Study On The Action Mechanism Of Hair Follicles Cells In Inner Mongolia Cashmere Goat

Inner Mongolia Cashmere Goat is a kind of goat that produces meat and cashmere.Its cashmere is known as "soft gold" and "fiber gem" because of its light,thin,soft,warm and bright cashmere fiber.It has important economic value in cashmere industry.The growth of Inner Mongolia cashmere secondary hair follicles has obvious periodic changes,which can be divided into three periods:telogen,anagen and catagen.At present,a large number of studies have shown that the yield and quality of cashmere are regulated by the secondary hair follicle,and there are many types of hair follicle stem cells in the hair follicle,among which the hair papilla cells,hair germ cells and bulge cells play the main role.These stem cells differentiate or apoptosis in different periods and interact,contact and influence each other to promote the cycle of cashmere goat's secondary hair follicle evolution.Therefore,to clarify the mechanism of these stem cells is helpful for us to further understand the hair follicle and cashmeree cycle,and lay a foundation for improving the economic benefits of cashmere.Based on the existing research results of Inner Mongolia cashmere goat,this study uses the adult Inner Mongolia cashmere goat(Albas type)to obtain and separate the skin hair follicle cells.Through single cell transcriptome sequencing,histomorphology,proteomics and polychromatic immunofluorescence,the expression characteristics of Inner Mongolia Cashmere Goat at different stages of individual tissue,cell,transcript and protein levels are analyzed After in-depth study,the main conclusions are as follows:1.In this study,18 cell clusters,7 cell types and 13 cell states were identified from Inner Mongolia cashmere goat skin cells by tSNE cluster analysis,the key genes and gene expression profiles of cells in different states were described.Among them,there is only one cell state under endothelial cells,melanocytes,hair bud cells and hair matrix,there are two intermediate state cells in dermal cells,four intermediate state cells in denmal papilla cells and seven intermediate state cells in carina region,including inner and outer root sheath cells.These cells interact with each other in the hair follicles,which together promote the production and atrophy of hair follicles,the growth and shedding of villi,and then promote the evolution of villi cycle.The sequence of differentiation in pseudo time differentiation was dermal cells,endothelial cells,melanocytes,dermal papilla cells,hair bud cells,carina cells and hair mother cells.2.In this study,new marker genes of major cell types were found through clustering markers analysised,among which the representative are:epithelial cell clusters highly expressed Collal and Lum,endothelial cell clusters highly expressed Pecam1 and Aqpl,Melanocyte cell clusters highly expressed Plpl and Sox6,dermal papilla cell clusters highly expressed Lefl and Msx1,hair germ cell clusters highly expressed Fcgbp and P-cad,Sox9 and Krt15 highly expressed in bulge cell clusters,Lrat and Gata3 highly expressed in hair matrix cell clusters,Fcerlg and Laptm5 highly expressed in immune cell clusters.3.This study combined the results of light length,histomorphology and single cell gene expression pattern clustering to find that when the light time reaches a certain threshold in anagen,melanocytes highly express Lmcd1,Fabp7,Iga6t Zfp36,Id4,Ramp1,Cryab And Hspe1,receptor tyrosine kinase signaling pathway is activated,and then Gpcl,Gsk3b,Igfbp5,Irs1,Itgav,Stmn1,Nck1,Pak2,Pik3cb,Porr2d,Porr2h,Itsn1,Tiam1,Nrp2,Actr3,Wasf2,Stam2,Cnks1,Nckap1,Cyfip1 and Ptpn18 gene expression increased to further activate WNT signaling pathway,MAPK signal and other signaling pathways(cell fate 1),that is,as the intensity of sunlight increases,the gene expression in melanocytes reaches a threshold and starts the hair follicle cycle.The expression of melanocytes in catagen and telogen also plays a guiding role.With the decrease in the intensity of sunlight in cell fate 2,the expression of melanin cells drops to another threshold,and the hair follicle cycle evoluted into cagagen.When the intensity of sunlight decreases further,the expression of melanocytes decreases to the lowest level,and the hair follicle cycle evoluted