Molecular Characterization, Phylogeny, and Expression Profiles of SoxE Subfamily in Scophthalmus maximus

SOXE transcription factors, including SOX8, SOX9


Introduction
Te Sox family encodes various transcription factors (TFs) in the animal kingdom that regulate diverse biological processes [1].Te Sox TF family is characterized by the presence of a sry-related high-mobility group (HMG).On the basis of the structural homology of the HMG domain together with partial regions outside the HMG-box, the Sox family is divided into 11 subfamilies (A-K) [2,3].More than 80% homology in HMG-box sequences is observed for diferent Sox genes in a subfamily; moreover, they exhibit similar biochemical properties and biological functions [4,5].Studies on the structure and function of SoxE subgroups were the most extensive when compared with other Sox subgroups [6,7].
In mammals and other higher vertebrates, the SoxE family is composed of three members, namely, Sox8, Sox9, and Sox10.For example, three SoxE genes were discovered in Homo sapiens, Mus musculus, and Gallus domesticus [7][8][9].Although fsh have a low evolutionary status among vertebrates, they are the most widely distributed and account for nearly half of the existing vertebrate species.Because teleost fsh underwent teleost-specifc whole-genome duplication (3R-WGD), a greater number of SoxE genes are present in fsh compared with other vertebrates.For example (as shown in Table 1), channel catfsh (Ictalurus punctatus) has 4 putative SoxE genes, zebrafsh (Danio rerio) has 5, tongue sole (Cynoglossus semilaevis) has 6, Japanese founder (Paralichthys olivaceus) has 6, puferfsh (Tetraodon fuviatilis) has 6, Nile tilapia (Oreochromis niloticus) has 6, and common carp (Cyprinus carpio) has 10 [3, 10-14].Because of its special phylogenetic status, the study of SoxE evolution and function in fsh is more attractive.SoxE modulates diferent bioprocesses in vertebrates, such as nervous system development [7,15], skeletogenesis [16], and sex determination and diferentiation [17,18].In fsh, Sox9 is the  Journal of Applied Ichthyology most studied SoxE that exerts a crucial efect on sex determination and diferentiation in P. olivaceus and medaka (Oryzias latipes) [19,20].In spotted sea bass (Lateolabrax maculatus), Sox8b, Sox9b, and Sox10 are upregulated in the brain, indicating that SoxE is a key regulator in central nervous system (CNS) development [21].Turbot (Scophthalmus maximus) is a valuable commercial farming fsh in the Chinese aquaculture industry, especially in Northern China [22][23][24][25].Te genome sequence of S. maximus has been published (GenBank Accession: PRJNA821077).Te systemic characterization of the SoxE subfamily has been completed in certain fsh species but not in S. maximus.Tis study comprehensively identifed genes, analyzed sequence structures, and evaluated the evolutionary characteristics for the systemic analysis of the turbot SoxE subfamily.Moreover, this work analyzed gene expression profles to investigate possible SoxE activities in adult tissues.Our fndings would contribute to a better understanding of SoxE-related biological activities in turbot and in other teleost species.

Animals and Sample Collection.
Turbot individuals (age: 2 years, mean length: 38 ± 3.62 cm, and mean weight 1.82 ± 0.21 kg) were obtained from an aquatic product market in Lianyungang (Jiangsu, China).Te turbot individuals were anesthetized using MS-222, followed by the collection of tissues, such as the brain, gill, liver, heart, spleen, stomach, kidney, muscle, intestines, and gonad (ovary or testis).Te collected tissues were frozen immediately in liquid nitrogen and stored at −86 °C until RNA purifcation.All animal-based experiments were approved by the Animal Research and Ethics Committee of Jiangsu Ocean University, and the detailed experimental operations were consistent with previous studies [26,27].

Identifcation of
SoxE in S. maximus.Te genes were identifed on the basis of the conserved HMG-box in the SoxE genes of zebrafsh (Danio rerio).Te sequences were obtained from Ensembl (https://asia.ensembl.org/Danio_rerio/Info/Index) and compared against the tblastn of BLAST in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi), with an E-value threshold level of 1e −6 .All gene candidates were analyzed to determine the presence of the core motif RPMNAFMVW, which verifed that the gene was Sox [2].

