Genome-wide discovery of lincRNAs with spatiotemporal expression patterns in the skin of goat during the cashmere growth cycle.
ABSTRACT: BACKGROUND:Long intergenic noncoding RNAs (lincRNAs) have been recognized in recent years as key regulators of biological processes. However, lincRNAs in goat remain poorly characterized both across various tissues and during different developmental stages in goat (Capra hircus). RESULTS:We performed the genome-wide discovery of the lincRNAs in goat by combining the RNA-seq dataset that were generated from 28 cashmere goat skin samples and the 12 datasets of goat tissues downloaded from the NCBI database. We identified a total of 5546 potential lincRNA transcripts that overlapped 3641 lincRNA genes. These lincRNAs exhibited a tissue-specific pattern. Specifically, there are 584 lincRNAs expressed exclusively in only one tissue, and 91 were highly expressed in hair follicle (HF). In addition, 2350 protein-coding genes and 492 lincRNAs were differentially expressed in the skin of goat. The majority exhibited the remarkable differential expression during the transition of the goat skin from the May-June to August-October time point, which covered the different seasons. Fundamental biological processes, such as skin development, were significantly enriched in these genes. Furthermore, we identified several lincRNAs highly expressed in the HF, which exhibited not only the co-expression pattern with the key factors to the HF development but also the activated expression in the August to October time point. Intriguingly, one of spatiotemporal lincRNAs, linc-chig1598 could be a potential regulator of distal-less homeobox 3 expression during the secondary hair follicle growth. CONCLUSIONS:This study will facilitate future studies aimed at unravelling the function of lincRNAs in hair follicle development.
Project description:<h4>Background</h4>Cashmere is a keratinized product derived from the secondary hair follicles (SHFs) of cashmere goat skins. The cashmere fiber stops growing following the transition from the actively proliferating anagen stage to the apoptosis-driven catagen stage. However, little is known regarding the molecular mechanisms responsible for the occurrence of apoptosis in SHFs, especially as pertains to the role of non-coding RNAs (ncRNAs) and their interactions with other molecules. Hair follicle (HF) degeneration is caused by localized apoptosis in the skin, while anti-apoptosis pathways may coexist in adjacent HFs. Thus, elucidating the molecular interactions responsible for apoptosis and anti-apoptosis in the skin will provide insights into HF regression.<h4>Results</h4>We used multiple-omics approaches to systematically identify long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs expressed in cashmere goat skins in two crucial phases (catagen vs. anagen) of HF growth. Skin samples were collected from three cashmere goats at the anagen (September) and catagen (February) stages, and six lncRNA libraries and six miRNA libraries were constructed for further analysis. We identified 1122 known and 403 novel lncRNAs in the goat skins, 173 of which were differentially expressed between the anagen and catagen stages. We further identified 3500 gene-encoding transcripts that were differentially expressed between these two phases. We also identified 411 known miRNAs and 307 novel miRNAs, including 72 differentially expressed miRNAs. We further investigated the target genes of lncRNAs via both cis- and trans-regulation during HF growth. Our data suggest that lncRNAs and miRNAs act synergistically in the HF growth transition, and the catagen inducer factors (TGF?1 and BDNF) were regulated by miR-873 and lnc108635596 in the lncRNA-miRNA-mRNA networks.<h4>Conclusion</h4>This study enriches the repertoire of ncRNAs in goats and other mammals, and contributes to a better understanding of the molecular mechanisms of ncRNAs involved in the regulation of HF growth and regression in goats and other hair-producing species.
Project description: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.
Project description:The Yangtze River Delta white goat is a unique goat species that can produce superior quality brush hair. CKLF-like MARVEL transmembrane domain-containing 3 (CMTM3), which influences the transcriptional activity of androgen receptor (AR), was identified as a candidate gene related to superior-quality brush hair formation. CMTM3 is generally expressed at low levels, but miR-149-5p is highly expressed in the skin tissues of these goats. The mechanism by which CMTM3 regulates the proliferation and apoptosis of goat hair follicle stem cells has not been elucidated. Here, RT-qPCR, western blotting, 5-ethynyl-2'-deoxyuridine (EdU), cell cycle, apoptosis, and dual-luciferase assays were used to investigate the role and regulatory mechanism of CMTM3 and miR-149-5p. Functional studies showed that CMTM3 overexpression inhibited proliferation and induced apoptosis in cultured hair follicle stem cells, whereas silencing CMTM3 markedly facilitated cell proliferation and deterred apoptosis in cultured hair follicle stem cells. Then, using bioinformatic predictions and the aforementioned assays, including dual-luciferase assays, RT-qPCR, and western blotting, we confirmed that miR-149-5p targets CMTM3 and preliminarily investigated the interaction between CMTM3 and AR in goat hair follicle stem cells. Furthermore, miR-149-5p overexpression significantly accelerated the proliferation and attenuated the apoptosis of hair follicle stem cells. Conversely, miR-149-5p inhibition suppressed the proliferation and induced the apoptosis of hair follicle stem cells. These results reveal a miR-149-5p-related regulatory framework for the miR-149-5p/CMTM3/AR axis during superior quality brush hair formation, in which CMTM3 plays a negative role.
