Comparative study on seasonal hair follicle cycling by analysis of the transcriptomes from cashmere and milk goats

Guard hair and cashmere undercoat are developed from primary and secondary hair follicle, respectively. Little is known about the gene expression differences between primary and secondary hair follicle cycling. In this study, we obtained RNA-seq data from cashmere and milk goats grown at four different seasons. We studied the differentially expressed genes (DEGs) during the yearly hair follicle cycling, and between cashmere and milk goats. WNT, NOTCH, MAPK, BMP, TGFβ and Hedgehog signaling pathways were involved in hair follicle cycling in both cashmere and milk goat. However, Milk goat DEGs between different months were significantly more than cashmere goat DEGs, with the largest difference being identified in December. Some expression dynamics were confirmed by quantitative PCR and western blot, and immunohistochemistry. This study offers new information sources related to hair follicle cycling in milk and cashmere goats, which could be applicable to improve the wool production and quality.     

Melatonin promotes Cashmere goat (Capra hircus) secondary hair follicle growth: a view from integrated analysis of long non-coding and coding RNAs

The role of melatonin in promoting the yield of Cashmere goat wool has been demonstrated for decades though there remains a lack of knowledge regarding melatonin mediated hair follicle growth. Recent studies have demonstrated that long non-coding RNAs (lncRNAs) are widely transcribed in the genome and play ubiquitous roles in regulating biological processes. However, the role of lncRNAs in regulating melatonin mediated hair follicle growth remains unclear. In this study, we established an in vitro Cashmere goat secondary hair follicle culture system, and demonstrated that 500 ng/L melatonin exposure promoted hair follicle fiber growth. Based on long intergenic RNA sequencing, we demonstrated that melatonin promoted hair follicle elongation via regulating genes involved in focal adhesion and extracellular matrix receptor pathways and further cis predicting of lncRNAs targeted genes indicated that melatonin mediated lncRNAs mainly targeted vascular smooth muscle contraction and signaling pathways regulating the pluripotency of stem cells. We proposed that melatonin exposure not only perturbed key signals secreted from hair follicle stem cells to regulate hair follicle development, but also mediated lncRNAs mainly targeted to pathways involved in the microvascular system and extracellular matrix, which constitute the highly orchestrated microenvironment for hair follicle stem cell. Taken together, our findings here provide a profound view of lncRNAs in regulating Cashmere goat hair follicle circadian rhythms and broaden our knowledge on melatonin mediated hair follicle morphological changes.     

High-throughput sequencing of hair follicle development-related micrornas in cashmere goat at various fetal periods

Inner Mongolia cashmere goat marks a precious gerplasm genetic resource due to its excellent cashmere traits. Therefore, it is of crucial importance to investigate the cashmere development mechanism of cashmere goat and to search for the important cashmere growth-related candidate genes. Fetal skin samples at 10 different periods of cashmere goat were collected in this research. Moreover, high-throughput sequencing was conducted on RNA samples from side skin of cashmere goat fetuses collected at three critical periods of skin hair follicle initiation, growth and development (namely, 45, 55 and 65?days) after balanced mix in line with the previous research results. Meanwhile, 3 samples at corresponding periods were used as the biological duplications. Data regarding microRNA and mRNA expression in skin and hair follicles of cashmere goats at various fetal periods were obtained using the high-throughput sequencing method. The results indicated that microRNAs in the oar-let-7 and oar-miR-200 families in 55?days and 66?days of pregnancy samples had been notably up-regulated relative to those in 45?days of pregnancy samples. This revealed that they might be the critical microRNAs in hair follicle development.     

