(1): 193-203. DOI:10.1007/s00424-005-1424-4
Pedersen S F, Owsianik G, Nilius B. 2005. TRP channels: an overview. Cell Calcium, 38(3-4): 233-252. DOI:10.1016/j.ceca.2005.06.028
Rui X, Xu X Z S. 2010. Mechanosensitive channels: in touch with Piezo. Current Biology, 20(21): R936-R938. DOI:10.1016/j.cub.2010.09.053
Ryskamp D A, Witkovsky P, Barabas P, Huang W, Koehler C, Akimov N P, Lee S H, Chauhan S, Xing W, Rentería R C, Liedtke W, Krizaj D. 2014. The polymodal ion channel trpv4 modulates calcium flux, spiking rate and apoptosis of mouse retinal ganglion cells. Journal of Neuroscience, 31(19): 7 089-7 101.
Sha Z X, Luo X H, Liao X L, Wang SL, Wang Q L, Chen S L. 2011. Development and characterization of 60 novel EST-SSR markers in half-smooth tongue sole Cynoglossus semilaevis. Journal of Fish Biology, 78(1): 322-331. DOI:10.1111/j.1095-8649.2010.02793.x
Strotmann R, Harteneck C, Nunnenmacher K, Schultz G, Plant T D. 2000. OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nature Cell Biology, 2(10): 695-702. DOI:10.1038/35036318
Suzuki M, Mizuno A. 2012. The molecular mechanism of multifunctional mechano-gated channel TRPV4. In: Amkin A, Lozinsky I eds. Mechanically Gated Channels and their Regulation. Springer, Dordrecht.
Thisse C, Thisse B. 2008. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nature Protocols, 3(1): 59-69. DOI:10.1038/nprot.2007.514
Voets T, Prenen J, Vriens J, Watanabe H, Janssens A, Wissenbach U, Bödding M, Droogmans G, Nilius B. 2002. Molecular determinants of permeation through the cation channel TRPV4. Journal of Biological Chemistry, 277(37): 33 704-33 710. DOI:10.1074/jbc.M204828200
Vriens J, Watanabe H, Janssens A, Droogmans G, Voets T, Nilius B. 2004. Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel trpv4. Proceedings of the National Academy of Sciences of the United States of America, 101(1): 396-401. DOI:10.1073/pnas.0303329101
Watanabe S, Seale A P, Grau E G, Kaneko T. 2012. Stretchactivated cation channel TRPV4 mediates hyposmotically induced prolactin release from prolactin cells of Mozambique tilapia Oreochromismossambicus. American Journal of Physiology Regulatory Integrative & Comparative Physiology, 302(8): R1 004-R1 011.
Wissenbach U, Bödding M, Freichel M, Flockerzi V. 2000. Trp12, a novel Trp related protein from kidney. FEBS Letters, 485(2-3): 127-134. DOI:10.1016/S0014-5793(00)02212-2
Zhang Y A, Okada A, Lew C H, Mcconnell S K. 2002. Regulated nuclear trafficking of the homeodomain protein otx1 in cortical neurons. Molecular and Cellular Neuroscience, 19(3): 430-446. DOI:10.1006/mcne.2001.1076
Zhang Z, Zhao Z, Margolskee R F, Liman E. 2007. The transduction channel TRPM5 is gated by intracellular calcium in taste cells. Neuroscience, 27(21): 5 777-5 786. DOI:10.1523/JNEUROSCI.4973-06.2007