into telogen.From this we speculate that melanocytes or the inducing factors of the cashmere cycle transformation.4.Dermal cells have two intermediate state cells,one was enriched the PID-AP1/IL67/ATF2/Fox pathway,the regulation of apoptosis signaling pathway,the negative regulation of cell differentiation,rhythmic process and other signaling pathways,The highly expressed genes were Colla1,Cd34,Fgf7,Jund and Tmem59 and their expression showed a downward trend,revealing their important role in hair follicle apoptosis during telogen.Another was enriched in receptor tyrosine kinase signaling,Wnt signaling pathway,MAPK signal transduction pathway,BMP signal transduction pathway and mesenchymal cell differentiation,adipocyte differentiation,osteoblast differentiation and other functions,genes that are highly expressed and have an upward trend are Ddx5,Rpl7,Zfp36l1,Eif4a2,Id1,Igf2 and Multicolor immunofluorescence results show that dermal cells are mainly located in the upper part of the hair papilla and hair shaft in mature hair follicles,while in the new hair follicles,they are marked at all positions in the entire hair follicle,further revealing the transformation of dermal cells in the hair follicle cycle Important functions.5.By constructing the differentiation trajectory of hair papillary cells,the key genes,signals and functions that determine the fate of hair papillary cells are revealed.The results showed that dermal papillary cells differentiated into four intermediate cell states in different cycles:Intermediate cell 10 showed important functions in the growth and maintenance of cashmere.Highly expressed genes were Sfn,Krt10,Krtl4,Tgm3,Krtap3-1,Cnfn,Ivl,etc.;intermediate cell 1 acts on apoptosis and cashmere shedding,mainly enriching the foxo signaling pathway,p53 signaling pathway,etc.;intermediate cell 0 initiates a new hair follicle cycle,maintains hair follicle migration and promotes the production of cashmere,and signaling pathways includes wnt signaling pathway,notch signaling pathway,mapk signaling pathway,etc.;intermediate cells 15 are considered dermal papillary progenitor cells.In addition,up to 84%of pseudogenes were found in the gene expression profile of intermediate cells 1.After analysis,most of them were ribosome-related pseudogenes,and they played an important role in the process of apoptosis.This conclusion promoted the theory of competitive endogenous RNA(ceRNA)of pseudogenes.6.In cell fate 1,the hair germ cells receive the MAPK signaling pathway transmitted by melanocytes and the Notch signaling pathway transmitted by DP cells to activate the hedgehog signal "on" state,and then start the Smad gene family,TGF-? gene family,and further Activate Wnt signaling pathway,Notch signaling pathway and MAPK signaling pathway.And highly expressed Hmgn1,Cap1,Nop58,Rab25,Hspel,Srsf10,Tfg,Stag1,Sp3,and Mtx2 genes.From the perspective of expression patterns,hair germ cells are more like signal starters and amplifiers to start the hair follicle cycle.The signal cascade is amplified and transmitted to the bulge cells,which further promotes the development of hair follicles and the growth of cashmere.And soon enters the apoptosis program after achieving its function,and the signaling pathways were enriched in the process include cell apoptosis,the regulation of cell death,and the regulation of programmed cell death in cell fate 2.7.The bulge cells are more like high-threshold response cells.Their gene expression did not change significantly in the early anagen,until the gene expression of the hair germ cells increased and activated the related signal pathways and cascaded to the bulge cells.After that,CTGF/Hcs24-actin(?/?)complex,CD98-LAT2-ITGB1 complex,ITGA6-ITGB1-CD151 complex,ITGA6-ITGB1-CYR61 complex,ETS2-FOS-JUN complex and FZD5-LGR5-LRP6 complex were produced in the bulge cells and high expression the genes of Achy,Atf4,Cdk4,Egr1/2/3,Hspa5,Id1/3/4,Jun,Mdk,Nfil3,Ntrk2,Ptn,Robo2,Slit3,Tgfb3,Klf10,Bhlhe40,Adamts1,Cbx3 and Crtcl,and then achieve cell differentiation,development,morphological changes,proliferation and hair follicle structure formation.In cell fate 2,signaling pathways in bulge cells are mainly enriched in the breakdown of ?-catenin destruction complex,?