2 University of Chinese Academy of Sciences, Beijing 100049, China;
3 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266000, China;
4 National & Local Joint Engineering Laboratory of Ecological Mariculture, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
5 Laboratory for Marine Fisheries and Aquaculture, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266000, China
Genes encoding Wnt ligands are restricted to multicellular animals and have important roles during the complicated developmental processes characteristic of these species (Nusse and Varmus, 1992; Logan and Nusse, 2004). Much has been learnt about Wnt genes in metazoan model animals (Gordon et al., 2005; Marikawa, 2006; Garriock et al., 2007; Lavery et al., 2008; Simons and Mlodzik, 2008; Fagotto, 2014), particularly in the Deuterostomia and Ecdysozoa, since the first report of Wnt1 as a protooncogene in mouse in 1982 (Nusse and Varmus, 1982); however, these genes have not been well studied in non-model animals, and functional studies in the Lophotrochozoa, the third main clade of Bilateria, are limited. In addition to some early reported genomes and Wnt complements, including that of the sponge Amphimedon queenslandica (Porifera) (Adamska et al., 2010; Srivastava et al., 2010), the cnidarian, Nematostella vectensis (Radiata) (Kusserow et al., 2005; Putnam et al., 2007), the echinoderm, Strongylocentrotus purpuratus (Croce et al., 2006; Sodergren et al., 2006), the chordate, Danio rerio (Garriock et al., 2007; Howe et al., 2013), the nematode, Caenorhabditis elegans (The C. elegans Sequencing Consortium, 1998; Korswagen, 2002), and the arthropods Drosophila melanogaster (Adams et al., 2000; Croce et al., 2006) and Daphnia pulex (Janssen et al., 2010; Colbourne et al., 2011), the recently published lophotrochozoan Wnt complements (including those of the Platyhelminthes species, Schistosoma mansoni (Berriman et al., 2009; Riddiford and Olson, 2011), Schmidtea mediterranea (Riddiford and Olson, 2011; Robb et al., 2015), Echinococcus granulosus, Echinococcus multilocularis, and Hymenolepis microstoma (Riddiford and Olson, 2011; Tsai et al., 2013); the annelids, Platynereis dumerilii (Janssen et al., 2010), Capitella teleta, and Helobdella robusta; and the mollusc, Lottia gigantea (Cho et al., 2010; Simakov et al., 2013) have greatly improved our understanding of Wnt gene evolutionary dynamics across the Eumetazoa. The rapid growth in the generation and analysis of genome data in recent years presents an opportunity to investigate the evolution of Wnt genes by thorough analyses of their distribution and function across a broad range of lophotrochozoans. These include species for which Wnt complement studies are lacking or only part of the Wnt genes has been identified: the platyhelminths Taenia solium (Tsai et al., 2013), Schistosoma japonicum (Zhou et al., 2009), and Schistosoma haematobium (Young et al., 2012); the bdelloid rotifer, Adineta vaga (Flot et al., 2013); the brachiopod, Lingula anatina (Luo et al., 2015); the annelid Spirobranchus (Pomatoceros) lamarcki (Kenny et al., 2015); and the quickly accumulating genomes of molluscs including Crassostrea gigas (Zhang et al., 2012), Pinctada fucata (Takeuchi et al., 2016), Patella vulgata (Kenny et al., 2015), Octopus bimaculoides (Albertin et al., 2015), Biomphalaria glabrata, and Aplysia californica.
