| Hair follicles(HF)in cashmere goats are divided into primary hair follicles(PHF)and cashmere secondary hair follicles(SHF).HF formation and development determine the morphological structure of hair and cashmere.The initial formation of hair follicles starts in the fetal phase and continues though several stages until mature.Due to the large number of key genes are involved in the developmental process of HF,the key functional genes and interaction networks of genes are still elusive.With the application of second-generation sequencing technology,RNA-seq technology has served the possibility of molecular mechanism study in HF of cashmere goat,and a great progress has been made in the studies of Inner Mongolia Cashmere goat.The study of the cashmere goat fetal hair follicle formation,especially the early stage of follicle formation,is largely unknow.In this study,we studied the morphological changes of HF during the early stage of hair follicle development by histological investigation of skin tissues from Shanbei cashmere goats.We further conducted RNA-seq analyses using skin tissues sampled from 60d(E60),120d(E120),and newly born(NB)individuals.Development-related candidate genes to fetal hair follicles were screened by functional prediction and literatures,which provides a theoretical basis to the study of regulation in the process of cashmere goats fetal hair follicle formation and development.On the basis of the previous studies,we verified the function of the key genes using the hair dermal papilla cells(DP)as experimental model.To provide technical reference to produce gene-modified cashmere goats,we further examined the function of the VDR gene with the perspective of the effect of the exogenous compounds on the mutated DP.This study has mainly achieved the following results:1.The morphological characteristics of hair follicles in E60,E120 and NB stages of fetal Shaanbei cashmere goats were observed for the first time.In E60 stage,the formation of visible PHF substrate happens,and the depth of follicle substrate in skin of eye edge and neck is greater than the skin of body sides;In E120 stage,a large number of redifferentiated SHF arise,SHF and PHF parallel to each other,and PHF has a complete structure and the hair shaft has begun to form;In NB stage,PHF hair extends out of the epidermis,indicating PHF has matured and part of the SHF have a complete structure,while there are still a large number of SHF is in the proceed of redifferentiation.2.The morphological characteristics of hair follicles in the eight stages(0,7,14,28,42,56,84,160-day-old)lambs were first observed in this study.The hair follicle group of the Shaan bei white cashmere goat has a three-dimensional composition,including three PHFs and several SHFs.During the period of 0-14 d,hair shaft has been observed in the structure of the original SHF,while the structure of many SHF in the redifferentiation process is uncomplete During the period of 28-84 d,the number of SHF in the hair follicle group increased rapidly,and the SHF in the proceed of redifferentiation continuously grow downward.Up to 160 days,the structure of the inner root sheath,outer root sheath and hair shaft of most SHFs became complete,which is similar to that of 84 d,While some hair shaft of SHF still did not appear,SHF hasn’t matured completely.3.We successfully construct the total RNA library of skin tissues in E60,E120 and NB stage.Sequenced by Illumina Hi Seq2000,each library received more than 2.06 million clean reads.There were 2,059 DEGs in three stages,1,024 DEGs in E120/E60,1,801 DEGs in NB/E60,no DEGs between E120 and NB.And 8 DEGs were detected by real-time quantitative PCR.The results showed that the expression of 8 genes was consistent with RNA-seq data.4.The clustering analysis of 2,059 DEGs expression trajectories in E60,E120 and NB showed that 1,975 DEGs were significantly clustered in four transcription traces,each of them might play different functions during hair follicle formation and development.GO enrichment analysis showed that DEGs were significantly enriched in cell location,molecular function,and biological processes(P < 0.05).80.6% of the DEGs in the biological process category were in the single tissue process category,followed by the single tissue cell process category(69.3%)and the biologic regulation category(48.1%).KEGG analysis found that 55.5% of DEGs(1142/2059)were involved in 218 signaling pathways,during which cancer pathway and ECM-receptor interaction signaling pathway were significantly enriched(P < 0.05).These DEGs present in these functional classifications and signaling pathways can be used as candidate key genes for maturation and formation of cashmere goat fetal hair follicles.5.We combined the results of DEGs KEGG analysis with gene expression trajectory analysis to screen the candidate genes related to hair follicle formation and maturation.10 DEGs was annotated in the interaction pathway of ECM and its receptor and had a significantly higher expression in E60 than that of E120 and NB(P< 0.05),which are candidate key genes for the initiation of hair follicle formation.10 DEGs were annotated to the cancer pathway,and seven transcription factors were involved in the synthesis of keratin,the expression of those DEGs in E120 and NB were significantly higher than those in E60(P < 0.05)and were the candidate genes related to hair follicle maturation.The results showed that VDR could be annotated to GO categories including the developmental process,cutin cell proliferation regulation and differentiation,and is related to the regulation of keratin.The expression of VDR in E120 and NB stage was significantly higher than that in controls(P < 0.001).6.The expression of VDR m RNA in the nine tissues of northern white cashmere goats in E60 and E120 was significant different,and the expression of VDR m RNA was the highest in E120.In NB and E120 stage,the VDR protein was highly expressed in the root sheath and hair ball region of the cashmere goat.7.We cloned complete sequence of VDR gene CDS area in the shaanbei white cashmere goat and constructed the VDR-deficient DP cell model by CRISPR/Cas9.The heterozygous mutated DPCs and homozygous mutated DPCs were produced successfully.After analysis of the function of VDR,It was found that growth rate of mutated DPCs is significantly lower than control(P < 0.05)and that the expression of VDR m RNA in DPCs was significantly decreased after VDR knockdown(P < 0.05).Further,the expression of VGF,Noggin,Lef1 and b-catenin were significant down regulated(P < 0.05).The treatment with different concentrations of CM and AA could significantly increase the expression of VDR(P < 0.05)and the activity of DP cells(P < 0.05),while this function will not affected by gene knockout.The CM could significantly increase the expression of Noggin,b-catenin and Lef1 in VDR-deficient DP cells(P < 0.05),which was not affected by VDR knockout,and CM could compensate the inhibitory effect of VDR knockout on the expression of hair follicle-related genes in DP cells.Taken together,the HF developmental process in cashmere goat fetus and lambs are continuous.Specifically,the PHF placode was generated at E60,while PHF were matures in NB.However,the SHFs were still unmatured at 160 d after birth.The comparative RNA-seq determined that the VDR gene play key roles in the DP cells,and this gene was further verified through the CRISPR/Cas9 approach. |
PDF Full Text Request