| Shanbei White Cashmere goat is one of the most important Cashmere goat varieties in China,especially in northern Shaanxi,where agriculture is relatively underdeveloped and has been one of the important economic sources to the local agriculture.Cashmere is a kind of amyelinic fiber produced from secondary hair follicles in Cashmere goat and has been one of the important research objects in the animal husbandry and breeding industry due to its economic performance.During early hair follicle development,the embryonic stage development of hair follicles is well known for controlling the hair traits in adult,therefore,recapitulating the molecular mechanisms underlying early hair follicle development will improve our understanding of hair follicle morphogenesis and will be instructive to the animal breeding that aimed at Cashmere in animal husbandry.The morphogenesis of hair follicles involves the specification of different kinds of cells and cellular communication,together with the asynchronous development of hair follicles,or,in other words,the inconsistency of time between different hair follicles,contributing to the paucity of information regarding the regulatory mechanisms underlying hair follicle morphogenesis during embryonic development.With the development of single cell RNA sequencing(scRNA seq)technology,the problem of cell heterogeneity during mammalian histogenesis and organogenesis has been perfectly solved.Since scRNA seq requires a high quality of single cells in the sample and scRNA seq sequencing has not been reported in isolated cells from a single hair follicle,our study here firstly established a technology platform for isolating hair follicle stem cells from a small number of hair follicle tissues and delineated single cell transcriptional profile of Shanbei White Cashmere goat during the induction stage,organogenesis stage and cytodifferentiation stage,besides,the main cell types involved in hair follicle morphogenesis were identified and their molecular characteristics were also described.Since there were few studies on the development of Cashmere goat hair follicle,we used scRNA seq to investigate the whole hair follicle morphogenesis process and cellular heterogeneity in mice.We also constructed pseudotime differentiation trajectory of epidermal and dermal cell lineage and described the detailed molecular mechanism underlying cell fate decisions.The main results were as follows:1.We successfully established single cell isolation and high throughput RNA sequencing platform from a small quantity of hair follicles in Shanbei White Cashmere goat.The hair follicle stem cells were successfully enriched by using conditioned media and we preliminary investigated the promoting effects of melatonin to Cashmere goat hair follicle growth based on single hair follicle in vitro culture.Based on these results,we demonstrated that melatonin promotes the elongation of hair follicles by regulating key signaling pathways in hair follicle stem cells,which further promotes the growth of the whole hair follicles.2.The single cell transcriptional atlas during Shanbei White Cashmere goat hair follicle development was successfully constructed at single-cell resolution.Based on tSNE clustering analysis,dermal cell lineage,epidermal cell lineage,dermal papilla cells,endothelial cells,keratinocyte,and pericyte cells were successfully identified,and for the first time,the detailed gene expression profiles for each cell type were described.Besides,we identified a series of cell-type-specific markers including LUM,COL1A1,POSTN in dermal cells;SOX9,KRT14,KLF5 l in epidermal cells;RSPO2,APOD,SOX18 in dermal papilla;KRT10,KRT1,SBSN,KRTDAP in keratinocytes,and SHH,HOXC13,MSX1 in hair shaft based on differential analysis.Based on the analysis of the differences between different cell types,a series of marker molecules with cell-type specificity were identified.Based on Monocle analysis,we successfully constructed dermal lineage and epidermal lineage pseudotime differentiation trajectory and analyzed pseudotime gene expression dynamics during the specification of dermal condensate cells,dermal papilla,epidermal cells,hair shaft,and keratinocytes.Taken together,we successfully recapitulated the pseudotime gene expression dynamics during the specification of different cell types in Shanbei White Cashmere goat.3.The main cell types in mouse hair follicle morphogenesis were successfully identified at single cell level.Based on tSNE analysis,epidermal cells,dermal cells,papilla cells,melanocytes,endothelial cells,and nerve cells were identified,and their detailed gene expression profiles were recapitulated.We also identified dermal cells(Col1a1,Lum,Twist2,and Mdk),epidermal cells(Krt14,Krt15,Pdgfra,and Epcam),DC cells(Sox2,Sox18,Trps1,and Foxd1),pericytes(Rgs5 and Acta2),and melanocytes(Plp1 and Fabp7)specifically expressed marker genes.Besides,we further performed gene expression enrichment analysis to cell type specifically expressed genes and demonstrated the detailed gene expression pattern for different cell types.4.Based on pseudotime differentiation trajectory analysis,we successfully constructed epidermal cell lineage and dermal cell lineage differentiation trajectory during hair follicle development and recapitulated pseudotime gene expression dynamics during the specification of dermal cell lineage dermal condensate,dermal papilla,and epidermal cell lineage matrix,hair follicle stem cell precursors,inner root sheath,hair shaft.Based on the SCENIC algorithm,we screened out key transcriptional regulators during the specification of different cells and delineated the transcriptional regulatory network during the cell fate decisions in different cell types.In conclusion,our study here,for the first time,recapitulated Shanbei White Cashmere goat and murine hair follicle morphogenesis process and characterized major cell types at single cell level by using scRNA seq.We also characterized major cell types at single cell level.Furthermore,we reported unparalleled gene expression profiles during Cashmere goat and murine hair follicle development and delineated dynamic gene expression profiles during dermal cell lineage and epidermal cell lineage specification,which uncovered the regulatory mechanisms controlling cell fate decisions.Our results here also enriched people's understanding on early hair follicle morphogenesis in Cashmere goats and provide a strong theoretical and technical support for the breeding researh of Shanbei white Cashmere goats. |
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