Here, we report the identification of Wnt genes based on genome data from 20 animals in the superphylum Lophotrochozoa, and we describe the evolutionary dynamics of Wnt gene families through comparisons among closely related species. We characterized the functional correlation of Wnt genes in Lophotrochozoans by comparing their time-course expression patterns. Finally, we verified our hypotheses by studying the dynamic expression patterns of Wnt genes in the Pacific oyster, C. gigas.2 MATERIAL AND METHOD 2.1 Animal materials and ethics statement
All of the Pacific oyster (C. gigas) specimens used in this study were collected from Qingdao, Shandong, China, and acclimatized in seawater at 22℃ for 7 days before use. No specific permissions were required for any of the experimental processes used, and all experiments were conducted according to local and national regulations. Fresh oyster tissues for RNA isolation were isolated, those from two animals mixed, and then frozen immediately and stored in liquid nitrogen. Three biological replicates were conducted for RNA isolation, i.e. a total of six animals were used in the study.2.2 Collection of genome assemblies and annotations
All genome assemblies and annotations were collected from the NCBI Eukaryotic GenomeAnnotation project (Pruitt et al., 2012) (accession numbers are listed in Table S1), except for the P. fucata genome from the genome database (http://marinegenomics.oist.jp/pearl/viewer/download?project_id=36), P. vulgata and S. (Pomatoceros) lamarcki genomes from (https://ora.ox.ac.uk/objects/uuid:6471e7d4-dd34-4eb3-883f-a5c438f11741). Additionally, we included Wnt genes from the annelid P. dumerilii, whose Wnt complement has been well studied (Pruitt et al., 2014), although no genome data are publically available. For species for which genomic-wide protein sets have been deposited in NCBI, the whole protein sets were downloaded in GenPept format. Wnt protein and domain sequences were retrieved by searching the protein database with conserved Wnt family domain accessions (CDD numbers 278536, 302926, and 128408). To confirm the completeness of Wnt complement or to identify all possible Wnt gene loci in the genome assembly, additional similarity searches against genomes were performed using TBLASTN with the domain sequences of all known Wnt proteins as queries. Wnt loci for species with un-annotated genomes were also identified with TBLASTN. The open reading frame (ORF) around a hit region was predicted with the NCBI ORFfinder service, and identified Wnt domains were assessed against the PFAM database (http://pfam.sanger.ac.uk/search) with an e-value threshold of 1e-10.2.3 Sequence alignment and phylogenetic analyses
A multiple alignment of the Wnt domain sequences was produced with MAFFT 7.221 (Katoh and Standley, 2013) using the L-INS-I algorithm. The alignment was trimmed with TrimAl, with parameters -gt 0.9 -st 0.001 -cons 40 (Capella-Gutiérrez et al., 2009). Then, molecular phylogenetic trees were constructed to support Wnt gene classification using three different phylogenetic methods: FastTree (Price et al., 2010), RAxML (Stamatakis, 2014), and MrBayes (Huelsenbeck and Ronquist, 2001). FastTree allows constructing approximately-maximum-likelihood phylogenetic trees quickly. Therefore, it was used to test alignment-trimming results and to preliminarily name Wnt genes. The resulting alignment belonging to the Wnt domain was used to perform maximum-likelihood (ML) phylogenetic analyses with the program RAxML, using the evolutionary model LG+Gamma+Invariant; 1 000 replicates were performed to obtain bootstrap support (BS) values to evaluate the nodal support. To confirm and compare the nodal support, Bayesian inference (BI) phylogenetic analysis was conducted, using MrBayes. Eight Markov chains were run for 1×106 generations, with sampling performed every 100 generations. We used the values, as well as congruence of statistical support (BS from RAxML and posterior marginal probabilities from MrBayes) as indicators of the reliability of different subfamilies. Two trees were constructed: a large tree containing all 360 Wnt genes identified from the 28 species, and a small tree containing well supported groups of Wnt genes from well annotated genomes (from 16 species, Table S1). Some known divergent Wnt genes (discussed later) from the chosen genomes were excluded during construction of the small tree. To further classify the divergent protein sequences mentioned above, e.g. Cel_wnt9_NP_505154, Aqu_ wnt16b_NP_001266204, Aqu_wnt11_XP_003388134, we defined a "standard" Wnt gene set by extracting representatives of Radiata, Lophotrochozoa, Ecdysozoa, and Deuterostomia from each well-supported Wnt subfamily, and then constructed a phylogenetic tree by adding them one by one (Fig.S1).2.4 Criteria for inference of evolutionary relationships
Orthology groups were defined from phylogenetic trees following the criteria adopted by other similar analyses (Gyoja, 2014) with some modifications. In brief, if genes from two or more organisms formed a single clade with a bootstrap value > 50% by ML or a posterior probability > 90% by BI, they were considered to constitute an orthologous subfamily most solidly. Members within the subfamily were then named according to the subfamily name. Where two or more members of a subfamily were identified in one species, they were named "a" (or "aa", "ab"), "b, " "c, " etc. For example, the two D. rerio members from the Wnt6 subfamily are named Wnt6a and Wnt6b, while the four Wnt7 subfamily members are named Wnt7aa, Wnt7ab, Wnt7ba, and Wnt7bb, to reflect the degree of relatedness (e.g., Wnt7aa and Wnt7ab are closer paralogs pair). Gene losses are revealed where a lineage is expected to have a gene based on the inferred ancestral gene subfamily complement, but the gene is not detected. However, this can also reflect missing data, and the genomes analyzed here vary in assembly or annotation quality. Accordingly, gene loss was modeled using Dollo parsimony, which allows multiple, independent losses (Riddiford and Olson, 2011), and we only inferred a loss where a gene was absent from two or more sister lineages.2.5 Time-course microarray or RNAseq data collection and analysis
