Exploring differentially expressed genes by RNA-Seq in cashmere goat (Capra hircus) skin during hair follicle development and cycling

Abstract

Cashmere goat (Capra hircus) hair follicle development and cycling can be divided into three stages: anagen, catagen and telogen. To elucidate the genes involved in hair follicle development and cycling in cashmere goats, transcriptome profiling of skin was carried out by analysing samples from three hair follicle developmental stages using RNA-Seq. The RNA-Seq analysis generated 8487344, 8142514 and 7345335 clean reads in anagen, catagen and telogen stages, respectively, which provided abundant data for further analysis. A total of 1332 differentially expressed genes (DEGs) were identified, providing evidence that the development of hair follicles among the three distinct stages changed considerably. A total of 683 genes with significant differential expression were detected between anagen and catagen, 530 DEGs were identified between anagen and telogen, and 119 DEGs were identified between catagen and telogen. A large number of DEGs were predominantly related to cellular process, cell & cell part, binding, biological regulation and metabolic process among the different stages of hair follicle development. In addition, the Wnt, Shh, TGF-β and Notch signaling pathways may be involved in hair follicle development and the identified DEGs may play important roles in these signaling pathways. These results will expand our understanding of the complex molecular mechanisms of hair follicle development and cycling in cashmere goats and provide a foundation for future studies.

Figure 1. Classification of total raw reads at different developmental stages.

After filtering the only adaptor sequences, containing N sequences and low quality sequences, the three RNA-Seq libraries still generated over 7.3 million clean reads in each library, and the percentage of clean reads among raw tags in each library ranged from 98.87% to 99.31%.

Figure 2. Percent of coverage representing the percentage of genes expressed at each of the stages.

The distribution of distinct reads over different read abundance categories showed similar patterns for all the three RNA-Seq libraries. The similarity distribution had a comparable pattern with about 20% of the sequences having an 80% similarity, while approximately 80% of the hits had a similar range.

Figure 3. The numbers of DEGs between different developmental stages.

Between the anagen and catagen libraries, there were 255 upregulated genes and 428 downregulated genes; Between the anagen and telogen libraries, there were 223 upregulated genes and 307 downregulated genes, while there were 94 upregulated genes and 25 downregulated genes between the catagen and telogen libraries.

Figure 4. Hierarchical cluster analysis of gene expression based on log ratio RPKM data.

The red color represents upregulated genes and the green color represents downregulated genes. There were clusters with relatively minor differences between anagen vs. catagen and anagen vs. telogen, but marked differences with catagen vs. telogen.

Figure 5. GO functional analysis of DEGs based on RNA-Seq data.

The results were summarised in three main categories: biological process, cellular component and molecular function. Among these groups, the terms cellular process, cell & cell part and binding were dominant in each of the three main categories, respectively.

Figure 6. The qPCR validations of DEGs characterised by RNA-Seq.

The results verified that these genes were differentially expressed at different hair follicle developmental stages, consistent with the RNA-Seq findings.