Lusha LIU, Xungang WANG, Mengjie ZHANG, Jianping JIANG

1Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, Sichuan, China

2University of Chinese Academy of Sciences, Beijing 100049, China

3 College of Life Sciences, Nanjing Normal University, Nanjing 210023, Jiangsu, China

1. Introduction

Amphibian metamorphosis is a complex process, in which various organs and tissues undergo dramatically remodeling to transform from larva to juvenile(Atkinsonet al., 1998; Brown and Cai, 2007; Duet al.,2017). Although complex, this development process is completely initiated and orchestrated by only one hormone, thyroid hormone (TH) (Buchholzet al., 2006).The TH signaling pathway in amphibians has been well studied: when TH is absent, the unliganded TR/9-cis-retinoic acid receptor (RXR) heterodimers recruit corepressors to repress the transcription of downstream target genes; once liganded with TH, the TR/RXR heterodimers undergo conformational change and thus allow to recruit coactivators to activate the same group of downstream genes (Morvan-Duboiset al., 2008; Grimaldiet al., 2013; Zhaoet al., 2016). Therefore,TRsplay important roles in the metamorphosis by acting as liganddependent transcriptional factors.

TRs, members of a large superfamily of nuclear receptors (NR), possess a similar domain structure as that found in the other NRs: an N-terminal A/B domain with binding sites for transcriptional coregulators, a central DNA-binding domain C (DBD) containing two“zinc fingers” for target gene recognition, a D domain(hinge region) containing the nuclear localization signal, and a C-terminal ligand-binding domain E/F (LBD) where thyroid hormone binds and activates the receptor (Chenet al., 2014). There are two closely related families ofTRscalledTRαandTRβin vertebrate(Yaoitaet al., 1990; Chenet al., 2014).TRαandTRβare differentially expressed in various tissues of different species (Kawaharaet al., 1991). Particularly, theTRαmRNA increases throughout the premetamorphosis stage of tadpole development, and falls after the climax of metamorphosis to a lower level in frogs (Yaoita and Brown, 1990). TheTRβmRNA is barely detectable during premetamorphosis. In synchrony with the onset of endogenous TH synthesis by the thyroid gland, the level ofTRβmRNA rises in parallel with endogenous TH, reaching a peak at the climax of metamorphosis and drops after metamorphosis (Choiet al., 2015). Although TH signaling pathway has been well studied, functional ofTRαandTRβduring metamorphosis have not been clearly characterized.TRα-deficient tadpoles developed faster with smaller body size than their wild-type siblings suggesting thatTRαplayed important roles in controlling the timing ofXenopus tropicalismetamorphosis (Choiet al., 2015; Wen and Shi, 2015). Furthermore, disruptedTRαhad different effect on the development of larval and juveniles and the metamorphosis of different organs (Choiet al., 2017, Wenet al., 2017). Different fromTRα-knockout tadpoles, significantly delayed tail regression, the reduction in olfactory nerve length and head narrowing by gill absorption were detected inTRβknockout tadpoles (Nakajimaet al., 2017). The different relative abundance levels ofTRαandTRβtranscripts induced by T3 where the general pattern wasTRα≥TRβinR. catesbeiana, whileTRα≤TRβinXenopus laevis(Veldhoenet al., 2014).TRβwas highly expressed during metamorphosis inM. fissipesandX.laevis, butTRαshowed especially low expression inM. fissipes, implying thatTRβis essential for initiating metamorphosis, at least inM. fissipes(Zhaoet al., 2016).

Microhyla fissipesis a small-sized anuran from the family Microhylidae suborder Neobatrachia (Figure 1). Due to the special expression pattern ofTRsinM.fissipesmentioned above, it is important to cloneTRsand understand the molecular mechanism of them in regulating metamorphosis. Furthermore, because of its characteristics (including wide distribution, fast development, developmentin vitro, strong survivability,biphasic life cycle, small body size, diploid and transparent tadpoles) and being induced to metamorphose by exogenous TH,M. fissipesmay be an ideal model to evaluate the possible effects of environmental compounds on the thyroid system (Liuet al., 2016).

