Cashmere growth is a seasonal and cyclic phenomenon under the control of photoperiod and multiple stimulatory and inhibitory signals. Beyond relevant coding genes, microRNA (miRNA) and long non coding RNA (lncRNA) play an indispensable role in hair follicle (HF) development and skin homeostasis. Furthermore, the influence of lncRNA upon miRNA function is also rapidly emerging. However, little is known about miRNAs, lncRNAs and their functions as well as their interactions on cashmere development and cycling. Here, based on lncRNA and miRNA high-throughput sequencing and bioinformatics analysis, we have identified 1108 lncRNAs and 541 miRNAs in cashmere goat skin during anagen and telogen. Compared with telogen, 1388 coding genes, 41 lncRNAs and 15 miRNAs were upregulated, while 1104 coding genes, 157 lncRNAs and 8 miRNAs were downregulated in anagen (adjusted P-value ≤0.05 and relative fold-change ≥2). Subsequently, we investigated the impact of lncRNAs on their target genes in cis and trans, indicating that these lncRNAs are functionally conserved during HF development and cycling. Furthermore, miRNA-mRNA and miRNA-lncRNA interaction were identified through the bioinformatics algorithm miRanda, then the ceRNA networks, miR-221-5p-lnc_000679-WNT3, miR-34a-lnc_000181-GATA3 and miR-214-3p-lnc_000344-SMAD3, were constructed under defined rules, to illustrate their roles in cashmere goat HF biology. The present study provides a resource for lncRNA, miRNA and mRNA studies in cashmere cycling and development. We also demonstrate potential ceRNA regulatory networks in cashmere goat HF cycling for the first time. It expands our knowledge about lncRNA and miRNA biology as well as contributes to the annotation of the goat genome.

Cashmere, also known as soft gold, is produced from the secondary hair follicles (SHFs) of cashmere goats. The number of SHFs determines the yield and quality of cashmere; therefore, it is of interest to investigate the transcriptional profiles present during cashmere goat hair follicle development. However, mechanisms underlying this development process remain largely unexplored, and studies regarding hair follicle development mostly use a murine research model. In this study, to provide a comprehensive understanding of cellular heterogeneity and cell fate decisions, single-cell RNA sequencing was performed on 19,705 single cells of the dorsal skin from cashmere goat fetuses at induction (embryonic day 60; E60), organogenesis (E90), and cytodifferentiation (E120) stages. For the first time, unsupervised clustering analysis identified 16 cell clusters, and their corresponding cell types were also characterized. Based on lineage inference, a detailed molecular landscape was revealed along the dermal and epidermal cell lineage developmental pathways. Notably, our current data also confirmed the heterogeneity of dermal papillae from different hair follicle types, which was further validated by immunofluorescence analysis. The current study identifies different biomarkers during cashmere goat hair follicle development and has implications for cashmere goat breeding in the future.

Studies in humans and mice have revealed that hair follicle morphogenesis relies on tightly coordinated ectodermal–mesodermal interactions, involving multiple signals and regulatory factors. DNA methylation and long non-coding RNA (lncRNA) play a critical role in early embryonic skin development by controlling gene expression. Acting as an indirect regulator, lncRNA could recruit DNA methyltransferases to specific genomic sites to methylate DNA. However, the molecular regulation mechanisms underlying hair follicle morphogenesis is unclear in cashmere goat. In this study, RNA-seq and whole-genome bisulfite sequencing (WGBS) in embryonic day 65 (E 65) and E 120 skin tissues of cashmere goat were used to reveal this complex regulatory process. The RNA-seq, qRT-PCR, and immunohistochemistry results showed that Wnt signaling played an important role in both hair follicle induction and differentiation stage; transcriptional factors (TFs), including HOXC13, SOX9, SOX21, JUNB, LHX2, VDR, and GATA3, participated in hair follicle differentiation via specific expression at E 120. Subsequently, the combination of WGBS and RNA-seq analysis showed that the expression of some hair follicle differentiation genes and TF genes were negatively correlated with the DNA methylation level generally. A portion of hair follicle differentiation genes were methylated and repressed in the hair follicle induction stage but were subsequently demethylated and expressed during the hair follicle differentiation stage, suggesting that DNA methylation plays an important role in hair morphogenesis by regulating associated gene expression. Furthermore, 45 upregulated and 147 downregulated lncRNAs in E 120 compared with E 65 were identified by lncRNA mapping, and then the potential differentially expressed lncRNAs associated with DNA methylation on the target gene were revealed. In conclusion, critical signals and genes were revealed during hair follicle morphogenesis in the cashmere goat. In this process, DNA methylation was lower in the hair follicle differentiation compared with the hair follicle induction stage and may play an important role in hair morphogenesis by regulating associated gene expression. Furthermore, potential lncRNAs associated with DNA methylation on target genes were delineated. This study enriches the regulatory network and molecular mechanisms on hair morphogenesis.