Time-course expression patterns of Wnt genes were determined for six species, as follows: microarray data from 61 developmental stages of D. rerio were derived from a previous report (Domazet-Lošo and Tautz, 2010); FPKM (fragments per kilobase of transcript per million mapped reads) values for D. melanogaster and C. elegans Wnt genes were retrieved from the modENCODE project (Li et al., 2014); and RNAseq data for the different developmental stages of three other species (N. vectensis, L. anatina, and C. gigas) were downloaded from the NCBI SRA database and analyzed with Hisat2, StringTie, and Ballgown (Pertea et al., 2016), using NCBI annotation information. Details of these data are provided in Table S2. Correlation coefficient for the Wnt expression matrix was calculated by cor function of the R software (Ihaka and Gentleman, 1996). The probability values of individual correlations were determined by t-test followed by Holm's adjustment for multiple comparisons.2.6 RNAisolation, cDNAsynthesis, and quantitative reverse transcription (qRT-)PCR
RNA isolation and cDNA synthesis from different oyster tissues were conducted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and a PrimeScript RT reagent kit (TaKaRa, Shiga, Japan), respectively, according to the manufacturers' instructions. qRT-PCR of the C. gigas Wnt genes was conducted as previously described, with three technical replicates in each of three biological replicates (Qu et al., 2014). The EF-1α gene was used as an internal control (Du et al., 2013) and the 2-ΔΔCt method was used to calculate the expression level of target genes (Livak and Schmittgen, 2001). All primers used for qRT-PCR are listed in Table S3.3 RESULT AND DISCUSSION 3.1 Identification of Wnt genes from selected Eumetazoa species
We screened 20 lophotrochozoan genomes, 12 of which had never been assayed for Wnt complement at the genome-wide level, together with the latest assemblies of seven other metazoan genomes. All thirteen known Wnt gene subfamilies, i.e., WntA, Wnt1-11, and Wnt16, were identified (Fig. 1). As previously reported, some flatworm Wnt proteins are highly divergent (Riddiford and Olson, 2011) and thus, failed to be grouped within the thirteen known subfamilies. Similar results have been reported for H. robusta (Cho et al., 2010), in which some Wnt genes show greater structural variability. For example, the H. robusta Wnt2 has only 13 cysteines and Wnt7 has 27, while most Wnt proteins have normal cysteine counts between 23–24 (Cho et al., 2010). Three Amphimedon Wnt genes (Srivastava et al., 2010; Holstein, 2012) as well as C. elegans mom-2 and egl-20 (Prud'homme et al., 2002; Janssen et al., 2010) have also proven difficult to classify.
All assayed flatworm Wnt genes clustered into five groups with strong support (Figs.S2, S3). One group was within the previously defined subfamily, Wnt5 (Riddiford and Olson, 2011). Another group was in subfamily Wnt11, with strong support. Among the other three groups, they also showed close relationships (with strong support) with the neighboring previously defined subfamilies, we are careful to name them after the corresponding subfamily because these groups were isolated from the known subfamilies, whose members are broadly from Cnidaria and Bilateria. At the same time, our results for these three groups seemed to conflict with a previous classification (Riddiford and Olson, 2011). As previous studies have made good classification on two of these groups through expression patterns and other characters (Gurley et al., 2010; Koziol et al., 2016), we thus kept the current classification. We named these groups "Wnt1, " "Wnt2, " and "Unclassified" accordingly. The Wnt1 group of flatworm is a sister of the Wnt10 subfamily in our tree (BS support 29%, Fig.S2, and BI support 0.44, Fig.S3); the Wnt2 is a separate group in both ML and Bayesian trees; the Unclassified group contains members of the annelid H. robusta, together with genes from the bdelloid rotifer A. vaga. As in the previous report, Wnt4 is a closely related subfamily (BS support 25%, Fig.S2, and BI support 0.64, Fig.S3). The flatworm Wnt genes indicate the problem of family classification through phylogeny analysis with the sequences, especially for highly divergent genes. More evolutionary and functional details are needed before correctly classifying such genes. The three sponge Wnt genes have been previously annotated; one was suggested to belong to the Wnt11 subfamily, another to Wnt16, and one remained unclassified (Adamska et al., 2010). Our results based on the phylogenetic tree to which these genes were added one by one support the classification of one Wnt11 (Fig.S1) and two Wnt16 (Fig. 1 and Fig.S1) genes. One-by-one phylogenetic analysis of C. elegans mom-2 classified it into the subfamilies Wnt9, as previously reported (Prud'homme et al., 2002).