Sequence Analysis and Structure Construction of SoxE.
We obtained DNA, mRNA, and protein sequences of the S. maximus SoxE subfamily by using NCBI (https://www.ncbi.nlm.nih.gov).Moreover, the exon-intron structures were acquired using reference genome-related annotation fles.On the basis of the principle of equivalence, introns, exons, HMGboxes, and open-reading frames were further identifed, followed by mapping of the SoxE subfamily gene structure.Subsequently, the EXPASY compute pI/MW approach (https://www.expasy.org/tools/) was used to measure the isoelectric point (pI), molecular weight (MW), and other attribute values of every SoxE protein.SMART (https://smart.embl.de/smart/show_motifs.pl) was adopted to predict conserved HMG sequences in SoxE proteins, while DNAMAN 8.0 was used to perform multiple alignments.WebLogo (https://weblogo.berkeley.edu/logo.cgi)was used to present six multiple sequence alignments of S. maximus.

Phylogenetic Analyses of SoxE.
A total of 59 full-length sequences of SoxE proteins were downloaded from NCBI and Ensembl.Te sequences of diferent organisms, such as M. musculus, H. sapiens, G. domesticus, X. laevis, O. Latipes, D. rerio, I. punctatus, P. olivaceus, C. semilaevis, T. rubripes, O. niloticus, and C. carpio, were downloaded.SMART was used to identify and retrieve HMG domains for phylogenetic analysis.ClustalW was used to align HMG-box sequences, followed by phylogenetic tree construction by using the neighbor-joining (NJ) approach.For this, the Poisson model from MEGA 7.0, with 1000 bootstrapping replicates, was used.

RNA Isolation and Real-Time Quantitative Reverse
Transcription PCR.Total RNA was extracted from the tissues by using TRIzol (Invitrogen, CA, USA), and frst-strand cDNA was synthesized using MMLV and oligo (dT)18 reverse transcriptase (Termo Fisher Scientifc, USA) according to the manufacturer's protocol.Specifc primers (Table 2) were designed using Oligo 7.0.Primer specifcities were examined through alignment against S. maximus transcriptomes (unpublished data) by using BLASTN, with the E-value being 1e −8 and β-actin being the endogenous control gene.Figure S1 displays the melting curves of different genes.Real-time quantitative reverse transcription PCR (qRT-PCR) was performed using the Light Cycler 480 Real-time PCR System (Roche Diagnostics, Mannheim, Germany) in triplicate.Te reaction volume was 20 μL and consisted of 10 μL TB Green Premix EX Taq Mix (Takara, Japan), 1.6 μL cDNA templates, 8 μL ddH2O, and 0.2 μL respective primers.PCR conditions were 30 s at 94 °C; 10 s at 94 °C; 30 s at 60 °C; and 30 s at 72 °C for 35 cycles.Te 2 −ΔΔCt approach was used to determine SoxE expression, which was consistent with the previous method of calculating gene expression [28,29].SPSS 19.0 was employed for statistical analysis through an independent sample t-test.P < 0.05 was considered statistically signifcant.

Results and Discussion
3.1.SoxE Identifed in the S. maximus Genome.Six SoxE genes were identifed in the S. maximus genome by using Ensembl and NCBI (Table 3).Te cDNA length in S. maximus ranged from 2211 to 4592 bp, while the corresponding gene-encoded proteins were 464-499 aa in length.In addition, the predicted MWs were 50.88-54.42kDa, and the pIs were 6.14-7.17.Te S. maximus SoxE number is similar to those of P. olivaceus, C. semilaevis, T. fuviatilis, and O. niloticus (Table 1).In these teleost mentioned above, members of the SoxE subfamily all evolved into two orthologs in their genome (including Sox8a, Sox8b, Sox9a, Sox9b, Sox10a, and Sox10b).Te reason for this Journal of Applied Ichthyology phenomenon may be that these bony fsh have undergone whole-genome duplication (3R-WGD events) during evolution, resulting in a signifcant expansion of members of the SoxE subfamily [30][31][32].A similar phenomenon of double copies of members of the SoxE subfamily has also been seen in other teleost, such as the large yellow croaker and spinyhead croaker [33,34].However, Cyprinus carpio has more SoxE (including CySox8a, CySox8b, CySox8c, CySox8d, CySox9a, CySox9b, CySox9c, CySox9d, CySox10a, and CySox10b) compared with S. maximus and other aforementioned fsh, implying that C. carpio underwent additional genome duplication events during evolution (4R-WGD events) [14].

