- 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.. - 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. - Keywords: Differentially expressed genes, Hair follicle cycle, RNAseq, Keratin 4. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - Full list of author information is available at the end of the article. - This growing time span is recognized as the anagen phase of the hair cycle. - The reshape of a new hair follicle (HF) from follicular keratinocytes is guided mainly from the dermal papilla cells (DPCs) that manage information generated by local and systemic hormons and molecules to promote hair growth [6]. - The role of Sonic Hedgehog, WNTs and β catenin as promoters of the hair growth are largely outlined, as the involvement of BMPs pathways in the regression phase of the hair cycle [8, 9]. - Lately, the principal canonical pathways of the HF cycle were assessed also in cashmere goats [10, 11]. - However, in double-coated animals, some other un- common pathways could be involved in the undercoat growth.. - Precise knowledge of genes and pathways involved in the HF cycle, and therefore the fine evaluation of mole- cules involved in the active growth and in the regressive phases of the fiber, can be used to plan the most favor- able harvest time and may improve cashmere yield. - Althought cashmere goats are reared mainly in Asia, mostly in China, Italy imports the largest amount of cashmere in the world for prestigious. - Thanks to histomorphological evaluations, the SHFs in the anagen phase are easily distinguishable for the typ- ical dilated and rounded morphology of the bulbs (Fig. - Quality control and trimming procedures retained the vast majority of the sequences produced (from 87 to 97% of the total) from an average of to reads. - Alignment was successful for 79 to 89% of the cleaned reads, and a good proportion of unique alignments was observed with an output of average mapped reads.. - An overview of trimming and mapping data is shown in the Add- itional file 1.. - After a statistical analysis with edgeR using a data set of 12,486 filtered genes, we found 214 differentially expressed genes (DEGs) in the isolated HFs, with a sig- nificance of q <. - Full results are shown in the Additional file 2.. - Nocelli et al. - b Histological section showing a group of secondary HFs in the anagen phase. - However, due to the limitation of Capra hircus gene an- notation, we evaluate them also in the vocabularies of Bos Taurus. - Among them, some are peculiar of the HF cycle activities such as “thermogenesis”, “circa- dian rhythm” and “regulation of pluripotency of stem cells”. - A selection of DEGs, have been evaluated by RT-qPCR for validation in the isolated HFs (two phases, anagen and catagen, Fig. - As expected, Plin4, a member of the perilipin family, involved in coat intracellular lipid storage drop- lets, is fairly expressed during the cold season. - 3 Smear plot of the total analyzed genes. - In this study, similarly to Su et al. - This finding underlines the major role of environmental temperature and points out the importance of fatty acid related pathways in the cashmere cycle especially during the cold winter season.. - Despite the fact that prolactin levels increase following ovulation, leading to a seasonal moult [20], it seems by our evidences that genes in the estro- gen pathway exceed the activity of the prolactin pathway and its cluster of genes. - This suggests a direct role of the estrogen in the control of HF cycle, modulating the 105 genes annotated in this pathway. - Although estrogen mediation is remarkable in the HF development, the im- pact of this pathway in the cashmere growth need to be further investigated. - 4 PIA gives a graphical output to facilitate the understanding of the pathway relationship amongst genes. - The intensity of the color of the red diamonds give an idea of the expression level of the pathway. - Furthermore, fixing anagen as reference, the color intensity of the red balloon highlights the up-regulation in catagen. - While the intensity of the green balloon gives information about the down-regulation in catagen. - The bar chart shows the expression between anagen and catagen in the HFs (a, b, c and d). - While in the skin biopsies (e, f, g and h) is possible to evaluate the same genes during early anagen, anagen, early catagen and catagen phases. - Regarding skin biopsies, the significance of the expression level ( P <0.005) is evaluated as ANOVA test and is visible in the Additional file 5. - Furthermore, genes in- volved in the pluripotency of stem cells are interactively linked to a series of canonical pathways, known to be implicated in the HFSCs fate, such as prolactin and es- trogen signalling [20], Hippo signalling [22] and other HF pathways like TGF-β, and Sonic Hedgehog [8]. - The strong expression of pluripotency of stem cells pathways, represented by genes expressed both during growth and regression, supports how stem cells cycle are deeply en- gaged in the cashmere regeneration. - One of them, PI3K- AKT-mTOR pathway, is crucial in the coordination of. - In our HFs, this pathway is connected, in addition to pluripotency of stem cells, with estrogen, cell cycle, and Notch signalling pathways, suggesting thath of PI3K-AKT-mTOR path- way is central in the cashmere growth. - 6 A detatiled picture of the K4 strong expression in HFs (a) and in skin biopsies (b) during growing phases (early anagen and anagen) and not less, the really low expression during regressing phases (early catagen and catagen). - The most of other genes of the thermogenesis are up-regulated in catagen, however some of them do not have a great differential expression among the HF phases. - Both K13 and K4 seems to be found as a protein in the exo- somes in human urine [30], but nothing is known about the role of K4 as a signalling molecule. - Interestingly, the most abundant molecules expressed in the study of Jaworski and colleagues [31], besides K4, are K13 and solute channels involved in mineral transport such as