In this study, the cashmere goat skin samples of the experimental group and the control group covered 12 months, that is, the entire cashmere growth cycle. Many studies have used RNA-seq to explore the differences in gene expression in different growth stages of cashmere. For example, Geng et al. conducted a functional analysis of the differences in gene expression between three developmental stages of hair follicles in cashmere goats, and identified key genes that are involved in the regulation of cashmere growth . Zhang et al. performed transcriptome sequencing analysis on hair follicles in four seasons and explored the regulation of seasonal variation genes on the cashmere growth cycle of the cashmere goat and milk goat . However, these studies only selected three or more stages determined by experiments or experience at the cellular level. There is still a lack of research on the in-depth exploration of the dynamic pattern of gene expression during different cashmere growth stages on the scale of whole cycles. Therefore, this study performed transcriptome sequencing on the skin samples covering the entire cashmere cycles for 12 months, which aims to explore the dynamics of gene expression in the cashmere growth cycle in more detail.
The gene expression pattern for 12 months can provide useful information for distinguishing different cashmere growth stages from the genetic and molecular levels. According to the cluster-month correlations in Figure 2C, we grouped the cashmere growth cycle into three main stages: (1) anagen (April-October); (2) catagen and telogen (October-December and January-April); (3) telogen-anagen regeneration (February-May). The corresponding gene clusters are DC1, DC2 and DC3, respectively. Some canonical pathways such as the Wnt, TGF-beta and Hippo signaling pathways are enriched in DC1. DC2 genes are mainly enriched in pathways such as Cell adhesion molecules (CAMs), Cytokine-cytokine receptor interaction, Jak-STAT, Fc epsilon RI, NOD-like receptor, Rap1, PI3K-Akt, cAMP, NF-kappa B and many immune-related pathways. Interestingly, due to the overlap of the early anagen and telogen-anagen regeneration stages, the genes of DC3 and DC2 were partially enriched in several same pathways like CAMs, Focal adhesion, extracellular matrix (ECM)-receptor interaction, PI3K-Akt and NF-kappa B signaling. Besides, by constructing a co-expression network of genes (that are enriched in key pathways) and lncRNAs in three clusters, we reveal the possible regulators for crosstalk between different signaling pathways, and unearthed novel lncRNAs that may participate in these pathways.
In addition to unraveling the gene expression regulation mechanisms of the transition between different stages of the hair follicle cycle, this study also helps to figure out the role of exogenous melatonin in the specific stages of the cashmere growth cycle. By identifying genes that exhibit different expression patterns during the cashmere growth cycle under the stimulation of melatonin, we also obtained three gene clusters (MC1, MC2 and MC3) that may affect the cashmere growth cycle. Among them, MC1 genes (BAMBI, BMP2, BMP8A, FZD10, LEF1, PPP2R1B, SMAD6 and WNT11) and MC2 genes (IL6R, IL7R, IL11RA, IL15, IL18, PDE1A, PDE1B and PDE3B) showed opposite periodicity in group D. However, after the melatonin treatment, this regular fluctuation has been disordered. MC3 genes (COL1A1, COL1A2, COL3A1, CHAD, CREB3L1 and THBS3) were expressed specifically in the anagen restart phase (Apr-May) in group D, but there was no similarly significant expression pattern in group M.
The relative expression levels of monthly DEGs (Additional file 13) show that the hair development related genes HOXC13, KRT25, KRT71, FOXN1 were generally expressed at higher levels at the beginning of fast anagen progressing period from April to May, implying that they may function to promote the initiation of anagen. Wnt genes (Wif-1, WNT11, FZD10, LEF1, NOTUM, SFRP2, WNT6) together with Hedgehog genes (SHH, PTCH1, PTCH2, FOXE1) showed higher expression levels between April and May, but decreased in August, which implied that Wnt-related genes may promote the rapid transition into anagen phase of hair follicles between April and May, and repress the growth of hair follicles on the eve of the second cashmere shedding period in August. Chemokines (CCL17, CCL22, CCL2, LYN, RAC2, LOC102170772, PIK3CG and VAV1) and NF-κB genes (ZAP70, LYN, BTK, CD40LG, LTB) were highly expressed in September. The NF-κB pathway may facilitate the progress of the subsequent cashmere growth phase. Meanwhile, chemokines such as LTN may promote the second cashmere shedding.
KEGG pathway could be used as a reference to demonstrate the regulatory relationships of differentially expressed genes. Taking the above results together and collating the relevant KEGG pathway visualization results (Additional file 14), here we proposed a signaling pathway diagram of melatonin influenced cashmere growth cycle (Figure 6), which covered the main differentially expressed genes related to cashmere growth in anagen phase from April to September. The anagen phase of melatonin-treated groups was composed of a fast anagen progressing stage and a second cashmere shedding stage. The fast anagen progressing stage was from April to July, and this period was characterized by the occurrence of the first massive cashmere shedding at the end of April, and the rapid transition into anagen phase of hair follicles from May to July, when the quick resumption of cashmere growth appeared instead of residing in a resting non-growth period. The rapid resumption of the anagen phase of hair follicles may be due to the high expression of KRT25, HOXC13 and HOXC13’s regulatory target FOXN1, high expression of FZD10, WIF1, LEF1, WNT11 in Wnt signaling pathway, and SHH, PTCH1, PTCH2, FOXE1 in sonic hedgehog signaling pathway. The second cashmere shedding period was from August to September. The appearance of the second cashmere shedding may not only be associated with the low expression of ECM signaling molecules such as FREM1, FREM2, FRAS1, COL1A1, COL6A3, THBS3 in June and July, sonic hedgehog signaling pathway genes such as SHH, PTCH2 and WNT signaling pathway genes such as NOTUM, SFRP2, WNT6 in August, but also with the high expression of chemokines such as CCL, LYN, PIK3CG, VAV1, RAC2 in August. In addition, the highly expressed genes in NF-kappa B signaling pathway such as CD40LG, LTB, ZAP, LYN, BTK in September may promote the subsequent growth of cashmere after the second cashmere shedding period.