Genomic Structure and Sequence Alignment of Turbot
SoxE.Te genomic structure of S. maximus SoxE was investigated and constructed using an online analysis tool (https://gsds.gao-lab.org).Relatively conserved exon-intron structures were observed in a SoxE subgroup [10].On the basis of the structural distribution characteristics of introns and exons, turbot SoxE can be divided into two categories, those consisting of two and three introns, respectively.Specifcally, SmSox8a, SmSox8b, SmSox9a, and SmSox9b have two introns, whereas SmSox10a and SmSox10b have three introns (Figure 1(a)).Intron insertion occurred in the HMG domain of all SmSoxE genes.Te presence of introns in HMG boxes has also been observed in SoxE of other fsh, such as Lateolabrax maculatus (LmSox8a, LmSox8b, and LmSox10), Danio rerio (DrSox10), and Paralichthys olivaceus (PoSox8a, PoSox8b, PoSox9a, PoSox9b, PoSox10a, and PoSox10b) [10,13,21].Because of the genetic diversity among species, only the insertion state of founder is completely consistent with that of turbot; however, only some SoxE in other fshes exhibit this phenomenon.We further aligned the six HMG domains and explored the corresponding intron positions in the domains (Figure 2).Te logo plots of the HMG-box domain revealed relatively conserved genes.Moreover, all HMG domains comprised a core motif of RPMNAFMVW, which recognized and bound to cis-regulatory elements in relevant target genes' promoters [12,35] located at residues 5 to 13 (Figures 2(a) and 2(b)).Te insertion locations of introns in the HMG domain of all SmSoxE genes were identical (Figure 2(a)).Terefore, the intron positions are evolutionarily conserved in the SoxE subgroup among species, a result consistent with those of previous studies [3,13,21].

Phylogenetic Analysis of Turbot SoxE.
To identify six SoxE members and their clade, we used MEGA 7.0 to construct a nonrooted phylogenetic tree with 59 full-length sequences of SoxE proteins in twelve species.Turbot SoxE were clustered with corresponding counterparts, and three clades, namely Sox8, Sox9, and Sox10, were identifed, conforming to prior classifcation results (Figure 3).Interestingly, most SoxE, including Sox9a, Sox9b, Sox10a, and Sox10b, can be clustered into their respective branches, indicating that SoxE of diferent species may have a common evolutionary origin.However, phylogenetic trees have some anomalies.For example, most Sox8a and Sox8b are clustered into diferent branches, which are close to Sox10 and Sox9 in the evolutionary tree structure  [13,36].Tis may be attributable to the third WGD event, leading to all members of the SoxE subfamily having two parallel homologous genes.Te Sox8a and Sox8b of founder fsh are more likely to act as substitutes for Sox10 and Sox9, respectively, thereby supplementing evolutionary adaptation and innovation.

