Mink is one of the most important fur animals in the world. Previous studies only focused on the diversity and formation mechanism of fur color, but in this study, we not only explored the formation mechanism of fur color but also focused on key genes and pathways in the process of fur development. Three young and three adults of the short-haired black mink breed and three young and three adults of red-eyed white mink were selected as our experimental samples. A comparison of skin transcriptome between different groups was performed to achieve our research objectives. Animal coat color is a qualitative character, which is determined by the content of eumelanin and pheomelanin in the hair[19]. In IFPCS (International Federation of Pigment Cell Societies), 661 genes related to coat color have been published so far[20]. The research on fur development mainly focuses on the molecular mechanism of hair follicle development, including keratin[21, 22], keratin-associated proteins[23], signal pathway[24], gene family[25], and growth factor[26, 27].
Transcriptome sequencing of 12 mink skin samples yielded a total of 538 million clean reads. Correlation analysis between the samples showed that the samples could be divided into two components by age, indicating that the difference in gene expression caused by different coat colors was very small. Further principal component analysis (PCA) on the expression levels of samples can find that there are differences in samples of a different colors. This shows that our experimental design is reasonable. Differential gene expression analysis showed that the differentially expressed genes shared by adult black and white minks (AB vs AW) and juvenile black and white minks (TB vs TW) were not related to coat color formation. At the same time, adult black and juvenile black minks (AB vs TB) shared too many differentially expressed genes (4172) with adult white and juvenile white minks (AW vs TW). The genes related to fur development could not be identified.
Therefore, we searched for genes related to hair color formation and fur development through GO enrichment and KEGG enrichment analysis. The analysis showed that differentially expressed genes were enriched in 73 GO terms, and the most significant term we looked at about fur development was Keratin Filaments (GO:0045095). The keratin filaments are intervening filaments made of keratin that are found in various epithelial cells[28]. These GO terms identified thirteen keratin-associated protein genes (KRTAP), all of which were down-regulated in adult minks. The KRT is the main protein that makes up the outer layers of hair and skin[29, 30]. Mutations in the KRT gene have been found to cause changes in hair shape in dogs[31], cats[32], and mice[33], resulting in curly hair. It has also been found that mutations in the KRT gene cause hereditary skin diseases in humans, such as ichthyosis, congenital tachycardia, and palmoplantar epidermolytic keratosis[34, 35]. KRTAP can be divided into homocysteine (HS-KAPS) and high glycine tyrosine (HGT-KAPS) according to the composition of amino acids[36, 37]. The thirteen KRTAP genomes we identified were HS-KAPS. It has been reported that the KRTAP gene is related to wool weight, strength, diameter, elongation, and other traits[38–41]. In addition, different numbers of cysteine-containing repeats in human KRTAP1 and KRTAP2 genes lead to length polymorphism, which changes the interaction between KRT and KRTAP and results in differences in hair traits among individuals[42]. From this, we can infer that these thirteen KRTAP genes play an important role in the development of mink fur.
KEGG enrichment analysis also found five developmental-related signaling pathways. In mammals, the JAK-STAT pathway is the main signal transduction mechanism for a variety of cytokines and growth factors[43]. Pluripotent stem cells (PSCs) are basic cells with an indefinite self-renewal capacity, which is associated with the TGF[44], and BMP[45] signaling pathways. Extracellular matrix (ECM) is a mixture of macromolecules with complex structure and function, which plays an important role in the morphogenesis of tissues and organs and the maintenance of cell and tissue structure and function[46]. Adhesion molecules, while catalytically inactive, can do this by binding to growth factor receptors, which can initiate, integrate, or feedback adhesion-based signals[47]. Ras proteins are GTPases, which act as molecular switches in signaling pathways that regulate cell proliferation, survival, growth, migration, differentiation, or cytoskeletal activity[48]. In conclusion, we speculate that these signaling pathways may be related to mink fur development.
In addition, the Tyrosine metabolism pathway in KEGG enrichment was related to hair color. TYR and DCT in genes enriched by this pathway are related to melanogenesis. The tyrosinase gene family that regulates melanin production mainly consists of TYR (Tyrosinase), TYRP1 (Tyrosinase Related Protein 1), and TYRP2 (Dopachrome Tautomerase, DCT)[49]. Tyrosinase is a rate-limiting enzyme in the process of melanin synthesis. The expression level and activity of TYR directly affect the expression of eumelanin and melanin and then affect the animal hair color[50]. TYRP1 and TYRP2 play a catalytic role in the last steps that control the type of melanin melanocytes produce[51]. In our results, the expression level of TYR in black young minks was higher than that in white young minks, but this phenomenon was not observed in adult minks. Interestingly, TYRP1 and TYRP2 were more expressed in white young minks than in black minks. In adult minks, only TYRP2 was highly expressed in black minks, while there was no significant difference in the expression of TYR and TYRP1. Therefore, it can be inferred that the regulatory genes of mink hair color have been playing a role in early childhood, but the difference in gene expression level in adults is not significant. In conclusion, the tyrosinase gene family plays an important role in the regulation of mink hair color.