Project description:Long intergenic non-coding RNAs (lincRNAs) regulate testicular development by acting on protein-coding genes. However, little is known about whether lincRNAs and protein-coding genes exhibit the same expression pattern in the same phase of postnatal testicular development in goats. Therefore, this study aimed to demonstrate the expression patterns and roles of lincRNAs during the postnatal development of the goat testis. Herein, the testes of Yiling goats with average ages of 0, 30, 60, 90, 120, 150, and 180 days postnatal (DP) were used for RNA-seq. In total, 20,269 lincRNAs were identified, including 16,931 novel lincRNAs. We identified seven time-specifically diverse lincRNA modules and six mRNA modules by weighted gene co-expression network analysis (WGCNA). Interestingly, the down-regulation of growth-related lincRNAs was nearly one month earlier than the up-regulation of spermatogenesis-related lincRNAs, while the down-regulation of growth-related protein-coding genes and the correspondent up-regulation of spermatogenesis-related protein-coding genes occurred at the same age. Then, potential lincRNA target genes were predicted. Moreover, the co-expression network of lincRNAs demonstrated that ENSCHIT00000000777, ENSCHIT00000002069, and ENSCHIT00000005076 were the key lincRNAs in the process of testis development. Our study discovered the divergent regulation patterns of lincRNA on spermatogenesis and testis growth, providing a fresh insight into age-biased changes in lincRNA expression in the goat testis.
Project description:BACKGROUND:Cashmere goat is known for its precious undercoat. Being photoperiod-dictated, cashmere growth has been studied focusing mainly on hair follicle cycle phases (anagen, catagen and telogen). An accurate molecular knowledge of the goat hair follicle cycle, disentangling gene expression changes during phases and recognizing timing boundaries, could be useful to improve cashmere goat management and ultimately cashmere production. RESULTS:To better describe goat's hair follicle transcriptome we applied RNA-sequencing to isolated hair follicles from five Italian cashmere goats, during the anagen and catagen phase, identifying total of 214 differentially expressed genes (DEGs): 97 were up-regulated while 117 were down-regulated in catagen with respect to anagen. Gene Ontology and pathway analysis were performed. We detected 144 significant pathways spanning from estrogen, pluripotency of stem cells, thermogenesis and fatty acid metabolism that were strongly expressed during the hair follicle phases analysed. Finally, we validated promising DEGs by RT-qPCR in the same set of samples as well as in hair follicles and entire skin biopsies of another cashmere goats cohort accounting for early anagen, anagen, early catagen, and catagen phases. CONCLUSIONS:As in the isolated hair follicles, some target genes were homogenously modulated during the four hair follicle phases. Ceruloplasmin (CP) and Keratin 4 (K4), confirmed their clear cut expression between growing and resting phase. In fact, K4 was almost absent in catagen phases while CP was barely expressed in anagen phases. In particular, the strong expression of K4 in early anagen makes it an eligible marker to track the beginning of a new hair cycle, and therefore defining the optimum time for cashmere harvesting.
Project description:The undercoat fiber of the cashmere goat, from the secondary hair follicle (HF), possesses commercial value. However, very few studies have focused on the molecular details of primary and secondary HF initiation and development in goat embryos. In this study, skin samples at embryonic day 45, 55, and 65 (E45, E55, and E65) were collected and prepared for RNA sequencing (RNA-seq). We found that the HF probably initiated from E55 to E65 by analyzing the functional pathways of differentially expressed genes (DEGs). Most key genes in canonical signaling pathways, including WNT, TGF-?, FGF, Hedgehog, NOTCH, and other factors showed clear expression changes from E55 to E65. We, for the first time, explored alternative splicing (AS) alterations, which showed distinct patterns among these three stages. Functional pathways of AS-regulated genes showed connections to HF development. By comparing the published RNA-seq samples from the E60, E120, and newborn (NB) stages, we found the majority of WNT/?-catenin signaling genes were important in the initiation of HF development, while other factors including FOXN1, GATA3, and DLX3 may have a consistent influence on HF development. Our investigation supported the time points of embryonic HF initiation and identified genes that have potential functions of embryonic HF initiation and development. We further explored the potential regulatory roles of AS in HF initiation, which extended our knowledge about the molecular mechanisms of HF development.
