The Wnt Gene Repertoire in Bovinae
To identify the Wnt family members, 45 verified Wnt amino acid sequences were used as the query. These belonged to cattle (Bos taurus, 7), human (Homo sapiens, 19) and mouse (Mus musculus, 19). We used query sequences for genome-wide detection of homologous sequences in Bos taurus, Bos indicus, Hybrid-Bos taurus, Hybrid-Bos Indicus, Bos grunniens, Bos mutus, Bubalus bubalis and Bison bison bison. In Bos taurus, 19 non-redundant Wnt protein sequences were identified that included Wnt1-4, Wnt7A-10A, Wnt7B-10B, Wnt2B, Wnt3A, Wnt5A, Wnt5B, Wnt6, Wnt11, and Wnt16 (Table 1). Wnt family proteins were also recognized in Bos indicus (17), Hybrid-Bos taurus (19), Hybrid-Bos Indicus (19), Bos grunniens (19), Bos mutus (19), Bubalus bubalis (19) and Bison bison bison (18). (Supplementary info File 1 and 2). Two unannotated genes of the Wnt family, ENSBMUG00000022627 and ENSBMUG00000022624, were identified in Bos mutus. Further analysis revealed that they both have the WNT conserved domain, whereas ENSBMUG00000022627 has an incomplete N terminal and ENSBMUG00000022624 has an incomplete C terminal. They showed the highest sequence similarity and a collinearity relationship with Wnt7B, and were named Wnt7B1 and Wnt7B2 preliminary (Supplementary info File 3).
The amino acid lengths of the 19 bovine Wnt proteins ranged from 333 (Wnt8B) to 585 (Wnt4), whereas molecular weight (Mw) ranged from 36.61 to 63.17 kDa, consistent with protein length. Except Wnt3, the rest Wnt family proteins had the isoelectric points (pI) that higher than 8.0, as they contained more basic amino acids than acidic amino acids. Wnt3 was neutral, with a pI of 7.73. All 19 Wnt proteins contained the WNT conserved domain (Supplementary info File 4).
Structural features of bovine Wnt family members
In order to learn about the structural characteristics of bovine Wnt proteins and genes, we projected the conserved motifs and gene structures based on their phylogenetic relationships (Fig. 1). According to the evolutionary clades, the 19 bovine Wnt family menbers clustered into six main subfamilies (Ⅰ-Ⅵ). All the Wnt family proteins shared six conserved domains termed motifs 1, 2, 4, 5, 6, and 7 formed by 50, 49, 41, 29, 28 and 9 amino acids, respectively (Supplementary info File 5). Wnt2, Wnt5A and Wnt5B of the first subfamily and Wnt3 of the third subfamily have all ten motifs. Wnt2B, Wnt3A and Wnt4A have nine motifs, since Wnt2B lacks 9, Wnt3A and Wnt4A lacks 10. Wnt7A, Wnt7B, Wnt10A, Wnt10B and Wnt1 (lacking 9 and 10) and Wnt1 (lacking 8 and 10) have eight motifs. Wnt8A, Wnt8B, Wnt11 and Wnt16 comprise seven motifs lacking 8, 9 and 10. Wnt9A and Wnt9B comprise six motifs lacking 3, 8, 9 and 10.
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 areas, 3’UTR and 5’UTR, were also variable. Despite this variability in CDS, introns and UTRs, the Wnt members in the same evolutionary subfamily tend to possess similar patterns in gene structures and conserved motifs.
Phylogenetic relationship of Wnt proteins in different organisms
Phylogenetic analysis can provide a reference for understanding functional diversification of the Wnt family in Bovinae. So, phylogenetic analysis was conducted, which included eight Bovinae species (Bos taurus, Bos indicus, Hybrid-Bos taurus, Hybrid-Bos Indicus, Bos grunniens, Bubalus bubalis, Bos mutus, and Bison bison bison) and Homo sapiens and Mus musculus. Since Wnt proteins in model organisms (human and mouse) have been studied extensively, they were also included in this study. Finally, 186 amino acid sequences from these ten species were aligned to generate a nonrooted Neighbor-Joining (NJ) tree (Fig. 2). It revealed that Wnt family proteins could be subdivided into 12 proposed subfamilies, including Wnt1–11 and Wnt16 subfamily. There were seven subfamilies that contain two Wnt members, they are subfamily 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), respectively.