Aquapo- rin 3 (AQP3) as well as the S100A family, which are also implicated in our research. - Recently K4, AQPs and the S100 cluster were inserted in the group of seasonal rhythm genes (SRGs) also in cashmere goat skin [21].. - The last-mentioned S100 seems to be a marker for hair follicle stem cells in mice, both in the bulge and in sec- ondary hair germ [21], while AQP3 is a water channel protein involved in neonatal skin inflammation and eyes morphogenesis [21, 32]. - These results in concert with Wu et al. - 2020 [21] study in whole goat skin, revealed that some molecules, also in the isolated HFs, could be potentially photosensitive. - Accordingly, some cells af- fected by those molecules could maintain the specific rhythm of the peripheral organs based on the. - In this context CP acts as a metalloprotein binding most of the copper in plasma and exert its role in iron peroxidation. - Despite not be- ing expressly named in the mineral pathway, CP was re- cently included in the KEGG Porphyrin and chlorophyll metabolism, which is clearly linked to the Ferritin and Apotransferitin pathway. - 8 The estrogen signalling pathway (a) is solid active instead, prolactin pathway (b) results are not in the group of the strongest expression level. - The cashmere goats samples used in this project are the same used in Pazzaglia et al. - Skin biopsies were collected from twelve (1 year) Italian cashmere does by punch biopsy method (0.8 cm diam- eter) from the Chianti Cashmere goat farm of the DVM Nora Kravis, Azienda Agricola La Penisola, 53017 Radda in Chianti, Siena,Tuscany, Italy N 11°23′. - The sampling of the five unrelated does was performed in August (during anagen phase) and January (catagen phase). - The sampling of seven skin biopsies were collected by punch biopsy method (0.8 cm diameter) from June to February, respectively in different periods: June (early anagen), September (anagen), December (early catagen) and February (late catagen) as previously described in the Pazzaglia et al. - Isolated HFs were accurately evaluated to determine the stage of the HF cycle. - To establish whether the collected follicles were in ana- gen, catagen or telogen, we performed a gross morpho- logical analysis of the whole isolated HFs and histomorphological evaluations on paraffin embedded HFs. - Eosin, Sacpic method [39], and Floxin B/Orange G/Alcian blue [34], and then they were accurately analyzed under a photomicroscope (Nikon Eclipse E800, Nikon Corp., Tokyo, Japan) to de- termine the stage of the follicular cycle.. - electrophoresis on the BioAnalyzer 2100 (Agilent Tech- nologies, California, USA) revealed the integrity and suit- ability of the RNA samples for NGS library preparation.. - HTSeq v.0.6.1 was used to quantify the mapping of the reads at each gene locus based on genomic features annotated in NCBI genomes of goat ARS1.. - Due to the scarcity of the Capra hircus gene annotation, a Pathway Interaction Analysis (PIA) [18] procedure has been carried out. - PIA is a tool that attempt to find a connection between genes using the interaction described in the KEGG database [43] starting from user-defined pathways of interest and the RNA-Seq results gene list.. - Reference genes were chosen in accordance with the authors Bai et al. - As in the most GenBank databases as NCBI, some genes of goats are predicted, primers were primarily validated with a mold tool [44] (http://. - The output of the reactions was an- alyzed by qbase+, a software developed by Biogazelle for processing RT-qPCR data through ANOVA test (skin bi- opsy) or t-test for paired samples (HFs). - HF: Hair follicle. - HFSCs: Hair follicle stem cells. - PHF: Primary hair follicle;. - SHF: Secondary hair follicle. - The datasets analysed during the current study are available in the additional files. - The accession numbers corresponding to the DEGs found in the isolated HFs can be found in Additional file 2 and were sourced from the ENSEMBL genome browser. - The accession numbers corresponding to the genes that were amplified in the RT-qPCR experiments can be found in Additional file 4 and were sourced from the NCBI database.. - Environmental influences on the hair follicle. - Determination of hair follicle characteristics, density and activity of Iranian cashmere goat breeds. - Ma S, Wang Y, Zhou G, Ding Y, Yang Y, Wang X, et al. - Synchronous profiling and analysis of mRNAs and ncRNAs in the dermal papilla cells from cashmere goats. - Single-Cell Transcriptomics Reveals that Differentiation and Spatial Signatures Shape Epidermal and Hair Follicle Heterogeneity. - In vitro models to study hair follicle generation. - Selection and validation of suitable reference genes in skin tissue of Liaoning cashmere goat during hair follicle cycle. - Gao Y, Wang X, Yan H, Zeng J, Ma S, Niu Y, et al. - Transcriptome analysis of fetal skin reveals key genes related to hair follicle morphogenesis in cashmere goats. - Wang X, Liu J, Zhou G, Guo J, Yan H, Niu Y, et al. - Cyclic expression of Lhx2 is involved in secondary hair follicle development in cashmere goat. - Transcriptomic analysis reveals critical genes for the hair follicle of Inner Mongolia cashmere goat from catagen to telogen. - Prolactin and its receptor are expressed in murine hair follicle epithelium, show hair cycle-dependent expression, and induce Catagen. - Circannual Rhythm in the Skin Gene Expression of Cashmere Goat bioRxiv. - Computational derivation of a molecular framework for hair follicle biology from disease genes. - Unveiling hair follicle stem cells. - Molecular genetics of the PI3K-AKT- mTOR pathway in Genodermatoses: diagnostic implications and treatment opportunities. - PDGFA in cashmere goat: a motivation for the hair follicle stem cells to activate. - Isolation and characterization of human hair follicle epithelial cells. - Isolation and characterization of stem cell-enriched human and canine hair follicle keratinocytes. - Epithelial expression of the hormone leptin by bovine skin
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