Figure 1 Photograph of Microhyla fissipes.

Therefore, it is necessary to characterizeTRsand evaluate responsiveness to TRs agonist (such as 3,3',5-Triiodo-L-thyronine, T3) inM. fissipes.In this study, we have isolated, characterized, and phylogenetically analyzedTRαandTRβgene inM. fissipes, examined their expression pattern after T3 treatment and explored the utility ofM. fissipesas a model species for assaying TH signaling disrupting effects.

2. Materials and Methods

2.1. Animals sampling and experimental treatmentsMature female and maleM. fissipeswere collected from Shuangliu, Chengdu, China (30.5825oN, 103.8438oE) in June, 2016. The male and female were injected luteinizing hormone-releasing hormone a (LHRHa) with 0.3 μg/g body weight resolving dosage. Fertilized eggs were obtained from one pair of frogs and incubated in the dechlorinated tap water. Five days later, tadpoles were fed with spirulina powder once daily and subjected to a 12:12 h light:dark cycle at 25 ± 0.6°C. The developmental stage of tadpole was recorded using theM. fissipesdevelopmental table (Wanget al., 2017). Tadpoles at stage 40 (metamorphosis climax) were selected for gene cloning. Tadpoles of stage 33 (premetamorphosis, oarshaped limb bud) were selected to treat with 10 nmol/L 3,3',5-triiodo-l-thyronine sodium (T3, Sigma-Aldrich,USA) for 48 h. The chemicals were renewed after 12 h of exposure when the medium was also refreshed. Tadpoles treated for 0 h, 12 h, 24 h, 36 h, and 48 h were collected for quantitative real-time (RT) PCR (n= 3 for each time point). Tadpoles treated 0 h were set as the control group.After anesthetization by MS222, tadpole sample was frozen immediately in liquid nitrogen, and then stored at–80°C for RNA extraction.

The care and treatment of animals in this study were performed according to the Guideline for the Care and Use of Laboratory Animals in China. The animal experiments were approved by the Experimental Animal Use Ethics Committee of the Chengdu Institute of Biology (Permit Number: 2016036).

2.2. Cloning and molecular characterization of TRsTotal RNA was extracted using TransZol (Transgen Biotech, Beijing, China), following the manufacturer’s instructions. Total RNA concentration was calculated using Nanodrop ND-1000 (Nanodrop, DW, USA).Partial cDNA sequence ofTRs(TRα: comp77374_c0 ;TRβ: comp124487_c3) were obtained fromM. fissipestranscriptome (Zhaoet al., 2016). According to the partial cDNA sequence, two specific primers for each gene,GSP3-1 and GSP3-2 (Table 1) were designed to amplify the 3' terminal regions by nested PCR. The 3' DNA ends were obtained using the SMART RACE cDNA Amplification Kit (Clontech, CA, USA) in accordance with the manufacturer’s instructions. Products of rapid amplification of cDNA ends (RACE) were cloned into pMD 18-T vector (TaKaRa, Japan) and sequenced using an automated DNA sequencer ABI3730 (Thermo Fisher Scientific, CA, USA) by Sangon Biotech Co. Ltd.(Shanghai, China).

The amino acid sequence was deduced from the coding region via DNAStar (version 6.13). The cDNA sequence and the deduced amino acid sequence were analyzed using BLASTN and BLASTP, respectively. Deduced amino acid sequences of amphibian were aligned for analysis of putative conserved functional residues by Clustal X. The relevant amino acid sequences were obtained from the NCBI GenBank database:Rugosa rugoseTRαBAM15695.1,Pelophylax nigromaculatus TRαAGT55994.1,Rana chensinensisTRαAIA98429.1,X. laevisTRαANP_001081595.1,X. laevisTRαBBAL70322.1,X. tropicalis TRαNP_001039261.1,X. tropicalis TRβNP_001039270.1,X. laevis TRβANP_001090182.1,X. laevisTRβBNP_001081250.1,P.nigromaculatusTRβAGT55995.1,R. chensinensisTRβAIA98430.1.