Chromosomal distribution and collinearity analysis of Wnt genes
Wnt genes were mapped on nine chromosomes in organisms of Bovinae (Fig. 3). The bovine Wnt genes showed a similar distribution with the other five species. However, the order of Wnt1 (30832513-30835596 bp) and Wnt10B (30841487-30847512 bp) in Chr 5 and Wnt3A (3035810-3087361 bp) and Wnt9A (3155441-3163239 Mb) in Chr 7 of Bos taurus was reversed from that in Bos grunniens. In addition, compared with Bos taurus, Wnt9B and Wnt16 were lacking in Bos Indicus.
Genome collinearity analysis revealed a satisfactory corresponding relationship between chromosomes of Bos taurus and Hybrid-Bos Indicus, Hybrid-Bos taurus, Bos indicus and Bos grunniens, respectively (Fig. 4a). Although the chromosome number is different between cattle (2 N = 60) and buffalo (2 N = 50), there is also large chromosome homology between these two species. Also, collinearity modules explain the difference in the position of Wnt gene family between cattle and the other five species in Bovinae. For instance, the position variation of Wnt2B, Wnt11, Wnt1 and Wnt10B between Bos taurus and Bos grunniens may be caused by complex intra-chromosomal translocation events (Fig. 4b), whereas Wnt3 and Wnt9B are distributed on different chromosomes between cattle and buffalo (bovine Chr 19 and buffalo Chr 3), which may be caused by inter-chromosomal rupture or fusion during the evolutionary process (Supplementary info File 6).
Expression analysis of Wnt genes in different tissue
The functionally related genes tend to show a co-expression patterns and these genes often regulate biological processes collaboratively. To explore the expression patterns of the Wnt gene family during adipogenic differentiation, 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 diverse 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 Wnt family and its receptors, FZD gene family, displayed similar and overlapping expression patterns in the 60 tissues, suggesting their broad and coordinated regulatory role in life activities.
PPARγ, a marker gene for adipocyte differentiation, showed high expression in Group a which included omental, intramuscula, subcutaneous and mammary gland fats. And CTNNB1, FZD1, FZD5, FZD6 and Wnt2B clustered and showed the most similar expression pattern with PPARγ. Further analysis of the five different fat tissues revealed that CTNNB1, a core gene of Wnt signaling pathway, showed high expression and similar expression patterns with PPARγ (Fig. 5b).
Isolation and induced differentiation of bovine primary adipocytes
To explore the expression patterns of the Wnt gene family during adipocyte differentiation, primary adipocytes collected from subcutaneous adipose tissue of calves were induced. The results of oil red O staining showed that lipid droplet gathered together in adipocytes induced for 10 days compared to preadipocytes (Fig. 6a). Further analysis revealed that the absorbance of the differentiated adipocytes at 260nm was significantly higher than that of the preadipocytes (Fig. 6b). Also, The adipogenic marker genes (PPARγ, CEBPβ, FABP4 and LPL) were up-regulated (Fig. 6c). These results indicate that an induced differentiation system of primary adipocytes was established successfully, which can be used in the subsequent gene expression analysis.
Expression analysis of Wnt genes during adipocyte differentiation
A qPCR analysis was conducted to detect the expression of Wnt genes and their receptors (FZD genes) at 0, 2, 6, and 10 days during adipocytes differentiation (Fig. 7). The members of Wnt8B, Wnt11, Wnt16 and their receptors Fzd1, Fzd2, Fzd3, Fzd4, Fzd6 showed a relatively high expression in preadipocytes and then reduced with the process of induced differentiation, suggesting that they may collectively play a role in keeping adipocytes undifferentiated. The members of Wnt2, Wnt6, Wnt9B, Wnt10A and their receptors Fzd9, Fzd10 showed an up-regulated tendency, indicating that they may play a regulatory role during adipocyte differentiation. Furthermore, Wnt2B, Wnt4, Wnt8A and Fzd5, Fzd8 reached the lowest expression on the second day and displayed a similar overall trend of expression.