The other Wnt genes were classified into known subfamilies with high support. All reported Wnt genes were identified in the genome of each species, except for D. rerio, for which 5 Wnt2 and 3 Wnt7 genes were previously reported (Garriock et al., 2007), while we identified 3 and 4, respectively (Table S4). Our gene counts correspond with the genome annotation data in the Zebrafish Model Organism Database (Howe et al., 2013). The previous report may have assayed only genes with a full Wnt domain, while we used all possible Wnt loci identified across the whole genome. Most of the gene products from the additional loci are characterized by a partial Wnt domain, which may be because of genome misassemblies or pseudogenes. In N. vectensis, S. purpuratus, L. gigantea and H. robusta, we identified four, one, one, and four additional possible Wnt loci with partial Wnt domain, respectively. Wnt counts for other, unreported species were between 4 and 39 (Fig. 1, Tables S1, S4). The genome sequences of the mollusc P. vulgata and the annelid S. lamarcki were of poor quality. There were only contigs, with N50 only 3 160 bp and 1 939 bp, respectively. We identified 34 and 39 possible Wnt loci in the genome assemblies of P. vulgata and S. lamarcki, respectively, which are obviously abnormal numbers when compared to other relative species. However, we included these genes in the analysis as they could provide information for verifying our gene loss hypothesis in certain lineages.3.2 Phylogenetic analyses and repertoire of Wnt genes
We adopted a commonly used approach for constructing Wnt gene phylogenetic trees based on the well-annotated gene sequences of vertebrate (D. rerio), and ecdysozoan (D. melanogaster and C. elegans) species. As no outgroup can be used for Wnt genes (Schubert et al., 2000), an unrooted phylogenetic tree was produced. Based on the tree generated, Wnt genes were subdivided into orthologous Wnt subfamilies and named according to the orthologous subfamily name (Fig. 2). As FastTree uses only the approximately-maximum-likelihood method to construct a tree within seconds, in this study, we mainly used RAxML and MrBayes, the topologies of which were largely the same (Figs. 2, S2, S3).
As previously reported, Wnt3 was identified only in species classified as Cnidaria and Deuterostomia (Prud'homme et al., 2002; Janssen et al., 2010; Riddiford and Olson, 2011), and Wnt9 was only found in Bilateria (Croce and McClay, 2008; Lengfeld et al., 2009; Cho et al., 2010). It has been speculated that Wnt8 has undergone lineage-specific loss in Mollusca (Cho et al., 2010; Riddiford and Olson, 2011); however, we identified Wnt8 in two mollusks, C. gigas (Bivalvia, Mollusca) and O. bimaculoides (Cephalopoda, Mollusca), suggesting that the loss may have been specific to Gastropod mollusks. Previous reports used only the gastropods P. vulgata and L. gigantea as representatives of mollusks; thus, the conclusion that mollusks are characterized by a loss of Wnt8 (Riddiford and Olson, 2011) on the basis of data lacking other classes of Mollusca has been refined by our study. Similarly, although we have shown the loss of Wnt8 in four species from both the Patellogastropoda (P. vulgata and L. gigantea) and Heterobranchia (A. californica and B. glabrata) clades of Gastropoda, our claim requires further validation in other mollusks.
