Genome-wide identification and expression profiling analysis of Wnt family genes affecting adipocyte differentiation in cattle

The Wnt family features conserved glycoproteins that play roles in tissue regeneration, animal development and cell proliferation and differentiation. For its functional diversity and importance, this family has been studied in several species, but not in the Bovinae. Herein we identified 19 Wnt genes in cattle, and seven other species of Bovinae, and described their corresponding protein properties. Phylogenetic analysis clustered the 149 Wnt proteins in Bovinae, and 38 Wnt proteins from the human and mouse into 12 major clades. Wnt genes from the same subfamilies shared similar protein motif compositions and exon–intron patterns. Chromosomal distribution and collinearity analysis revealed that they were conservative in cattle and five species of Bovinae. RNA-seq data analysis indicated that Wnt genes exhibited tissue-specific expression in cattle. qPCR analysis revealed a unique expression pattern of each gene during bovine adipocytes differentiation. Finally, the comprehensive analysis indicated that Wnt2B may regulate adipose differentiation by activating FZD5, which is worthy of further study. Our study presents the first genome-wide study of the Wnt gene family in Bovinae, and lays the foundation for further functional characterization of this family in bovine adipocytes differentiation.

preadipocytes differentiation by inhibiting the expression of C/EBPα and PPARγ 8,13,14 . In porcine adipose-derived mesenchymal stem cells (AMSCs), Wnt3A inhibits the potential of adipogenic differentiation and alters the cell fate from adipocytes to osteoblasts 15 . In murine embryonic mesenchymal cell line C3H10T1/2, the Wnt3-Fz1 chimera is an inhibitor of differentiation into the adipocyte lineage and a potent activator of differentiation into osteoblasts 16 . In addition, the Wnt gene family also functions via the non-canonical Wnt signaling pathway. In hMSCs, the inhibition of Wnt3A suppressed the non-canonical Wnt/JNK pathway and enhanced adipocyte differentiation whereas its activation enhanced osteoblast differentiation 11 . In 3T3-L1 preadipocytes, Wnt4 and Wnt5A positively regulated adipogenesis at the initial stage of the differentiation process by activating PKC and calcium/calmodulin-dependent kinase II 17 .
So far, the Wnt family has been extensively studied in some species, e.g., Drosophila melanogaster, Tribolium castaneum, Acyrthosiphon pisum, Anopheles gambiae, and Apis mellifera [18][19][20][21][22] . Spatiotemporal expression profile revealed that some Wnts might participate in early embryonic development as well as in adult organ/ tissue morphogenesis and homeostasis, whereas others may be involved in coping with challenging intertidal environments 20 . Similarly, research on Wnts and Wnt signaling pathway has mainly focused on regulating embryonic development in cattle 23,24 . For instance, Wnt11 activate JNK to improve the competence of the embryo to develop to the blastocyst stage 25 . The expression of Wnt6 was upregulated in bovine trophectoderm 25 , consistant with the previous study which found that it can promote differentiation of primitive endoderm 26 . Wnt7A inhibited the PCP pathway to improve blastocyst development, without affecting the amount of CTNNB1 27 . These findings stimulated our interest and guided us to explore the evolution of the Wnt gene family in Bovinae and function in adipocyte differentiation.
In the present study we have performed a genome-wide identification and evolutionary analysis of the Wnt gene family in eight species of Bovinae. And the expression profiles in different tissues and stages during adipocytes differentiation were also analyzed in cattle based on transcriptome data and qPCR. Our study provides a basis for understanding the distribution of Wnt genes and will contributes to further elucidate their potential function in adipocytes differentiation.
Two unannotated genes from the Wnt family, ENSBMUG00000022627 and ENSBMUG00000022624, were also identified in Bos mutus. Further analysis revealed that they both possessed the WNT conserved domain; however, ENSBMUG00000022627 had an incomplete N-terminus and ENSBMUG00000022624 had an incomplete
Introns, coding sequences (CDS) and untranslated regions (UTR) were variable among the Wnt gene family. For instance, the length of Wnt genes ranged from 3,084 nt (Wnt1) to 64,231 nt (Wnt7A), mainly due to intron variation. The number of CDS varied from 3 to 6, and the length and layout of the noncoding regions (3'UTR and 5'UTR) were also variable. Despite this variability, the Wnt members in the same evolutionary subfamily tend to possess similar gene structures patterns and conserved motifs.
