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. 2024 Jul 28;25(15):8248.
doi: 10.3390/ijms25158248.

Transcriptomic and Metabolomic Analyses Reveal Molecular Regulatory Networks for Pigmentation Deposition in Sheep

Mancheng Zhang  1 , Xiaoli Xu  1 , Yuan Chen  1 , Chengqi Wei  1 , Siyuan Zhan  1 , Jiaxue Cao  1 , Jiazhong Guo  1 , Dinghui Dai  1 , Linjie Wang  1 , Tao Zhong  1 , Hongping Zhang  1 , Li Li  1
Affiliations

Transcriptomic and Metabolomic Analyses Reveal Molecular Regulatory Networks for Pigmentation Deposition in Sheep

Mancheng Zhang et al. Int J Mol Sci. .

Abstract

Domestic animals have multiple phenotypes of skin and coat color, which arise from different genes and their products, such as proteins and metabolites responsible with melanin deposition. However, the complex regulatory network of melanin synthesis remains to be fully unraveled. Here, the skin and tongue tissues of Liangshan black sheep (black group) and Liangshan semi-fine-wool sheep (pink group) were collected, stained with hematoxylin-eosin (HE) and Masson-Fontana, and the transcriptomic and metabolomic data were further analyzed. We found a large deposit of melanin granules in the epidermis of the black skin and tongue. Transcriptome and metabolome analysis identified 744 differentially expressed genes (DEGs) and 443 differentially expressed metabolites (DEMs) between the pink and black groups. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses revealed the DEGs and DEMs were mainly enriched in the pathways of secondary metabolic processes, melanin biosynthesis processes, melanin metabolism processes, melanosome membranes, pigment granule membranes, melanosome, tyrosine metabolism, and melanogenesis. Notably, we revealed the gene ENSARG00020006042 may be a family member of YWHAs and involved in regulating melanin deposition. Furthermore, several essential genes (TYR, TYRP1, DCT, PMEL, MLANA, SLC45A2) were significantly associated with metabolite prostaglandins and compounds involved in sheep pigmentation. These findings provide new evidence of the strong correlation between prostaglandins and related compounds and key genes that regulate sheep melanin synthesis, furthering our understanding of the regulatory mechanisms and molecular breeding of pigmentation in sheep.

Keywords: melanin; metabolome; sheep; skin; tongue; transcriptome.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Morphological and melanin deposition characteristics of skin and tongue of sheep. (A) The skin stained with HE and Masson–Fontana stains. (B) The tongue stained with HE and Masson–Fontana stains. Scale bars: 100 μm. Ep: epidermal layer, De: dermal layer, Hf: hair follicle, Sg: sebaceous gland, Ml: muscular layer. Purple arrow: melanin. (C) Statistical analysis of skin epidermis and dermis. (D) Statistical analysis of tongue epidermis. (E) Black skin and black tongue melanin diameter size. Section count: 15 fields of view per section were selected for counting (n = 5). In the figure: “ns” indicates no significant difference, “**” indicates p < 0.01, “****” indicates p < 0.0001.
Figure 2
Figure 2
DEGs in skin and tongue between pink and black sheep. (A) PCA plot of 4 groups of samples. (B) Violin plot of gene expression in skin and tongue samples. (C) Volcanic map of DEGs in skin group and tongue group. (D) Venn diagram of DEGs in skin group and tongue group. (E) Amino acid sequence comparison between ENSOARG00020006042 and YWHAs family. Note: ENSOARP00020028330 is the protein ID of ENSOARG00020006042.
Figure 3
Figure 3
GO and KEGG enrichment analysis of DEGs associated with sheep pigmentation. (A) Top 20 GO enrichment terms for upregulated and downregulated genes in the skin. (B) Top 20 GO enrichment terms for upregulated and downregulated genes in the tongue. (C) Top 10 KEGG metabolic pathways for upregulated and downregulated genes in the skin and tongue tissues.
Figure 4
Figure 4
Pigmentation-related metabolomics of sheep skin and tongue. PCA maps of skin and tongue tissue samples in positive (A) and negative ion modes (B). PLS-da score plots for skin and tongue tissue samples in positive (C) and negative ion mode (D). Volcano plots of DEMs in the Black_vs_Pink_skin (E) and Black_vs_Pink_tongue (F) groups, respectively. (G) Venn diagram of DEMs in the Black_vs_Pink_skin and Black_vs_Pink_tongue groups.
Figure 5
Figure 5
KEGG functional enrichment analysis of DEMs. (A) Enrichment bubble plots of KEGG metabolic pathways for DEMs. (B) Heatmap of metabolites enriched in the tyrosine metabolic pathway across samples. (C) Boxplots of the content of several metabolites in different tissues. In the figure: “ns” indicates no significant difference, “*” indicates p < 0.05, “**” indicates p < 0.01, “***” indicates p < 0.001, “****” indicates p < 0.0001.
Figure 6
Figure 6
Integrative analysis for transcriptome and metabolome. (A) Joint pathway analysis of DEGs and DEMs in skin group and tongue group. (B) Network diagram of genes, metabolites, and metabolic pathways in the skin and tongue groups. (C) Heatmaps of genes and metabolites correlate in the skin group and tongue group.
Figure 7
Figure 7
Network of pigmentation-associated genes and metabolites in the skin (A) and tongue group (B). (|r| > 0.75, p < 0.05).
Figure 8
Figure 8
Data analysis flowchart.
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