A Single-cell Transcriptome Atlas of Cashmere Goat Hair Follicle Morphogenesis / 基因组蛋白质组与生物信息学报·英文版

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.

LncRNA-599554 sponges miR-15a-5p to contribute inductive ability of dermal papilla cells through positive regulation of the expression of Wnt3a in cashmere goat

BACKGROUND: Long non-coding RNAs (lncRNAs), as post-transcriptional regulators, were thought to function in the inductive property of dermal papilla cells (DPCs) in cashmere goat. Previously, lncRNA-599554 was identified in secondary hair follicle (SHF) of cashmere goat, but its functional significance is unknown. RESULTS: In the present investigation, we verified that lncRNA-599554 had significantly higher expression at the anagen dermal papilla of cashmere goat SHF than that at telogen. Based on overexpression and knockdown techniques, we found that lncRNA-599554 contributes the inductive property of DPCs of cashmere goat, which was assessed by detecting the changes in the expression of several typical indictor genes in DPCs including ET-1, SCF, Versican, ALP, Lef1 and Ptc-1. Based on RNA pull-down assay, we verified that lncRNA-599554 directly interacted with chi-miR-15a-5p. Also, we showed that lncRNA-599554 positively regulated the Wnt3a expression in DPCs but which did not appear to involve its modulating of promoter methylation. Based on the use of Dual-luciferase reporter assays, our data indicated that lncRNA-599554 regulated the Wnt3a expression through chi-miR-15a-5p-mediated post-transcriptional level. CONCLUSIONS: We showed that lncRNA-599554 contributes the inductive property of DPCs in cashmere goat which might be achieved through sponging chi-miR-15b-5p to promote the Wnt3a expression. The results from the present investigation provided a novel insight into the functional mechanism of lncRNA-599554 in the SHF regeneration of cashmere goat along with the formation and growth of cashmere fiber.

Discovery and molecular analysis of conserved circRNAs from cashmere goat reveal their integrated regulatory network and potential roles in secondary hair follicle

Background: Circular RNAs, a novel class in the eukaryotic transcriptome, are characterized by the 3' and 5' ends that are covalently joined in a covalently closed loop without free ends. Circular RNAs are considerably stable molecules and act as microRNA sponges with regulatory potential to the protein-coding genes. Results: Eight circular RNAs were found to be significantly upregulated at anagen skin tissue of cashmere goat compared with their counterparts at telogen. Rich and complex regulatory patterns were revealed among the eight upregulated circular RNAs at anagen and related miRNAs with their potential regulatory genes. The potential regulatory genes of eight upregulated circular RNAs at anagen were involved in several pathways related to the main physiological process of hair follicle, such as histone acetylation and axon. For chi_circ_1926, chi_circ_3541, chi_circ_0483, chi_circ_3196, and chi_circ_2092, overall, the relative expression in secondary hair follicle exhibited highly similar trends with their corresponding host genes during the different stages of the hair follicle cycle. However, the expression trends of chi_circ_0100, chi_circ_2829, and chi_circ_1967 were found to diverge from their corresponding host genes during the different stages of the hair follicle cycle. Conclusions: A total of eighteen circular RNAs were identified and characterized from skin tissue of cashmere goat. The eight upregulated circular RNAs at anagen might have significant roles in the secondary hair follicle of cashmere goat. Our results would provide a novel regulatory layer to elucidate the molecular mechanisms underlying the development of secondary hair follicle and the growth of cashmere fiber in cashmere goat.

Effects of all-trans retinoic acid on goat dermal papilla cells cultured in vitro

Background: All-trans retinoic acid (ATRA), a vitamin A-derived active metabolite, exerts important functions in hair biology. Previous studies indicated that excess ATRA hampered hair follicle morphogenesis and cyclic regeneration in adulthood, but other studies stated that ATRA promoted hair growth. Dermal papilla (DP), a cluster of specialized fibroblasts, plays pivotal roles in controlling development and regeneration of hair follicle. Several lines of evidence indicated that DP might be the target cells of ATRA in the hair follicle. To confirm this hypothesis, the present study was performed to explore the biological effects of ATRA on goat dermal papilla cells (DPCs) and clarify the roles of ATRA in hair biology. Results: Our experimental results indicated that key signaling transducers of ATRA were dynamically expressed in distinct stages of goat cashmere growth cycle, and high-dose ATRA treatment (10-5 M) significantly impaired the viability of goat DPCs and lowered the ratio of proliferating cells. Otherwise, goat DPCs were stimulated to enter apoptosis and their cell cycle progression was severely blocked by ATRA. Moreover, the expression of fibroblast growth factor 7 (Fgf7), one of the potent hair growth stimulators secreted by DPCs, was transcriptionally repressed following ATRA treatment. Conclusion: DPCs are the targets of ATRA in the hair follicle, and ATRA negatively regulates hair growth by the targeted suppression of cell viability and growth factor expression of goat DPCs. Through these observations, we offer a new mechanistic insight into the roles of ATRA in hair biology.

