Study Of Genes Regulating Of Proliferation And Differentiation Of Hair Follicle And Idetification Of Wool Follicle Tissue-specific Promoter

The wool is one of important products in the sheep industry,and plays a vital role in textile manufacture.The quality and yield of the wool directly affect its competitiveness in the market economy.Wool follicle is the most important organ to regulate the growth of wool.Especially,the expression and interaction of the genes in the wool dermal papilla,germ matrix and inner root sheath play an important role in the proliferation and differentiation of wool follicle.So studying the molecular mechanism of proliferation and differentiation in the development of wool follicle will improve wool quality and economic traits.We screened out the differentially expressed genes(DEGs)from human hair dermal papilla cells(HHDPC),human hair germ matrix cells(HHGMC)and human hair inner root cells(HHIRSC)and identified the genes and pathways which affected the proliferation and differentiation of wool follicle.These results will provide evidences for molecular breeding of sheep.In addition,we have identified and cloned the wool follicle tissue-specific promoter,the activity of which was validated by the transgenic technology.Thus,it will provide molecular elements to improve the wool quality in the future.The results are as follows:1.Screening and analyzing the differentially expressed genes in different cells.(1)The selection of DGEs among HHDPC,HHGMC and HHIRS.Two methods were used to analyze the DEGs of different cells:?the DEGs were selected between HHDPC vs HHGMC and HHGMC vs HHIRSC resepectively according to their expression position and the way they interact in the hair follicles(P<0.01,Fold chage?2).1004 DEGs were obtained between HHDPC vs HHGMC and 369 DEGs were upregulated in HHDPC.1218 DEGs were obtained between HHGMC vs HHIRSC and 774 DEGs were upregulated in HHGMC.?In order to further analyze the molecular signature genes in each cell,we compared one type of cell with another two types of cells(P<0.01,Fold chage>2).We found 85 upregulated DEGs in HHDPC,337 upregulated DEGs in HHGMC and 85 upregulated genes in HHIRSC respectively.(2)GO analysis of the upregulated genes in HHDPC,HHGMC and HHIRS.The GO analysis of the HHDPC molecular signature genes revealed that the upregulated DEGs might be related to the "regulation of response to external stimulus","regulation of inflammatory response","negative regulation","negative regulation of multicellular organismal process" and "regulation of growth";The GO analysis of the HHGMC molecular signature genes indicated that the upregulated DEGs might be related to cell proliferation and growth,such as "regulation of transcription","regulation of cell proliferation","regulation of programmed cell death","cell adhesion" and so on;The GO analysis of the HHIRSC molecular signature genes suggested that the up-regulated expressed genes might be related to cell proliferation,differentiation,morphology and migration,such as "cell surface receptor linked signal transduction","cell adhesion'","Wnt receptor signaling pathway","cell-matrix adhesion" and so on.Some DEGs were validated by quantitative polymerase chain reaction(qPCR).For example,ADHIB,CNR1,STMN2,WIPS2,CIDEC were the upregulated genes of HHDPC,MMP1,MMP3,TXNDC5,DNER,DPP4,SERPINB2,FGF2,EGR1 were the upregulated genes of HHGMC,SOX2,IFITMI,CRISPLD2,WNT5A,COL5A1,ECM2 were the upregulated genes of HHIRSC.The results were consistent with the microarray,and they also proved the results of mircroarray were reliable.(3)The analysis of the expression patterns of DEGs in Tibet sheep wool follicle.The expression patterns of proteins encoded by the DEGs were indentified in the wool follicle.WNT5A and MMP1 were expressed in the ORS.The expression of SOX2 was in the ORS,IRS and CTS.And then,the expression of FGF7 was in dermal papilla,matrix,precortex,ORS and medulla.The expression of EGR1 was demonstrated in dermal papilla,matrix,precortex,ORS and IRS.(4)The research of STK36 mediating BMP singal pathway during the differentiation in mouse and Tibet sheep hair follicile.The expression patterns of Stk36,Smurfl and pSmad5 were detected during the different phases of the mouse hair follicle.The proteins of Stk36,Smurfl were distributed in the matrix,precortex and inner root sheath in anagen(postnal 7 day).The pSmad5 which is the downstream gene of BMP signaling pathway was not detected in the nucleus of these cells.These results indicated that BMP signaling pathway was not activated.Stk36,Smurfl were weakly expressed in the hair germ and ORS of the mouse hair follicle during catagen(postnal 21 day)and telogen(postnal 28 day),whereas pSmad5 was significantly expressed in the nucleus of the hair germ and ORS during the same pahses.These results suggested BMP signaling pathway was activated during catagen and telogen.The expression patterns in the wool follicles transforming from anagen to catagen were similar to those in catagen of mouse hair follicles.Thereofore,it was predicted that STK36 may mediate the BMP signaling pathway to regulate the differentiation of wool/hair follicle cells.(5)Investigating the influence of ERK signal pathway activated by PDGF-BB on the proliferation of HHGMC.PDGFR? was found to be differentially expressed in HHDPC,HHGMC and HHIRSC by qPCR.The proliferation in HHGMC was promoted significantly and the phosphorylation of PDGFR? and ERK1/2 were increased when HHGMC was treated with PDGF-BB.The results suggested that the exogenous PDGF-BB might stimulate proliferation of matrix cells via phosphorylating PDGFR? and then activating the ERK signaling pathway.2.The validation of the activity of the wool follicle tissue-specific promoterThe expression vector was constructed by using the 6404 base pairs of 5' upstream sequence of KRTAP3-3 and a promoterless vector ZsGreenl.We used the expression vector to construct transgenic mouse and found that strong but scattered green fluorescent signals were observed in the sheared dorsal hair from transgenic mice.These results indicated that the KRTAP3-3 promoter region can drive the the expression of exogenous genes in the mouse hair fiber.In conclusion,this study investigated the DEGs of HHDPC,HHGMC and HHIRSC.We also analyzed the expression patterns of the proteins enconded by the DEGs,which suggested that these genes may affect the proliferation and differentiation of wool/hair follicles.The relevant signaling pathway was also found in our study.The results would provide evidences to further understand the molecular mechanism which regulated the hair/wool development and growth.In addition,the cloned wool tissue-specific promoter could drive the exogenous genes expressed in the hair fiber,which could enable this promoter to be a useful tool in improving wool quality through transgene technology in the future.