During hair growth, cortical cells emerging from the proliferative follicle bulb rapidly undergo a differentiation program and synthesize large amounts of hair keratin proteins. In this process, HOXC13 is one critical regulatory factor, proved by the hair defects in HOXC13 mutant mice and HOXC13 mutant patients. However, inconsistent conclusions were drawn from previous researches regarding the regulation of HOXC13 on different keratins. Whether HOXC13 has extensive and unified regulatory role on these numerous keratins is unclear. In this study, firstly, RNA-seq was performed to reveal the molecular mechanism of cashmere cycle including anagen and telogen. Subsequently, combining the sequencing with qRT-PCR and immunofluorescent staining results, we found that HOXC13 showed similar expression pattern with a large proportion of keratins except for KRT1 and KRT2, which were higher in anagen compared with telogen. Then, the regulatory role of HOXC13 on different keratins was investigated using dual-luciferase reporter system and keratin promoter-GFP system by overexpressing HOXC13 in HEK 293 T cells and dermal papilla cells. Our results demonstrated that HOXC13 up-regulated the promoter activity of KRT84 and KRT38, while down-regulated the promoter activity of KRT1 and KRT2, which suggested HOXC13 had an ambivalent effect on the promoters of different KRTs. Furtherly, the regulation on HOXC13 itself was investigated. At transcriptional level, the binding sites of HOXC13 and LEF1 were found in the promoter of HOXC13. Then, through transfecting corresponding overexpression vector and dual-luciferase reporter system into dermal papilla cells, the negative-feedback regulation of HOXC13 itself and positive regulation of LEF1 on HOXC13 promoter were revealed. In addition, melatonin could significantly increase the promoter activity of HOXC13 under the concentration of 10 μM and 25 μM by adding exogenous melatonin into dermal papilla cells. At post-transcriptional level, we investigated whether chi-miR-200a could target HOXC13 through dual-luciferase reporter system. At epigenetic level, we investigated the methylation level of HOXC13 promoter at different stages including anagen, telogen and 60d of embryonic period. As a result, miR-200a and methylation were not regulatory factors of HOXC13. Interestingly, we found two SNPs (c.812A > G and c.929A > C) in the homeodomain of HOXC13 that could deprive the regulatory function of HOXC13 on keratins without changing its protein expression. HOXC13 had an inconsistent effect on the promoters of different keratins. Two SNPs (c.812A > G and c.929A > C) in the homeodomain of HOXC13 deprived its function on keratin regulation. Besides, the negative-feedback regulation by HOXC13 itself and positive regulation by LEF1 and melatonin on HOXC13 promoter were revealed. This study will enrich the function of HOXC13 on keratin regulation and contribute to understand the mechanism of hair follicle differentiation.

Lambskin of the Hu sheep exhibits high economic value due to its water-wave pattern. Wool curvature is the key factor of the pattern types and quality of lambskin, and it is formed by the interaction between dermal papilla cells and hair matrix cells in the hair follicle, which is regulated by various genes and signaling pathways. Herein, three full-sibling pairs of two-day-old healthy lambs (n = 6) were divided into a straight wool group (ST) and small waves group (SM) with three repetitions. RNA-seq was applied to determine the expression profile of mRNAs and lncRNAs in Hu sheep hair follicles. 25 differentially expressed mRNAs and 75 differentially expressed lncRNAs were found between SM and ST. FGF12, ATP1B4, and TCONS_00279168 were probably associated with hair follicle development. Then, Gene Ontology (GO) and KEGG enrichment analysis were implemented for the functional annotation of target genes of differentially expressed lncRNAs. The results showed that many genes, such as FGF12 and ATP1B4, were found enriched in PI3K-Akt signaling, MAPK signaling, and Ras signaling pathway associated with hair follicle growth and development. In addition, the interaction network of differentially expressed lncRNAs and mRNAs showed that a total of 6 differentially expressed lncRNAs were associated with 12 differentially expressed mRNAs, which may be as candidate mRNAs and lncRNAs. TCONS_00279168 may target ATP1B4 and FGF12 to regulate MAPK, PI3K-Akt, Ras signaling pathways involved in the sheep hair follicle development process. These results will provide the basis for exploring hair follicle development.

Hair follicle development is closely associated with wool curvature. Current studies reveal the crucial role of microRNAs (miRNAs) in hair follicle growth and development. However, few studies are known regarding their role in wool curvature. To reveal the potential roles of miRNAs in Hu sheep lambskin with different patterns, a total of 37 differentially expressed (DE) miRNAs were identified in hair follicles between small waves (SM) and straight wool (ST) groups using RNA-seq. Through functional enrichment and miRNA-mRNA co-expression analysis, some key miRNAs (oar-miR-143, oar-miR-200b, oar-miR-10a, oar-miR-181a, oar-miR-10b, oar-miR-125b, etc.) and miRNA-mRNA pairs (miR-125b target CD34, miR-181a target FGF12, LMO3, miR-200b target ZNF536, etc.) were identified. Though direct or indirect ways affecting hair follicle development, these miRNAs and mRNAs may have possible effects on wool curvature, and this study thus provides valuable insight on potential pattern formation.