Effect of miR-125b on dermal papilla cells of goat secondary hair follicle

Background: MicroRNAs (miRNAs) are endogenous noncoding RNAs that regulate various biological processes. miR-125b is a miRNA that has been reported to be critical for hair follicle (HF) morphogenesis and development. We identified that the expression of miR-125b varies during an individual hair cycle (anagen, catagen, and telogen) in the skin of cashmere goats. We constructed a gain model (by overexpressing miR-125b) and a loss model (by inhibiting endogenous miR-125b) based on dermal papilla cells (DPCs) to further investigate the role of miR-125b in HF cycle. In addition, we used a dual-luciferase system to highlight the predicated target genes of miR-125b. Results: We found that miR-125b affects the expression of FGF5, IGF-1, SHH, TNF-α, MSX2, LEF-1, FGF7, NOGGIN, BMP2, BMP4, TGF-ß1, and ß-catenin. The dual-luciferase assay further validated a direct interaction between miR-125b and FGF5 and TNF-α. Conclusion: miR-125b affects the expression levels of genes related to hair cycle and may also play a critical role in regulating the periodic development of HF.

Estimating the copy numbers of exogenous gene in transgenic cashmere goats by real-time fluorescence quantitative PCR / 中国兽医学报

The copy numbers of exogenous gene in transgenic animals is always regarded as an important information of transgenic animals.Thus,simple and sensitive methods are required for the detection of the copy numbers of exogenous gene.Three kinds of transgenic Shanbei white cashmere goats,containing Tβ4-GFP,FGF5s-GFP and VEGF164-GFP,has been obtained by using PiggyBac(PB) transposon system.Fluorescence quantitative PCR was carried out to detect the copy numbers of copGFP.Using Gluc as reference gene,the double standard curves of exogenous gene and reference gene were mapped and the genomic DNA of transgenic goats were analysized by real-time fluorescence quantitative PCR.Moreover,the copGFP/Gluc ratio in the samples was calculated as the copy numbers of copGFP.In addition,Tβ4-GFP transgenic cashmere goats were selected to detect the integration sites by using the genomic walking kit.The results showed that the standard curve equation of copGFP was y=-3.230 6x+39.216 (R2 =0.998 8) and the standard curve equation of Gluc was y=-3.564 8x+38.440 (R2 =0.996 0).The copy numbers of exogenous gene in the transgenic cashmere goats were obtained and the numbers of integration sites in the selected Tβ4-GFP transgenic goats were consistent with the copy numbers of copGFP.As a conclusion,the high throughput,fast and sensitive real-time fluorescence quantitative PCR is an efficient and convenient method for the copy number of exogenous gene in transgenic cashmere goats.

Polymorphisms in The 5' flanking Region of IGF-Ⅰ Gene are Associated With Cashmere Fibre Traits in Liaoning Cashmere Goats / 生物化学与生物物理进展

Insulin-like growth factorⅠ(IGF-Ⅰ) is essential for the growth and differentiation of hair follicles which is an important part of wool and cashmere.But there is no report on polymorphisms of the IGF-Ⅰ gene in cashmere goat,and also few candidate genes for cashmere production traits have been reported in cashmere goats.The objectives of this work were to detect the single nucleotide polymorphism(SNP) in the 5' flanking region of IGF-Ⅰ gene and to determine their association with fibre traits in Liaoning cashmere goats.The fibre traits data investigated in the experiment were combed cashmere weight,cashmere fibre length and cashmere fibre diameter.A few individuals of the Liaoning cashmere goats,selected according to phenotypic character,were used for SNPs detection in the 5'flanking region of IGF-Ⅰ gene,and four point mutation G→C(388 bp),A→G(668 bp),A→ C(719 bp),G→A(752 bp) were identified,which result in a CdxA bonding-site lack,and score increase of C/EBP site in the 5' flanking region compared with the wild type.Then different genotypes were detected in 520 Liaoning cashmere goats using create restriction site(CRS) and polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP),three genotypes,AA,AB and BB were observed coded for by two different alleles A and B for each SNP,and 13 diplotypes were identified in the groups of the four SNPs.The relationships between the genotypes and combed cashmere weight,cashmere fibre length and cashmere fibre diameter were analyzed.The statistical analysis showed that animals with the genotype AA of SNP2 in Liaoning cashmere goats had the thinnest cashmere fiber diameter compared with those with the AB and BB genotype(P

Immunohistochemical technology of labeling skin of inner mongolia cashmere goats with BrdU / 中国免疫学杂志

Objective:The purpose of this study is to investigate the feasibility of labeling skin of inner mongolia cashmere goats with BrdU,and to optimize its immunohistochemical conditions.Methods:We injected different dose of BrdU into the neck vein of cashmere goats,then we took samples from the back skin of the goats,fixed in 4% paraformaldehyde.Next the samples were embedded with paraffin and sectioned.We examined the samples with immunohistochemical technology to test the effect of the influencing factors to labeling results as the BrdU injection,such as dosage,the concentration of second antibody and streptavidin-peroxidase,the way of antigen pretreatment,and DNA denature temperature.Results:We observed red BrdU positive cells in labeled goats whereas not in the control.Meanwhile,in the samples of the unlabeled goats or those incubated with PBS,no nuclear staining is observed.Conclusion:BrdU can be used for labeling of inner mongolia cashmere goats in vivo,which have no obvious dosage effect.The best condition of labeling is that puttig the section in 2 mol/L HCl 30 min at 37℃,in trypsin 30 min at 37℃.The most appropriate concentration of biotin-conjugated second antibody and streptavidin-peroxidase is 1∶100,under which the results of immunohistochemical are the best.