Effects of SLC45A2 and GPNMB on Melanin Deposition Based on Transcriptome Sequencing in Chicken Feather Follicles

Abstract

As an essential genetic and economic trait, chicken feather color has long been an important research topic. To further understand the mechanism of melanin deposition associated with coloration in chicken feathers, we selected feather follicle tissues from the neck and wings of chickens with differently colored feathers (yellow, sub-Columbian, and silver) for transcriptome analysis. We focused on genes that were expressed in both the wings and neck and were expressed with the same trends in breeds with two different plumage colors, specifically, SLC45A2, GPNMB, MLPH, TYR, KIT, WNT11, and FZD1. GO and KEGG enrichment analyses showed the DEGs were enriched in melanin-related pathways, such as tyrosine metabolic pathway and melanogenesis, and PPI analysis highlighted the genes SLC45A2 and GPNMB as associated with melanin deposition. Verification experiments in chicken melanocytes demonstrated that these two genes promote melanocyte melanin deposition. These data enrich our knowledge of the mechanisms that regulate chicken feather color.

Keywords: GPNMB; SLC45A2; melanin; melanocytes; transcriptome.

Figure 1

The feather colors of different colored hens, as well as their neck and wing feathers. (A) line chicken, sub-Columbian feathers: almost white with black barring only in the hackles, primary and secondary feathers, and tail; (B) Gushi chicken, yellow feathers: yellow with black spots all over the body feathers; (C) Hy-Line chicken, silver feathers: pure white feathers all over the body.

Figure 2

Analyses of DEGs in four comparisons. (A–D) YW vs. HW, YN vs. HN, SW vs. HW, and SN vs. HN groups, respectively. Red and green points indicate the genes with significantly increased or decreased expression, respectively (FDR < 0.05). The x-axis shows the log2-fold change in expression, and the y-axis shows the log10-fold likelihood of a gene being differentially expressed. (E) Venn diagram showing the overlap of the DEGs in four comparisons.

Figure 3

RT–qPCR validation of RNA-seq results. The left Y-axis displays the FPKM derived from the RNA-seq, while the data from RT–qPCR are shown on the Y-axis on the right. The data are represented as the means ± SEDs; red represents the FPKM value of RNA-seq, blue represents the RT–qPCR with GAPDH as the internal reference gene; * indicates p < 0.05; ** indicates p < 0.01.

Figure 4

GO classification of DEGs. (A) YW vs. HW groups. (B) YN vs. HN groups. (C) SW vs. HW groups. (D) SN vs. HN groups. The x-axis indicates the secondary classification in the GO database; the y-axis on the left side indicates the ratio of the number of genes annotated with this GO classification to all genes. The y-axis on the right side indicates the number of genes annotated with this GO entry. The denominator of ‘Selected_gene_number’ is the number of DEGs, and the denominator of ‘AllGene_number’ is the total number of genes.

Figure 5

Scatterplot of the top 20 pathways in KEGG enrichment. (A) YW vs. HW groups. (B) YN vs. HN groups. (C) SW vs. HW groups. (D) SN vs. HN groups. The x-axis represents the rich factor, which is the ratio of the DEGs annotated with the pathway term to the total number of genes annotated with the pathway term. The greater the rich factor is, the greater the degree of enrichment. The y-axis shows each KEGG pathway name. Each round point represents a specific KEGG pathway. The circle size indicates the number of DEGs associated with each significantly enriched pathway. The circle color indicates the significance level (q-value). A q-value < 0.05 was considered to indicate significant enrichment. Light purple indicates the least significant, and orange represents the most significant.

Figure 6

The networks of DEGs in the melanogenesis pathway. Proteins and DEGs in the same families are represented by the same color.

Figure 7

PPI network of DEGs. (A,B) represent Y vs. H and S vs. H, respectively. Red nodes represent upregulated DEGs, and green nodes represent downregulated DEGs.

Figure 8

SLC45A2 and GPNMB promote the deposition of melanin in chicken melanocytes. (A) Marker gene identification in chicken primary melanocytes (scale 50 μm). (B,C) The mRNA expression of SLC45A2 and GPNMB after 48 h of transfection of the SLC45A2 overexpression vector pcDNA3.1-SLC45A2-EGFP and the GPNMB overexpression vector pcDNA3.1-GPNMB-EGFP in melanocytes. (D,E) Effects of SLC45A2 and GPNMB overexpression on the expression of genes related to pigmentation melanocytes. (F,G) Effects of SLC45A2 and GPNMB overexpression on MITF protein levels. (H,I) Effect of SLC45A2 and GPNMB overexpression on the tyrosinase content of melanocytes. (J,K) Effect of SLC45A2 and GPNMB overexpression on the melanin content of melanocytes. * p < 0.05; ** p < 0.01.