Multiple cDNA sequences ofTRαandTRβfrom 17 species represented vertebrate (Mammalia, Aves, Reptilia,Amphibia, Pisces) and invertebrate were used in the sequence alignment by Clustal X. A phylogenetic tree was constructed by using the maximum likelihood (ML)method with the MEGA 6 (Tamuraet al., 2013), and the reliability of the tree was assessed by the bootstrap method with 1,000 replications. The gene accession numbers are:Alligator mississippiensisTRαNM_001287278.1,Branchiostoma lanceolatumTRαEF672345.1,Crassostrea gigas TRαKP271450.1,Gallus gallus TRαNM_205313.1,Homo sapiens TRαAB307686.1,Oryzias latipesTRαAB114860.1,Rattus norvegicusTRαM18028.1,P. nigromaculatus TRαKC139354.1,R. catesbeiana TRαL06064.1,R. chensinensis TRαKJ579109.1,R. rugose TRαAB683466.1,Schistosoma mansoni TRαAY395038.1,S. mansoni TRβAY395039.1,X. laevis TRαAM35343.1,X. laevis TRαBAB669465.1,X. tropicalis TRαNM_001045796.1,Nanorana parkeri TRαXM_018569566.1,Oryzias latipesTRαNM_001104705.1,Danio rerio TRαNM_131396.1,R. rugosa TRβAB683467.1,R. chensinensis TRβKJ579110.1,R. catesbeiana TRβL27344.1,P.nigromaculatus TRβKC139355.1,N. parkeri TRβXM_018570223.1,X. laevis TRβANM_001096713.1,X. laevis TRβBNM_001087781.1,X. tropicalis TRβAB244214.1,G. gallus TRβNM_205447.2,A.mississippiensis TRβNM_001287311.2,H. sapiens TRβM26747.1,R. norvegicus TRβJ03933.1,O. latipes TRβAB114861.1,D. rerioTRβNM_131340.1.

2.3. RNA isolation and gene expression analysisForTRsmRNA expression analysis after exposure of T3,we conducted qRT-PCR. Total RNA was extracted from tadpoles with TranZol reagent and first strand cDNA was synthesized from the same amount of RNA (1 μg) for each sample via TranScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen, Beijing, China)with oligo (dT) primer. The expression ofTRαandTRβmRNA was analyzed by qRT-PCR with the primerTRαandTRβ(Table 1).Rpl37gene was used as a reference gene to normalize mRNA expression ofTRs. PCR was performed in a reaction volume of 20 μl containing 10 μl ofTransStart? Tip Green qPCR SuperMix (2×), 0.4 μl of Passive Reference Dye (50×), 0.4 μl each of forward and reverse primer (10 μmol/L), 8.2 μl of ddH2O, and 1 μl of cDNA. Amplification was carried out in 7300plus(ABI, CA, USA), including 5 min at 95°C and 45 cycles of 5 s at 95°C and 31 s at 60°C, followed by a melting curve analysis. Each sample was run in triplicates. Each reaction was verified to contain a single product of the correct size by agarose gel electrophoresis. Quantitative data were shown as mean ± SD (n= 3). The fold change ofTRsexpression after T3 treatment was determined by 2?ΔΔCt(cycle threshold, Ct). Data were then subjected to one-way analysis of variance (ANOVA) with SPSS Statistics 13.0 (SPSS Inc., Chicago, IL, USA).P＜ 0.05 was regarded as statistically significant.