Project description:<h4>Background</h4> Hair follicle mesenchymal stem cells (HF-MSCs) have great potential for cell therapy. Traditional method to isolate whisker HF-MSC is time-consuming and few in cell numbers. How to quickly and conveniently obtain a large number of HF-MSC for experimental research is a problem worth exploring. <h4>Methods</h4> Two-step Ficoll Density Gradient Sedimentation (FDGS) was performed to isolate pelage HF-MSC from adult mice. The characteristic of the isolated cells was identified and compared with whisker HF-MSC by immunofluorescence staining, flow cytometry, three-lineage differentiation and hair follicle reconstruction. Pelage HF-MSC and exosomes were injected into the dorsal skin of mice as well as hair follicle organ culture to explore its role in promoting hair growth. The cells and exosomes distribution were located by immunofluorescence staining. <h4>Results</h4> Isolated pelage HF-MSC expressed similar markers (ALP, Versican, NCAM, Nestin), showed similar growth pattern, possessed similar mesenchymal stem cells function and hair follicle induction ability as whisker HF-MSC. A large number of cells can be obtained with fewer mice compared to traditional method. Injected pelage HF-MSC promoted hair growth by secreting exosomes. <h4>Conclusion</h4> A large number of Pelage HF-MSC can be isolated by FDGS, which can promote hair growth by secreting exosomes which may target the dermal papilla and hair matrix region of host hair follicle. <h4>Supplementary Information</h4> The online version contains supplementary material available at 10.1186/s13287-022-03051-3.
Project description:The study confirmed a batch of goat skin and hair follicles miRNA, by high-throughput sequencing methods.316 sequences miRNAs were obtained by the means of analysis and we confirmed the authenticity of 68 known miRNAs and discovered 248 novel miRNAs, as well as 22 miRNAs that havenM-bM-^@M-^Yt been reported before. Through the miRNAs family analysis, we found the co-expressed miRNAs in goat and sheep located in the same region of chromosome, which may play an essential role in skin and follicle development. In addition, the prediction of novel target miRNAs followed by the analysis of target gene pathways indicated that MAPK pathway may have an important effect to the fast growth of skin follicle cell. We sequenced a mixed sample which contains three goat skin in anagen of cashmere
Project description:In this study, the skin tissues were harvested from the three stages of hair follicle cycling (anagen, catagen and telogen) in a fiber-producing goat breed. In total, 63,109,004 raw reads were obtained by Solexa sequencing and 61,125,752 clean reads remained for the small RNA digitalization analysis. This resulted in the identification of 399 conserved miRNAs; among these, 326 miRNAs were expressed in all three follicular cycling stages, whereas 3, 12 and 11 miRNAs were specifically expressed in anagen, catagen, and telogen, respectively. We also identified 172 potential novel miRNAs by Mireap, 36 miRNAs were expressed in all three cycling stages, whereas 23, 29 and 44 miRNAs were specifically expressed in anagen, catagen, and telogen, respectively. Gene Ontology and KEGG pathway analyses indicated that five major biological pathways (Metabolic pathways, Pathways in cancer, MAPK signalling pathway, Endocytosis and Focal adhesion) accounting for 23.08% of target genes among 278 biological functions, indicating that these pathways are likely to play significant roles during hair cycling. the skin tissues were harvested from the three stages of hair follicle cycling (anagen, catagen and telogen) in a fiber-producing goat breed
Project description:The growth of Inner Mongolian Cashmere goat skin hair follicle exhibits a periodic growth pattern. The hair growth cycle is distinguished as telogen, anagen, and catagen stages. The role of vimentin in the growth process of hair follicles is evident. To elucidate the mechanism underlying the vimentin activity in the growth cycle of hair follicles, transcriptome sequencing and liquid chromatography-tandem mass spectrometry were used to obtain the nucleic acid and amino acid sequences of VIIM gene and vimentin. The amino acid and nucleic acid sequences were analyzed by comparison. Real-time quantitative PCR, Western blot, and immunohistochemistry analyzed the expression level and sites of vimentin in the three growth stages of the Inner Mongolia Cashmere goat skin samples.VIM gene cDNA, obtained by transcriptome sequencing, was aligned against that of the Capra hircus VIM gene. The amino acid sequence of vimentin revealed a high similarity rate across other species. The expressions of both VIM gene and vimentin were highest during the growth period and lowest in the rest period. Furthermore, vimentin was primarily expressed in the outer root sheath of the hair follicle as assessed by staining.The sequences of the gene and protein are similar to that of other species and identical to Capra hircus. However, the expression of VIM and vimentin was proportional to that of the growth of hair follicles. And vimentin expressed only in the outer root sheath of hair follicles. Thus, vimentin was speculated to participate in the regulation of the hair follicle growth cycle by affecting the outer root sheath.