MicroRNA对皮肤毛囊发育的调控机制

MicroRNA是参与转录后水平表达调控的重要因子, 在病理上成为药物作用的潜在靶点, 在生理上成为表型调控的潜在位点。目前, 对于microRNA的功能已有一定了解, 但其在皮肤毛囊发育中的作用机制还不完全清楚。近年来, 高通量测序技术为microRNA的鉴定提供了更准确、快速的途径, 研究发现一些microRNA能够影响皮肤毛囊细胞的分化和增殖, 其相关靶基因在调控毛囊周期性生长的过程中充当重要角色。文章综述了近年来microRNA在皮肤毛囊生长发育调控机制研究领域所取得的成果, 以期为后续开展绒山羊毛囊生长相关microRNA作用机制研究提供借鉴。     

The role of Sox9 in maintaining the characteristics and pluripotency of Arbas Cashmere goat hair follicle stem cells

In our previous work, we isolated Arbas Cashmere goat hair follicle stem cells (gHFSCs) and explored the pluripotency. In this study, we investigated the expression and putative role of Sox9 in the gHFSCs. Immunofluorescence staining showed that Sox9 is predominantly expressed in the bulge region of the Arbas Cashmere goat hair follicle, and also positively expressed in both nucleus and cytoplasm of the gHFSCs. When the cells were transfected using Sox9-shRNA, cell growth slowed down and the expression of related genes decreased significantly, cell cycle was abnormal, while the expression of terminal differentiation marker loricrin was markedly increased; cells lost the typical morphology of HFSCs; the mRNA and protein expression of gHFSCs markers and stem cell pluripotency associated factors were all significantly decreased; the expression of Wnt signaling pathway genes LEF1, TCF1,c-Myc were significantly changed. These results suggested that Sox9 plays important role in gHFSCs characteristics and pluripotency maintenance.     

Molecular Cloning and Expression Analysis of Prostaglandin E Receptor 2 Gene in Cashmere Goat (Capra hircus) Skin during Hair Follicle Development

As a member of the four subtypes of receptors for prostaglandin E2 (PGE2), prostaglandin E receptor 2 (PTGER2) is in the family of G-protein coupled receptors and has been characterized to be involved in the development and growth of hair follicles. In this study, we cloned and characterized the full-length coding sequence (CDS) of PTGER2 gene from cashmere goat skin. The entire open reading frame (ORF) of PTGER2 gene was 1047 bp and encoded 348 amino acid residues. The deduced protein contained one G-protein coupled receptors family 1 signature, seven transmembrane domains, and other potential sites. Tissue expression analysis showed that PTGER2 gene was expressed strongly in the skin. The general expression tendency of PTGER2 gene at different hair follicle developmental stages in the skin was gradually decreased from anagen to catagen to telogen. After comparing with the expression of BMP4 gene and related reports, we further presume that it seems to have a relationship between the hair follicle cycle and the expression level of PTGER2 gene in cashmere goat skin.     

山羊microRNA研究进展

小RNA(micro RNA,mi RNA)是真核生物中发现的一类内源性的具有调控功能的非编码RNA,长度约20~25个核苷酸。研究表明,mi RNA参与真核生物的许多生命过程,包括个体发育、新陈代谢、细胞生长和凋亡等方面。目前,关于山羊(Capra aegagrus hircus)的mi RNA数据相对较少。文章综述了山羊mi RNA对皮肤与毛囊发育、肌肉生长发育、泌乳及生殖的调控作用,为利用mi RNA调控和改善绒山羊毛绒品质、生长性能及繁殖性能等提供理论基础和研究思路。     

Effect of melatonin administration to lactating cashmere goats on milk production of dams and on hair follicle development in their offspring

Melatonin treatment in adult cashmere goats can increase cashmere yield and improve cashmere fibre quality by inducing cashmere growth during cashmere non-growth period, of which time cashmere goats are in the mid and late stages of lactation. However, whether melatonin treatment in adult cashmere goats affects their offspring’s growth performance remains unknown. Therefore, the objectives of the current study were to determine the effects of melatonin treatment in adult cashmere goats on cashmere and milk production performance in dams and on hair follicle development and subsequent cashmere production in their offspring. Twenty-four lactating Inner Mongolian Cashmere goat dams (50 ± 2 days in milk, mean ± SD) and their single-born female offspring (50 ± 2 days old, mean ± SD) were randomly assigned to one of two groups supplemented with melatonin implants (MEL; n = 12) or without (CON; n = 12). The melatonin implants were subcutaneously implanted behind the ear at a dose of 2 mg/kg live weight on two occasions – 30 April and 30 June 2016. The results demonstrated that melatonin treatment in adult cashmere goats increased cashmere production and improved cashmere fibre quality as indicated by greater cashmere yield, longer cashmere fibre staple length, finer cashmere fibre diameter and thicker cashmere fibre density. The milk fat content was higher in MEL compared with CON cashmere goats. The daily yields of milk production, milk protein and milk lactose were lower in MEL compared with CON cashmere goats. Serum melatonin concentrations were greater, serum prolactin concentrations were lower and milk melatonin concentrations and yields were greater in MEL compared with CON cashmere goats. With regard to offspring, there were no differences in cashmere yield, fibre staple length, fibre diameter and fibre density at yearling combing, and the primary and secondary hair follicles population and maturation between treatments. In conclusion, melatonin treatment in adult cashmere goats during cashmere non-growth period is a practical and an effective way in cashmere industry as indicated by not only increasing cashmere yield and improving cashmere fibre quality in adult cashmere goat dams but also having no impairment in hair follicle development and the subsequent cashmere production in their single-born offspring.     