3. Results

3.1. Molecular characterization of TRs in M. fissipesThe full-length cDNA sequences ofTRαandTRβwere obtained by RNA-seq and 3'-RACE strategies. The full-length ofTRαcDNA was 1 706 bp in length and contained an open reading frame (ORF) of 1 257 bp, which encoded a peptide of 418 amino acids (Figure 2a). TheTRβcDNA was 1 422 bp with an ORF of 1 122 bp, which encoded a peptide of 373 amino acids (Figure 2b). And the 3'untranslated region ofTRαandTRβwere 449 bp and 300 bp, respectively. Two sequences were submitted to the GenBank (GenBank accession numbers: MG596879 and MG596880). The homologies of nucleotide sequences and deduced amino acid sequences betweenTRαandTRβinM. fissipeswere 61% and 72%, respectively. The calculated molecular weight of TRα polypeptide was 47.7 kDa, and the theoretical isoelectric point (pI) was 7.08,while the calculated molecular weight of TRβ polypeptide was 42.4 kDa with pI 6.76.

Table 1 Primers used in this study. F and R denote forward and reverse primer, respectively.

A multiple alignment ofTRsdeduced amino acid sequences was performed in amphibians (Figure 3).The deduced amino acids of both TRα and TRβ were composed of the N-terminal hypervariable region (A/B domain), a conserved DNA-binding domain (DBD domain), a hinge region (D domain), and a ligand-binding domain (LBD domain). TRα and TRβ inM. fissipeshad high similarity in the DBD, D and LBD domains, whereas there was a deletion of 42 amino acids in TRβ compared with TRα in the A/B domain (Figure 3). These deletion between TRα and TRβ inM. fissipescorresponded to the differences found inX. laevis,X. tropicalis,R.chensinensisandP. nigromaculatus. Although there were several different sites in the A/B domain among TRs, the N-terminal signature sequence (NTSS) was well conserved. The conserved cysteine residues that comprise the two zinc fingers and the regulatory elements P-box as well as the T-box and A-box in the DBD were conserved. Furthermore, the consensus motif I (spanning helix 3–6) and motif II (from helix 8 to helix 10) and the putative AF2 activation domain core (helix 12) were also identified. Therefore, this highly-conserved feature is likely to indicate pivotal significance ofTRsin thyroid signaling pathway.

3.2. Evolutionary relationships of TRsBLASTP and BLASTN in NCBI indicated thatTRαandTRβshared different levels of homology with other species. The amino acid sequences ofM. fissipesTRαhad highest homolgy withR. rugosa,N. parkeri,R. chensinensis(98%, 10 different sites out of 418 amino acids),whileM. fissipesTRβhad highest homolgy withR.catesbeiana(98%, 8 different sites out of 373 amino acids). Nucleotide sequences ofM. fissipesTRαandTRβshared high homology withN. parkeri(92% and 93%),P. nigromaculatus(92% and 92%),R. rugosa(93% and 92%),X. tropicalis(88% and 87%),X. laevis A(88% and 80%),X. laevisB(88% and 78%) and had a lower homology withG. gallus(65% and 74%),A.mississippiensis(63% and 63%),H. sapiens(58% and 47%),D. rerio(57% and 49%),B. lanceolatum(47% and 49%), respectively.

A phylogenetic tree constructed by the ML method from a multiple alignment of nucleotide sequences ofM.fissipes TRαandTRβand a wide range of counterparts in various species including invertebrate, reptiles,birds, mammals and other amphibian (Figure 4). The phylogenetic tree showed thatTRαandTRβgrouped into two highly consistent and separate clades. In bothTRαandTRβclades,TRsfromM. fissipeshave similar positions in the phylogenetic tree. Furthermore, phylogenetic tree constructed based onTRscDNA sequences was consistent well with the taxonomic positions of these organisms.

Figure 2 cDNA and deduced amino acid sequences of Microhyla fissipes thyroid hormone receptor gene. (a) TRα; (b) TRβ. The start codon(ATG) and the stop codon (TAG or TGA) are in bold.