Phylogenetic relationship of Wnt proteins in different organisms. Phylogenetic analysis can provide a reference for understanding the functional diversification of the Wnt family in Bovinae. Our phylogenetic analysis included eight species of Bovinae. We also included the Wnt proteins from well-studied model organisms (human and mouse). Of these ten species, 186 amino acid sequences were aligned to generate a non-rooted Neighbor-Joining (NJ) tree ( Fig. 2), which showed 12 proposed subfamilies, including Wnt1-11 and Wnt16. There were seven subfamilies containing two Wnt members: I (Wnt7A and Wnt7B), III (Wnt3 and Wnt3A), VI (Wnt2 and Wnt2B), VII (Wnt5A and Wnt5B), IX (Wnt10A and Wnt10B), XI (Wnt9A and Wnt9B), XII (Wnt8A and Wnt8B).
Chromosomal distribution and collinearity analysis of Wnt genes. Wnt genes were mapped on nine chromosomes of cattle (Fig. 3) and the distribution was found to be similar in the other five species. However, the order of Wnt1 (30, Genome collinearity analysis revealed a satisfactory corresponding relationship between the chromosomes of Bos taurus and Hybrid-Bos Indicus, Hybrid-Bos taurus, Bos indicus, and Bos grunniens (Fig. 4A). Although the chromosome number differed between cattle (2 N = 60) and buffalo (2 N = 50), the level of chromosome homology was high between these two species. Also, collinearity modules explained the difference in the position of the Wnt gene family in cattle relative to the other five species in Bovinae. For instance, the position variation of Wnt2B, Wnt11, Wnt1 and Wnt10B between Bos taurus and Bos grunniens might have been caused by complex intra-chromosomal translocation events (Fig. 4B). Wnt3 and Wnt9B are distributed on different chromosomes between cattle and buffalo (bovine Chr 19 and buffalo Chr 3). This may be caused by inter-chromosomal rupture or fusion during the evolution (Supplementary Info File 6).

Expression analysis of Wnt genes in different tissues.
Functionally related genes tend to show a coexpression patterns and often regulate biological processes collaboratively. To explore the expression patterns of the Wnt gene family, we investigated their expression levels in 163 samples of 60 tissue types. The Wnt genes along with other 13 closely related genes can be classified into four groups (I to IV) ( Fig. 5A) according to their differential expression patterns in tissues. Accordingly, the 60 bovine tissue types also clustered into four main clades (a-d) based on the expression patterns of all the 31 genes including Wnt family. The members of the Wnt family and their receptors, the FZD gene family, displayed overlapping expression patterns, suggesting a coordinated regulatory role. PPARγ, a marker gene for adipocyte differentiation, showed high expression in Group a (omental, intramuscular, subcutaneous and mammary gland fats). The cluster formed by CTNNB1, FZD1, FZD5, FZD6 and Wnt2B showed a similar pattern of PPARγ in expression.
Further analysis of the five different fat tissues revealed that CTNNB1, a core gene of Wnt signaling pathway, showed high expression and similar expression pattern as PPARγ (Fig. 5B). High expression was observed for Wnt7B, Wnt9B and FZD3 (adult mammary gland fat) and Wnt2B and FZD2 (embryonic subcutaneous fat). Expressions of C/EBPα, FZD1 and FZD8 were higher in some tissues (omental fat, intramuscular fat (IMF) and subcutaneous fat of adult cattle) than in others (mammary gland fat of adult cattle and subcutaneous fat of embryo). Knowledge of these patterns will provide useful information in bovine fat research. Meanwhile, the clustering analysis of tissue expression pattern revealed that intramuscular fat and subcutaneous fat of adult cattle got together firstly, indicating that they were the most similar among the five types of fat. This also suggests that primary adipocytes isolated from subcutaneous fat can be used for preliminary expression pattern validation of the Wnt gene family. www.nature.com/scientificreports/ Isolation and induced differentiation of bovine primary adipocytes. Meat tenderness and juiciness are affected by IMF content, whereas it is too limited to be sampled. Subcutaneous fat is significantly associated with IMF 28 , which is consistent with our clustering results (Fig. 5B). To explore the expression patterns of the Wnt gene family during adipocyte differentiation, primary adipocytes collected from subcutaneous adipose tissue of cattle were induced. Ten days after induction, Oil red O staining showed a greater extent of lipid droplet accumulation in adipocytes than in preadipocytes (Fig. 6A). The absorbance at 510 nm was significantly higher in differentiated adipocytes than in preadipocytes (Fig. 6B). Furthermore, the adipogenic marker genes (PPARγ, C/EBPα and FABP4) were all up-regulated (Fig. 6C). These results indicate that the induced differentiation of primary adipocytes was successful and could be used in the subsequent gene expression analysis.