Based on genome analysis of the ancient sponges, Wnt genes appeared in the metazoan last common ancestor, followed by gene duplication in the period between demosponge-eumetazoan and cnidarian-bilaterian last common ancestors (Adamska et al., 2010). However, the evolutionary relationships between subfamilies are difficult to infer through phylogenetic analysis because of insufficient resolution (Prud'homme et al., 2002). For example, the BS support for most sister groups is lower than 20% (Fig. 2). Therefore, it is difficult to infer evolutionary trajectories since the origin of Wnt genes, although studies have suggested that the three Amphimedon Wnt genes belong to Wnt11 and Wnt16 subfamilies. Meanwhile, some sister groups showed relatively strong BS support, including Wnt4–Wnt11 (51%), Wnt1–Wnt6 (76%), and Wnt9–Wnt10 (46%), in accordance with a previous report (Cho et al., 2010). These sister subfamilies have been hypothesized to have arisen from specific gene duplications (Prud'homme et al., 2002; Cho et al., 2010). Linked genes are considered as evidence of ancestral arrangements when they are shared by more than one lineage. Moreover, the closer genes are located to each other in a genome, the more likely they are a pair evolved by tandem duplication. In this context, the Wnt10-Wnt6-Wnt1-Wnt9 cluster, which widely exists in Metazoan genomes (Kusserow et al., 2005; Cho et al., 2010), should represent a Wnt tandem duplication from an ancestral gene. Such tandem duplication within a single Wnt subfamily has been reported in zebrafish Wnt8 (Ramel et al., 2004). In this study, we identified as many as 8 potential Wnt8 loci tandemly distributed in the scaffold NW_001834285.1 of N. vectensis. However, the ORFs of six loci are interrupted by a stop codon and produce only a partial Wnt domain, suggesting these may be misassemblies or pseudogenes. Possible tandem-duplicated Wnt loci have been also identified in Wnt7 of N. vectensis, Wnt10 of L. gigantea, Wnt1 and Wnt10 of L. anatina, Wnt13 of S. mediterranea and S. haematobium (Fig.S4).3.3 Expression patterns of clustered Wnt genes
To investigate the functions of Wnt genes further, we explored their expression patterns by analyzing published expression data from six animals (N. vectensis, D. rerio, D. melanogaster, C. elegans, L. anatina, and C. gigas) during their ontogeny. The results indicated that few Wnt genes are expressed maternally (Table S2). In contrast, expression of the majority of Wnt genes began during embryogenesis, corresponding with their primary function in the control of cell-cell interactions during development and adult tissue homeostasis. Additionally, we calculated the correlation between expression patterns of different Wnt genes in six species (Fig. 4). In this way, gene functional relationships could be inferred from the time-course expression patterns.
In oyster, the genes in the Wnt10-Wnt6-Wnt1-Wnt9 cluster showed an obviously consistent expression pattern (Fig. 4c), indicating the possible interaction between these adjacent genes. Although clusters in Lingula are more diverse because of the duplication of Wnt10 and Wnt1 (Fig. 3), a high correlation was observed between Wnt10 and Wnt6 (Fig. 4b).
Expression correlation was also indicated from the tissue expression pattern of oyster Wnt genes. We assayed the expression levels of the 13 oyster Wnt genes through real-time qPCR on seven main tissues. The results indicated that the linked Wnt10, Wnt6, Wnt1, Wnt9a all had high level expression gill and mantle (Fig. 5). However, differences were also observed between these four genes. For example, Wnt1 showed specific expression in gill and mantle, while Wnt6 also expressed in gonad. Additionally, Wnt9a also showed high expressed in transparent adductor muscle and gonad respectively. In this context, the clustered Wnt genes may be coordinately regulated during certain development stages or in some tissues, but also have diverged biological functions in other biological processes.
At the same time, no strong correlation between clustered genes was detected in fruit fly, C. elegans, and zebrafish (Fig. 4d, e, f). If the coexpression of Wnt10-Wnt6-Wnt1-Wnt9 genes in lophotrochozoans reflects their functional correlation and the members indeed arose by tandem duplication, the diverged expression pattern in other animals should reflect subfunctionalization of the duplicated genes as predicted by the duplication degeneration complementation model (Force et al., 1999) or neofunctionalization.4 CONCLUSION
Members of the Wnt gene family are widely distributed in the Eumetazoa. Wnt genes arose rapidly during the early evolution of animals. Loss of members of some Wnt subfamilies occurred in certain clades later in evolution, including Wnt3 from the Ecdysozoa and Lophotrochozoa, Wnt8 from the Gastropoda, and the simultaneous loss of Wnt2 and Wnt11 from D. melanogaster, C. elegans, and S. purpuratus. A number of genes exhibited elevated diversification relative to the main subfamilies, and were therefore assigned to the temporary groups, Unclassified. Other than these highly dynamic subfamilies, Wnt genes tended to appear in clusters, suggesting the occurrence of multiple tandem duplication events during Wnt family evolution. A number of Wnt orthologs exhibited similar time-course expression patterns in different animals, indicating their possible similar functions. The correlation of expression for genes of the Wnt10-Wnt6-Wnt1-Wnt9 cluster was identified in oyster, suggesting functional conservation of these genes.5 DATA AVAILABILITY
The authors declare that all data supporting the findings of this study are available within the article and its supplementary files.6 ACKNOWLEDGEMENT
We thank the High Performance Computing Center (HPCC) at the Institute of Oceanology, Chinese Academy of Sciences for providing the supercomputer cluster to conduct gene expression analyses.Electronic supplementary material
Supplementary material (Supplementary Figs.S1–S4 and Tables S1–S5) is available in the online version of this article at https://doi.org/10.1007/s00343-018-7132-7.
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