Figure 3 Alignments of TR amino acid sequences from Microhyla fissipes with other amphibian species. Asterisk (*) indicated conserved amino acids and hyphens (?) represented spaces inserted to maximize similarity. Colon (:) indicates conservation between groups of strongly similar properties, and period (.) indicates conservation between groups of weakly similar properties. The black vertical lines indicate the borders of four domains: the N-terminal hypervariable region (A/B domain), DNA-binding domain (DBD domain), hinge region (D domain)and ligand binding domain (LBD domain). Triangle indicated the conserved cysteine residues that comprise the two zinc finger with red box of the DBD. The conserved N-terminal signature sequence (NTSS), DR-box, P-box, T-box, A-box and Helices (H3-H12) are figured out.Motif I and Motif II are boxed by blue while activation domain (AF2-AD) is purple double underlined.

3.3. Responsiveness of M. fissipesTRs expression to exogenous T3In the presence of exogenous T3,the morphology ofM. fissipestadpoles has changed dramatically, and the characteristic of morphology change trend was similar to that during the natural metamorphosis(data not shown). To determine whether exogenous T3 could potentially regulate the expression pattern ofTRαandTRβ, we exposed the premetamorphic tadpoles to T3 and analyzed by qPCR. These results showed thatM.fissipes TRαandTRβmRNA expression were significantly increased by T3 at 12 h and 24 h, respectively. And thenTRαandTRβexpression decreased to the lower level at 48 h than 0 h (Figure 5). Furthermore, the same gene expression pattern ofTRβhas been detected between exogenous T3 induced and natural metamorphosis (Zhaoet al., 2016). Therefore, different responsiveness ofTRαandTRβto T3 indicated different functions of them in the metamorphosis.

Figure 4 Phylogenetic tree of nucleotide sequences of TRs genes of different species using the the maximum likelihood method. Numbers at the branches represent the bootstrap support values. Microhyla fissipesTRα and TRβ are highlighted in the box, respectively.

4. Discussion

This study was designed to determineTRαandTRβsequences and analyze their expression patterns after T3 exposure to gain further insights as to how these genes may function in metamorphosis. Therefore,TRαandTRβgenes ofM. fissipeswere cloned by RNA-seq and RACE. Phylogenetical analysis showedM. fissipesTRαandTRβgene had high homology with the corresponding genes of other amphibians at both the nucleotide and the amino acid level, respectively, confirming their identities. The expression patterns after T3 treatment indicated the important roles ofTRαandTRβduring the metamorphosis.

Figure 5 Relative expression levels of TRs genes after exposure to 10 nmol/L T3. Data were expressed as the mean fold difference(mean ± SD, n = 3) from the control group. Significant differences between treatment groups and the controls were indicated by* (P ＜ 0.05).

Both TRα and TRβ amino acid structure identified inM. fissipespossessed the typically functional domains of the NR superfamily. All of them had a conserved DBD domain with two zinc fingers, which determined the binding specificity of nuclear receptors (Natalia and Thorsten, 2004), and a LBD domain containing the typical 12 helices (Marchandet al., 2001). The P-box determining DNA binding specificity interacted with the specific response element AGGTCA, and the T-box and A-box regions contributed to dimerization and DNA binding stabilization, respectively (Manchadoet al., 2009). The conserved NTSS (GYIPS(Y/H) L(D/N)KDE(P/L)) which was the TR specific motif was also detected in the C-terminus of the variable A/B domain ofM. fissipesTRs (Wuet al., 2007). The deletion of 42 amino acids in the A/B domain of TRβ indicated its different function from TRα. The conserved AF2-AD motif (LFLEVF) played an important role in recruiting a coactivator (Nagyet al., 1999; Nelson and Habibi, 2009).The conservation of structure and functional roles of the above mentioned sites in TRs would be consolidated by their high identity throughout the evolution of vertebrates.