-catenin independent WNT signaling,negative regulatory pathway of BMP signaling and external apoptosis signaling pathway,stem cells communication in cell-to-cell,cell differentiation,revealed the branching of the bulge cells into root sheath cells.The related genes extracted were:Krt19,Abhd12,Ier5,Lydd3,Dsc1,Jag1.Mgp,Npdc1,Gypc,Rnd1,Sbsn and Ube2s.8.There are two directions about the origin of hair matrix cells.One is the hypothesis of the origin of stromal cells(the hypothesis of hair follicle predication),they believe that the hair matrix cells are differentiated from the branches of the hair germ cells in the late telogen which did not undergo apoptosis during the apoptosis process.Another thinks that the hair matrix cells are differentiated from the cells in the bulge area in the early anagen after receiving the signal from the hair germ cells,and the gene expression profile and signal pathway of the hair matrix cells are enriched,although the PID ECADHERIN NASCENT AJ pathway is enriched.By the way,P-cad is a marker gene of hair germ cells,but more of the signaling pathways and complexes that are enriched are more similar to those of cells in the bulge area,so although the evidence is insufficient,the results of this study still promote hypothesis that hair matrix cells differentiate from bulge cells.9.Through pseudo-time differentiation trajectory,cell enrichment signal pathway and gene expression profile,combined with multicolor immunofluorescence results,this study summarizes the sequence of signal transmission of different cells in different periods:melanocytes reach a certain length in the light After the threshold,the hair follicle cycle signal is started,which marks the start of the hair follicle cycle,and then the signal is transmitted to the dermal cells and the dermal papillary cells.The dermal papillary cells combine the melanocytes to transmit the signal to the hair germ cells.The signal is transmitted to bulge cells,and then bulge cells differentiate into root sheath cells and hair matrix cells.In the early telogen,as the root sheath cells and hair matrix cells undergo the apoptosis process,the cells in the convex area also follow apoptosis,but the process of apoptosis is not complete,one of the branches differentiates into hair germ cells and realizes its function in the next hair follicle cycle.10.Through proteomics,a total of 1903 proteins were detected in this study,including 1377 proteins in telogen,687 proteins in anagen,and 1344 proteins in catagen.By visualizing the relationship between the proteins,it was found that only 366 proteins were expressed in the telogen,and these proteins were mainly related to the apoptosis process of hair follicle cells.There are only 363 proteins expressed in anagen,mainly related to the hair follicle development process,and only 280 proteins are expressed in the catagen.During the evolution from the telogen to anagen,we enriched 20 kinds of high value Proteins,including DLDH,EF2,KPYM,GRP75,MDHC,TNNT3,FHL,etc.,they are mainly involved in the cycle start process;and from anagen to catagen,we enriched 63 kinds of proteins,including H4,QCR,CAZA,CH60 CAZA2,MIME,FLNC,etc.They are related to the growth and maintenance of cashmere.From catagen to telogen,there are 750 common proteins,which reveals that the hair follicles and cashmere in catagen and telogen have the same change trend.11.The expression of marker genes in cells is relatively stable.Western blot experiments were conducted on marker genes of different stem cells in telogen,anagen and catagen to verify the changes in the number of cells of different cells in each period.The results showed that the number of dermal papillary cells,dermal cells and bulge cells continued increased from telogen to anagen,which revealed the development of hair follicles in the process.From anagen to catagen,the number of these cells did not increase significantly,indicating that the hair follicle structure is well developed,And this result validates the conclusion that the number of dermal papillary cells determines the type of hair,and the expression levels of wnt10b and ?-catenin show a significant increase in the three periods,revealing that they play a key role not only in the development of hair follicles but also plays a vital role in the growth of cashmere and the maintenance of cashmere attached to the epidermis at a later stage.