Expression analysis of Wnt genes during adipocyte differentiation. qPCR analysis was conducted to detect the expression of Wnt genes and their Fzd receptors at different time points (0, 2, 6, and 10 days) during adipocytes differentiation (Fig. 7). Wnt8B, Wnt11, Wnt16 and their receptors (Fzd1, Fzd2, Fzd3, Fzd4, Fzd6) showed high levels of expression in preadipocytes. These levels were reduced after induction, suggesting a collective involvement in keeping adipocytes undifferentiated. Wnt2, Wnt6, Wnt9B, Wnt10A and their receptors (Fzd9, Fzd10) were significantly up-regulated, indicating a regulatory role during adipocyte differentiation. Furthermore, Wnt2B, Wnt4, Wnt8A and Fzd5, Fzd8 reached the lowest expression at the second day and displayed a similar overall trend of expression.

Discussion
Structural features of bovine Wnt family proteins and genes. The core motifs and domains of a protein determine its function and activity 29 . Gene families usually encode proteins that share similar motifs and act synergistically 30 . All the 19 bovine Wnt members have six conserved amino acid sequences (Motifs 1, 2, 4, 5, 6 and 7), pointing to a common functional site. Four members (Wnt2, Wnt3, Wnt5A, and Wnt5A) have ten motifs, whereas the other 15 members lack 1 to 4 of these motifs. Thus, it is likely that these four motifs (Motifs 3, 8, 9 or 10) are not located at the core of the Wnt protein domain.
Since the introns and UTRs vary in length and layout, the distribution of CDSs in the Wnt genes was also variable. Their sequences and conserved motifs were similar, and all possessed the WNT conserved domain. This might play a role in maintaining their three-dimensional structure and binding function.
The phylogenetic relationships of Wnt family proteins. Phylogenetic analysis provides a credible way to explore the relationship between amino acid sequence similarity and function of proteins in the same family 31 . In multicellular eukaryotes, the Wnt family proteins is divided into 13 subfamilies. For instance, a total of 11 (Zhikong scallop), 12 (Yesso scallop, Pacific oyster) and 13 (Lingula anatine, Plathynereis dumerlii, Lottia gigantean, Crassostrea gigas, etc.) subfamilies have been identified previously 20 . In the Bovinae, Wnt proteins were classified into 12 subfamilies but lacked WntA. This was consistent with previous studies reporting that vertebrates all have reserved subfamilies except for WntA 32,33 .
Although the function of the Wnt family is highly conserved, several members have been lost in many species after the complete set of Wnt genes emerged in cnidarians 34,35 . Among the eight species of Bovinae, Wnt7B was missing in Bison, while Wnt9B and Wnt16 were missing in Bos indicus. The Wnt9 subfamily specific to Bilateria was also found to be absent from Chlamys farreri 20 . It remains unclear whether these genes were not identified due to limitations in genome assembly or whether they were lost during evolution.
In the phylogenetic relationship, two genes from distinct species that are located in the same clade are defined as orthologs 36 . The orthologous gene pairs among cattle and the other five bovine species were identified based on homologous relationships. Orthologous Wnt members first clustered in a single clade, indicating that they were conserved among different species.

Collinearity analysis of Wnts in Bovidae.