Gene Patterns for Turbot SoxE in Diferent Tissues.
SoxE TFs are involved in diferent physiological and biochemical events through the activation or inhibition of specifc targets, depending on tissue and development [6,21].Tis study analyzed gene patterns for six turbot SoxE genes in 11 adult tissues.Expression patterns were observed for all turbot SoxE genes (Figures 4(a)-4(f )).Results showed that turbot Sox9a and Sox9b had higher levels in the gill, while the other SoxE genes had very low or negligible expression levels in the liver and gill.Notably, most SoxE genes expressed relatively higher in brain and intestine, and Sox8b as well as Sox10b expressed highest in brain compared with their expression in other tissues.In addition, expression of Sox8a in muscle, Sox9a in gill, Sox9b in stomach and Sox10a in testis were all relatively higher in their own expression profles.We also observed the weak expression levels of all SoxE genes in the heart, spleen, and kidney, indicating the role of SoxE genes in these organs' development needed further investigation.Generally, Sox8, Sox9, and Sox10, the TFs belonging to the subgroup E Sox protein family, exert crucial efects on numerous nervous system developmental processes in vertebrates.Tese TFs participate in the original neural crest occurrence and ensure pluripotency maintenance and survival in migratory neural crest stem cells.Moreover, they are crucial regulatory factors for glial norms in the CNS and peripheral NS [7,37].Terefore, several SoxE genes are simultaneously expressed in an organ, suggesting their coordinating activity in performing a function.Such activity is possibly essential for neurogenesis or for maintaining bioprocesses in the turbot brain.
In vertebrates, all three SoxE proteins (SOX8, SOX9, and SOX10) from diverse species exhibit high similarity in HMG nonbox structural conservation and box homology, with 95% similarity in amino acid sequence.Tis may be the reason behind the conserved function of SoxE among species [6,7].For example, SoxE proteins play multiple roles in the specifcation and diferentiation of mammalian sex.Among the SoxE proteins, Sox9 is the most important and conserved sex-determining protein.After sex determination, a complicated positive feedback pathway exists among Sox8, Sox9, and Sox10; the pathway is necessary to maintain spermatogenesis and fertility in males [38].SoxE paralogs Sox8, Sox9, and Sox10 have been isolated from many fsh, and the research on gonad function has also made some progress.In adult Japanese founder, Sox8b, Sox9a, Sox9b, and Sox10a are expressed in the testis and also in the ovary to a certain extent [13].In adult spotted sea bass, Sox8a, Sox8b, Sox9b, and Sox10 are highly expressed in the testis and ovaries,   Journal of Applied Ichthyology whereas Sox9a is almost not expressed in both testis and ovaries [14].According to our results, Sox8a, Sox9a, and Sox10a are upregulated in testis.Te Sox9b upregulation in the ovary indicated its critical regulatory efect on testis and ovary development.Te role of SoxE in turbot sex determination and development requires further exploration in gonads at diferent development stages and should be verifed using gene knockout or RNAi.In addition, almost all SoxE genes were expressed at low levels in the spleen and kidney, indicating that these genes may not participate in spleen or kidney development and function maintenance, a result consistent with those of previous reports [3,13,14].

Conclusion
Te present work identifed six SoxE genes in S. maximus.Gene expression patterns in adult tissues provide crucial data regarding turbot SoxE activities, and the data should be verifed using technologies such as gene knockout and RNAi.Tese fndings would assist in the understanding of fsh SoxE subfamily activity and evolution.

Figure 1 :Figure 2 :
Figure 1: Exon-intron structures in Sox together with motif analysis in Scophthalmus maximus.(a) Exon-intron structures in SoxE from Scophthalmus maximus.Yellow boxes, black horizontal lines, and blue boxes indicate the exons, introns, and noncoding regions, respectively.Te fuchsia box indicates the HMG-box domain.(b) Motif structures in SoxE from Scophthalmus maximus.MEME was employed to identify conserved motifs.Diferent colors indicate diferent motifs.

Figure 4 :
Figure 4: Spatial expression patterns for six SoxE genes in 11 tissues.Te expression of one gene was determined on the basis of its minimum expression level across diverse tissues.Vertical bars indicate the mean ± S.E.P values were determined using the SPSS 20.0 through an independent sample t-test.Diferent letters represent statistical signifcance (P < 0.05) or the common letters across diverse groups represent nonsignifcance.

Table 1 :
SoxE copy numbers across Scophthalmus maximus and other vertebrates.

Table 3 :
Basic information on the turbot SoxE subfamily.
branches.Similar results were also reported in the closely related species of Japanese founder (P.japonicus) and tongue sole (C.semilaevis)