TRαandTRβhave been demonstrated to have high homology across vertebrates (Okaet al., 2013). In this study, the amino acid and nucleotide sequence homologies ofTRαandTRβbetweenM. fissipesand other amphibians were over 90%, but lower homology with invertebrates. In the phylogenetic tree, vertebrateTRαandTRβwere located in two clearly separated clades, in accordance with the fact thatTRαandTRβmay be the products of an ancient gene duplication event during evolution (Chenet al., 2014). Furthermore, the homologies ofTRαandTRβto the corresponding genes from other species accorded with their evolutionary relationship. Only oneTRαandTRβgene were identified in amphibian except inX. laeviswhich is tetraploid. The homologies of nucleotide sequences and deduced amino acid sequences betweenTRαandTRβinM. fissipeswere 61% and 72%, respectively. The homology ofTRαtoTRβinM. fissipeswas lower thanR. nigromaculata(72%and 86%) andX. laevis(74% and 85%). Low sequence identification ofTRαandTRβmay indicate their different regulated function inM. fissipesmetamorphosis, which is also implied by their different expression pattern during natural metamorphosis (Zhaoet al., 2016).

Metamorphosis is a critical developmental stage mediated by TH in amphibian (Wanget al., 2008).The function ofTRsas transducers of TH responses has converted NRs in targets to clarify the molecular mechanisms that govern metamorphosis (Manchadoet al., 2009). To understand the potential importance of these two receptors inM. fissipesmetamorphosis and function of exogenous ligand for the receptor systems, we examined their mRNA expression levels by qPCR after T3 exposure. T3 not only inducedM. fissipespremetamorphic tadpoles metamorphosis at the morphology and histology level (data not shown), but also inducedTRαandTRβexpression, which have also been reported inX. laevis,X. troplisandR. catesbeiana(Shi, 1999; Wanget al.,2008). During the natural metamorphosis,TRβexpression increased dramatically and correlated with the endogenous THs, whileTRαexpression slightly increased; and all of them decreased at the end of metamorphosis (Shi,1999; Zhaoet al., 2016). In this study,TRαexpression reached peak at 12 h and then decreased from 12 h to 48 h. While dramatically up-regulatedTRβexpression was observed after exposure of T3 within 24 h, and it was down-regulated from 24 h and with the lowest expression observed at 48 h of T3 treatment. These results suggested thatTRβexpression pattern after T3 treatment is the same as that during natural metamorphosis, and the expression ofTRβin tadpoles treated with T3 for 24h also resembled its expression in the tadpoles at the climax of metamorphosis (Zhaoet al., 2016), which correlated with the morphological and histological results. Therefore,we can use T3 to simulate metamorphosis for further research on metamorphosis which will be high-efficiency and time saving. Moreover,M. fissipescould also serve as the model to assay environmental compounds on TH signaling disruption, while expression ofTRs(in particularTRβ) has been also used as a molecular biomarker for assaying TH signaling disrupting actions inX. laevisandR. nigromaculatabecause of their response to TH (Opitzet al., 2006; Veldhoenet al., 2006; Louet al., 2014). Due to the specific expression level ofTRsduringM. fissipesnatural metamorphosis and dramatically up-regulatedTRβmRNA expression after T3 treatment,we will use genome editing tools such as CRISPR/Cas9 to illustrate the mechanism ofTRαandTRβinM. fissipes.In conclusion,TRαandTRβfromM. fissipeswere cloned and characterized for the first time. Functional site and phylogenetic analysis indicated the conserved function ofTRsfrom invertebrate to vertebrates. Premetamorphic tadpoles treated with T3 for 24 h resembled the climax of metamorphosis tadpoles during natural metamorphosis,andTRβmRNA expression analysis could serve as a sensitive molecular testing approach to study effects of environmental compounds on the thyroid system inM. fissipes.

AcknowledgementsThis study was funded by the Important Research Project of Chinese Academy of Sciences (KJZG-EW-L13), 2015 Western Light Talent Culture Project of the Chinese Academy of Sciences(Y6C3021) and the Basic Application Project of Sichuan Province (2017JY0339). We thank Lanying ZHAO for the data analysis, and we also thank Liezhen FU and Shouhong WANG for the manuscript revision.

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