Generation of VEGF knock-in Cashmere goat via the CRISPR/Cas9 system

Cashmere is a rare and specialised animal fibre, which grows on the outer skin of goats. Owing its low yield and soft, light, and warm properties, it has a high economic value. Here, we attempted to improve existing cashmere goat breeds by simultaneously increasing their fibre length and cashmere yield. We attempted this by knocking in the vascular endothelial growth factor (VEGF) at the fibroblast growth factor 5(FGF5) site using a gene editing technology and then studying its hair growth-promoting mechanisms. We show that a combination of RS-1 and NU7441 significantly improve the efficiency of CRISPR/Cas9-mediated, homologous-directed repair without affecting the embryo cleavage rate or the percentages of embryos at different stages. In addition, we obtained a cashmere goat, which integrated the VEGF gene at the FGF5 site, and the cashmere yield and fibre length of this gene-edited goat were improved. Through next-generation sequencing, we found that the up-regulation of VEGF and the down-regulation of FGF5 affected the cell cycle, proliferation, and vascular tone through the PI3K-AKT signalling pathway and at extracellular matrix-receptor interactions. Owing to this, the gene-edited cashmere goat showed impressive cashmere performance. Overall, in this study, we generated a gene-edited cashmere goat by integrating VEGF at the FGF5 site and provided an animal model for follow-up research on hair growth mechanisms.     

A subset of skin-expressed microRNAs with possible roles in goat and sheep hair growth based on expression profiling of mammalian microRNAs

The microRNAs (miRNAs) are an extensive class of small noncoding RNAs (18-25 nucleotides) with probable roles in the regulation of gene expression. Due to the fact that miRNAs are conserved in closely related eukaryotes and some are also conserved across a larger evolutionary distance, their potential functions in mammalian development are under active study. In order to identify mammalian miRNAs that might function in hair growth, we characterized the expression of 159 miRNAs in adult body side skin and ear skin from goat and sheep using microarray analysis. Of these, 19 miRNAs were specifically expressed or greatly enriched in body side skin in goats and sheep. This suggests hair growth-specific functions for miRNAs. Of the coexpressed 105 miRNAs, the degree of correlation within species is higher than interspecies. Nine of the expressed miRNAs were detected exclusively in the goat body side skin area where more cashmere was grown than coat hair; mmu-miR-720 and mmu-miR-199b were expressed primarily in goat skin. The identification of 105 of skin-expressed miRNAs whose expression behavior is conserved in goats and sheep differentiating hair follicles implicates these miRNAs have functions in mammalian hair follicles growth and development. We demonstrate that miRNAs previously associated with hair follicles in the mouse are also expressed in the adult skin of goats and sheep. In addition, 69 more conserved miRNAs cross-species were discerned in the study. Of them, the let-7, mir-17, mir-30, mir-15, and mir-8 gene families were expressed in high frequency. These results reveal critical roles of them in skin and hair follicle development and function.     