Genome-wide collinearity analysis of Wnt genes provided key information on the function and evolution in the Bovidae. Gene duplication events can cause gene family expansion during genome evolution 37 . Indeed, both tandem and segmental duplications are responsible for the expansion of the Wnt family in Bovinae (Figs. 3, 4). The members of the Wnt gene family were distributed across nine chromosomes in the six selected species. WNT9B and WNT3 were tandem repeats on chromosome 19 of Bos Taurus, while WNT9B was missing on chromosome 19 of Bos Indicus (Fig. 3). This deletion may be due to the absence of a tandem duplication event or the loss of WNT9B after the tandem repeats occurred in Bos indicus during evolution. However, the causes, processes, and outcomes of this evolutionary event are still unclear and need more research; such work may further help to clarify the function of WNT9B.
Due to the different starting points of chromosome annotation among species, the arrangement of genes might be totally reversed. For instance, in Bos taurus and Hybrid-Bos taurus, the order of Wnt6, Wnt10A and Wnt4 in Chr2, Wnt2 and Wnt16 in Chr4, and Wnt3A, Wnt9A and Wnt8A in Chr 7 was opposite.
Intra-chromosomal translocation and rearrangement during species evolution also lead to the changes in gene arrangement 38,39 . For example, the position of Wnt2B was altered in Bos taurus and Bos grunniens due to the inversion of large segments within the chromosome (Fig. 4B). Furthermore, the locations of Wnt genes change from collinear (conserved in the same order) to syntenic (not necessarily in the same order) 40 42 . For instance, Wnt3 formed a chimera with FZD1 to regulate the canonical Wnt signaling pathway 16 . The observed overlaps in the expression of 19 Wnt and 10 Fzd members in 60 tissue types suggest a coordinated and selective regulatory role (Fig. 5). Group IV (PPARG , CTNNB1, FZD1, FZD5, FZD6 and WNT2B) was highly expressed in four fat tissues (omental, intramuscular, subcutaneous and mammary gland). Meanwhile, previous studies, carried out in humans, showed that Wnt2B and FZD5 exhibited physical interactions and coexpression relationships ( Table 2) and displayed similar expression patterns during the differentiation of bovine adipocytes (Fig. 7). Since PPARG is a marker gene for adipocyte differentiation and CTNNB1 is a core gene in the www.nature.com/scientificreports/ canonical Wnt signaling pathway, WNT2B might bind to its receptor FZD5 to regulate adipogenic differentiation through the canonical Wnt signaling pathway. Adipogenic differentiation is a well-organized and complicated process regulated by various genes. Analysis of the interactions between the Wnt and the Fzd family is essential to explore their roles. An integrated network for the Wnt and Fzd gene family and their interacting genes were constructed by STRING (https:// string-db. org/) 46 and visualized by Cytoscape (Supplementary Info File 7) 44 . To ensure the accuracy of this interaction network, only sources from literature mining and experimental verification were selected. Analysis showed there were extensive and complex direct or indirect relationships between the Wnt and Fzd gene family. Since such studies have not been carried out in cattle, we used GeneMANIA (https:// genem ania. org/) to mine their relationship in human. We observed clear bias in the Wnt family members in terms of their ability to recognize their Fzd receptors (Table 2) 16,42 . Collectively, these results revealed that the Wnt and Fzd genes interact and activate the canonical and/or non-canonical Wnt signaling pathway, thus regulating adipocyte differentiation. The results provide a foundation for further study Wnt genes and the regulation of adipocyte differentiation in cattle.  (7), human (19) and mouse (19) were obtained from the UniProt database (https:// www. unipr ot. org/) and used to query potential Wnt genes via BLASTP with a threshold e-value of 10 -5 . The HMM of Wnt (PF00110) was down-    Isolation, culture, and induction differentiation of bovine primary adipocytes. Primary adipocytes were isolated and cultured from subcutaneous adipose tissue of the cattle in the Zerui Ecological Breeding Farm using the Type I collagenase digestion method. Induction of primary adipocytes differentiation 62 and Oil red O staining 63 were performed as previous described. The absorbance of the substance extracted from adipocytes at 0 day and 10 day after induction was also measured at 510 nm with isopropanol as a control.