| Shanbei white cashmere goat is an excellent cashmere goat breed in Shaanxi province,and its cashmere industry is one of the leading industries in northern Shaanxi,playing an important role in revitalizing the regional economy and helping farmers increase their income.The cashmere production,the length of cashmere and the fineness of fiber are all advanced in China and have important economic value.The development of hair follicles in the embryonic period of the goat is decisive for the fleece production traits in adulthood.Therefore,the molecular regulation mechanism of hair follicle development during embryonic period is important for the morphogenesis of hair follicle and molecular assisted breeding of cashmere goats.The structural complexity of hair follicles involves multiple cell types and signal exchanges between different cell types and the spatio-temporal expression of numerous genes,which are the result of the interaction of numerous regulatory factors.With the development of single cell transcriptome sequencing technology,related single cell sequencing technology single cell chromatin open region sequencing technology and other rapid development,scATAC-seq(single cell assay for transposase accessible chromatin with high-throughput sequencing)provide powerful technical support for mining key genes and signaling pathways in the process of hair follicle morphological structure change.In this study,the chromatin accessibility map of mouse hair follicle morphogenesis was firstly constructed using scATAC-seq technique and verified by ex vivo tissue culture,and then the chromatin accessibility map of cashmere goat hair follicle morphogenesis was constructed using the chromatin accessibility map of mouse hair follicle morphogenesis as a reference.Finally,the 10× Genomic chromium spatial transcriptome sequencing technology was used to map the spatial location of important genes in hair follicles during hair follicle morphogenesis in white cashmere goats in northern Shaanxi Province and validated in immunofluorescence staining of cashmere goat skin tissues.The main results of this study are as follows:(1)Construction of a chromatin accessibility map on mouse hair follicle morphogenesis at the single cell level.The main cell types involved in the induction,organogenesis and differentiation of mice,including fibroblasts,keratinocytes and blood vessel cells,were successfully identified using gene activity and anchored withc RNA-seq,and differential chromatin accessibility and gene activity between different cell types were analyzed,and differentially expressed genes between different cell types were analyzed jointly with gene activity to obtain genes whose gene expression was homogeneous with chromatin for further analysis.In hair follicle induction,Col1a1,Col1a2,Lum,Dcn,Twist1 and Twist2 were highly expressed in fibroblasts with high gene activity,and Krt14,Krt15,Krt17,Krt5,Krt7 and Krt8 were highly expressed in keratinocytes with high gene activity.In hair follicle organogenesis,Twist2,Tcf4,Prrx2,Tbx18,Col1a1,Prrx1,Col1a2 and Twist1 were highly expressed and had high gene activity in fibroblasts,and Gata3,Wnt7 b,Wnt10a,Wnt10 b,Lef1,Pou2f3,Rxra and Sox15 were highly expressed and had high gene activity in keratinized cells.At hair follicle differentiation stage,Col1a1,Col1a2,Ebf1,Fgfr1,Notch2,Prrx1,Prrx2 and Rspo3 were highly expressed in fibroblasts with high gene activity,and Bmp2,Dmkn,Krt5,Krt14,Krt15,Wnt7 b,Lhx2 and Wnt10 b were highly expressed and had high gene activity.Further,by transcription factor motif enrichment analysis and motif footprinting analysis,the motif MA0633.1 of Twist2 was screened for consistent gene expression and gene activity during hair follicle induction,organogenesis and differentiation,and consistent motif activity,and differential peaks visualization also revealed the existence of differential peaks sites in the Twist2 promoter region.Based on the scATAC-seq results of mouse hair follicle morphogenesis,the expression of candidate genes such as Enpp2,Twist2,Wnt10 b and Edar were interference in ex vivo cultured skin tissues,and the immunofluorescence staining results revealed that Enpp2,Twist2,Wnt10 b and Edar play an important role in the structure formation of hair follicles.(2)Chromatin accessibility analysis and motif enrichment analysis of fibroblasts and keratinocytes at different stages of hair follicle morphogenesis were successfully enriched for Twist2,Junb,Nfatc1,Lef1,Lhx2 and Sox9,which are transcription factors related to dermal and epidermal development.The integrated fibroblasts and keratinocytes were repopulated and chromatin accessibility analysis was performed on the fibroblast nuclear keratinocyte subpopulation.(3)Construction of a chromatin accessibility map on goat hair follicle morphogenesis at the single cell level.The major cell types in the induction,organogenesis and differentiation of cashmere goats successfully identified,including fibroblasts,keratinocytes and blood vessel cells,and analyzed the differential chromatin accessibility and gene activity among different cell types.In the induction of hair follicles in cashmere goats,Col1a1,Col1a2,Pdgfra,Col6a2,Tcf4,Dcn,Tbx18,Enpp2 and Notch2 had high gene activity in fibroblasts,Krt14,Krt71,Krt17,Vdr,Ovol1,Wnt6,Wnt10 a,Wnt4,Krt80,Krt15,Krt7,Krt19,Krt85,Wnt7 a,Wnt7b and Edar had high gene activity in keratinocytes.In the organogenesis,Col1a1,Col1a2,Col5a2,Col6a3,Dkk2,Enpp2,Tcf4 and Ebf2 had higher gene activity in fibroblasts,and Krt14,Krt71,Krt17,Wnt6,Wnt7 a,Wnt7b,Krt80 and Krt5 had higher gene activity in keratinized cells.In the differentiation stage,Col1a2,Col6a2,Col6a3,Lox,Tcf4,Dcn,Tbx18,Enpp2 and Notch2 had higher gene activity in fibroblasts,and Krt14,Krt71,Krt17,Vdr,Ovol1,Lhx2,Wnt6,Wnt10 a and Wnt4 had higher gene activity in keratinized cells with high gene activity.(4)Construction of a single cell spatial transcriptome map on goat hair follicle morphogenesis.The UMAP dimensionality reduction,clustering and sub-clustering were used to visualize the spatial distribution location of alleles in the hair follicles and the obtained differential results were validated using tissue immunofluorescence.The Col1a1,Dcn,Dlk1,Thbs2,Thbs4 and Zfhx4 genes were widely expressed in the skin tissue of cashmere goats during hair follicle induction.In the induction stage,Krt1 was highly expressed near the epidermis,Krt15 was highly expressed around the hair pegs,CD34 and Postn were also significantly expressed around hair follicles,and Col1a1 and Col1a2 were highly expressed in the hair follicle mesenchyme.In the differentiation stage,Hes1 was highly expressed in the TAC,Krt1,Krt10 and Krt17 were highly expressed in epidermis,and Krt40,Krt71,Krt14,Krtap3-1,Sox9,Lhx2,Lef1 and VDR were highly expressed around hair follicle.We also verified the tissue localization of key genes as Lef1,Krt15,Ctnnb1,Bmp2,Vdr and PCNA using tissue immunofluorescence.The localization results are consistent with previous studies.In summary,we constructed the chromatin accessibility map of hair follicle morphogenesis in mice and Shaanxi white cashmere goats,further constructed the spatial transcriptional map of Shaanxi white cashmere goats,combined with our previous sc RNA-seq results to construct the transcriptional regulatory map and molecular characteristics of hair follicle morphogenesis,and verified the expression and spatial distribution of the key genes.This study enriched the regulatory mechanism of hair follicle morphogenesis and provided a theoretical basis for the analysis of the molecular regulatory mechanism of hair follicle development during embryonic stage and molecular assisted breeding of cashmere goats. |
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