<Emphasis Type="Italic">Tβ4</Emphasis>-overexpression based on the piggyBac transposon system in cashmere goats alters hair fiber characteristics

Increasing cashmere yield is one of the vital aims of cashmere goats breeding. Compared to traditional breeding methods, transgenic technology is more efficient and the piggyBac (PB) transposon system has been widely applied to generate transgenic animals. For the present study, donor fibroblasts were stably transfected via a PB donor vector containing the coding sequence of cashmere goat thymosin beta-4 (Tβ4) and driven by a hair follicle-specific promoter, the keratin-associated protein 6.1 (KAP6.1) promoter. To obtain genetically modified cells as nuclear donors, we co-transfected donor vectors into fetal fibroblasts of cashmere goats. Five transgenic cashmere goats were generated following somatic cell nuclear transfer (SCNT). Via determination of the copy numbers and integration sites, the Tβ4 gene was successfully inserted into the goat genome. Histological examination of skin tissue revealed that Tβ4-overexpressing, transgenic goats had a higher secondary to primary hair follicle (S/P) ratio compared to wild type goats. This indicates that Tβ4-overexpressing goats possess increased numbers of secondary hair follicles (SHF). Our results indicate that Tβ4-overexpression in cashmere goats could be a feasible strategy to increase cashmere yield.     

Wnt通路中的部分基因在绒山羊胚胎皮肤毛囊中的表达分析

已知绒山羊毛囊的发育受Wnt等信号通路控制,但Wnt通路相关基因在绒山羊胚胎毛囊启动和生长发育过程中的表达及作用机制尚不清楚。本文采用RNA-Seq技术对45 d,55 d和65 d的绒山羊胚胎体侧皮肤进行了转录组测序,鉴定Wnt通路相关基因的表达。 RNA- Seq技术结合blast搜索,将转录组有效测序数据与云南黑山羊参考基因组序列(http://goat. kiz.ac. cn/GGD/download.htm)比对,获得了已知的Wnt通路（pathway hsa04310）中的123个相关基因（86.0%）。进而采用实时荧光定量PCR技术检测,验证了差异表达的Sfrp4、Wnt3、Wnt10a（上调）和Apc2（下调）基因在绒山羊胚胎不同时期皮肤中的表达量,初步探索了绒山羊毛囊在胚胎期启动、发育过程中,Wnt通路部分基因的表达模式,为进一步研究Wnt通路部分基因在绒山羊胚胎毛囊启动、发育过程中的作用机制提供了有意义的线索。     

山羊不同组织来源EST片段的生物信息学分析

利用生物信息学方法,对GeneBank中截至2006年9月收录的全部来源于山羊基因的共计637条表达序列标签（EST）进行了综合及分类分析。结果显示,在来源于绒山羊含毛囊皮肤的392条EST序列中有48条为编码角蛋白或角蛋白关联蛋白的基因;在乳腺来源的245条EST中则无此类基因,而是如免疫球蛋白基因、维生素转运蛋白基因、MHC等诸多种类基因,以免疫和酶类居多。两类不同组织来源的FST相应基因相似之处最显著的是编码核糖体蛋白的基因,其中含毛囊皮肤组织的EST中有15．1％,乳腺组织为21．6％,并且有两种不同组织来源的EST组成的26条基序（contigs）中,编码核糖体蛋白的有17条,高达65．4％。     

Differential Expression of microRNAs and their Regulatory Networks in Skin Tissue of Liaoning Cashmere Goat during Hair Follicle Cycles

MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules that negatively regulate gene expression. Herein, we investigated a selective number of miRNAs for their expression in skin tissue of Liaoning Cashmere goat during hair follicle cycles, and their intracellular regulatory networks were constructed based on bioinformatics analysis. The relative expression of six miRNAs (mir-103-3p, -15b-5p, 17-5p, -200b, -25-3p, and -30c-5p) at anagen phase is significantly higher than that at catagen and/or telogen phases. In comparison to anagen, the relative expression of seven miRNAs (mir-148a-3p, -199a-3p, -199a-5p, -24-3p, -30a-5p, -30e-5p, and -29a-3p) was revealed to be significantly up-regulated at catagen and/or telogen stages. The network analyses of miRNAs indicated those miRNAs investigated might be directly or indirectly involved in several signaling pathways through their target genes. These results provided a foundation for further insight into the roles of these miRNAs in skin tissue of Liaoning Cashmere goat during hair follicle cycles.