| Cashmere is unmyelinated fiber produced by the secondary follicle in the skin of cashmere goat with substantial economic value.Hair follicle morphogenesis begins at embryonic period and finishes around birth.To produce new hairs,existing follicles undergo cycles of growth(anagen),regression(catagen)and rest(telogen).As the number and quality(fineness and length)of cashmere is depend on hair follicle morphogenesis and development,it is imperative to illuminate the regulation mechanism of hair follicle morphogenesis.The study is of great significance to improve the yield and quality of cashmere and is the current work in cashmere goat breeding.Although multiple signals and genes have been revealed in mice using transgenic technology,the related studies are seldom in cashmere.Besides,there are differences in morphology and physiology of hair follicle between mice and cashmere.For example,the cashmere is unmyelinated fiber under the control of photoperiod.Hence,it is imperative to illuminate the unique regulation mechanism of hair follicle morphogenesis and development on cashmere.Hair morphogenesis and development is a process of temporal-spatial expression of genes under the control of epigenetics.However,existing studies focused on regulation by coding genes,seldom on epigenetic regulation.Besides,dramatic increase in knowledge has demonstrated that long noncoding RNA(lncRNAs)are involved in multiple biological processes through the regulation on transcriptional and post-transcriptional as well as epigenetics.However,it is not clear of the function and regulation mechanism of lncRNA in hair follicle morphogenesis and development.According the above issues,our study aims to reveal the molecular mechanism of hair follicle morphogenesis and development from genetic and epigenetic respects.We revealed the dynamic change on transcriptome and genome DNA methylation in hair follicle morphogenesis and development using RNA-seq and whole genome bisulfite sequencing(WGBS).Hoxc13 functions on hair keratin and its regulation mechanism was detected using IF,gain and loss of function,dual luciferase reporter assay and site-directed mutagenesis.Through combining the RNA-seq and WGBS results,we illustrated the regulation mechanism of DNA methylation on hair differentiation and revealed that lncRNA participated in DNA methylation.At last,we studied the function of lnc-000552 using RNAi.The main results were as follows:1.Screening and identification of key signals and genes during hair follicle morphogenesis and development The signals including Wnt and Edaplayed an important role in hair induction while Wnt,Notch signals and transcriptional factors including Hoxc13,Sox21,Junb,Lhx2 and Gata3 played a critical role in hair differentiation according RNA-seq.Among them,Wnt signaling in the epidermal hair placode was activated by secretory Wnt ligands from hair placode and had a critical role in hair induction.Meanwhile,Wnt,TGF-? and ECM signals that function in telogen to anagen transition was revealed by RNA-seq.Compared the results between hair induction/differentiation and anagen/telogen,it was found that foxq1 was important in hair medulla and gata3 and cux1 had a regulatory role on hair IRS.2.Regulation of Wnt/?-catenin signaling on hair follicle development through Hoxc13Hoxc13 was not expressed in hair induction and telogen in which no development on hair shaft,while it expressed in hair differentiation and anagen in which hair shaft was growing.And t Hoxc13 had a similar expression pattern with the majority of keratins while had an opposite expression pattern with the minority of keratins.Using gain and loss of function,dual luciferase reporter assay and site-directed mutagenesis,it revealed that Hoxc13 could promote the promoter activities of hair KRT38 and KRT84 while depress the promoter activity of KRT1 and KRT2.The time-specific expression of Hoxc13 was under the control of Lef1 mediated by Wnt signaling.Besides,negative self-feedback regulation was existed.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,while Hoxc13 was not regulated by mi R-200 a and methylation.3.Regulation of DNA methylation on hair follicle development in cashmere goat The m C level in genome scale in hair indution was slight higher than that in hair differentiation detected by WGBS.DNA methylation had an important role in hair induction and hair differentiation through changing gene expression.The differentiation gene of hair follicle including signal genes such as Notch1,Notch3,Jag1,Fzd1 and transcriptional fators,such as Vdr,Gata3,Cux1 and Runx1,were highly methylated in hair induction while hypomethylated in hair differentiation.4.Regulation of lncRNAs on hair follicle morphogenesis and development in cashmere goat The lncRNAs in hair induction,hair differentiation,anagen and telogen had been identified using RNA-seq.The result showed that lncRNA might play an important role in hair morphogenesis and regeneration.Besides,combining the RNA-seq and WGBS results,lncRNA could regulate target gene expression mediated by DNA methylation.Lnc-000552 expressed significantly higher in anagen compared with that telogen.And it was mainly expressed in nucleus.The induction activity of dermal papilla depressed when interfering the lnc-000552 expression,suggesting lnc-000552 played an important role in dermal papilla cell induction.In conclusion,this study revealed the molecular mechanism on hair follicle morphogenesis and development from signaling,transcriptional factor regulation and DNA methylation respects.And the function and regulation mechanism of Hoxc13 and lnc-000552 were also systematically studied.This study will enrich the regulatory network in hair morphogenesis and development and play a foundation for precision breeding and has a great significance for promoting cashmere yield and quality in cashmere goat. |
PDF Full Text Request