Single-Cell Transcriptome Sequencing-based Study Of Secondary Hair Follicle Morphogenesis In Ordos Fine-wool Sheep Based

Ordos fine-wool sheep is a high-quality variety that can produce high-quality natural textile raw material wool and mutton,but there is still a big gap in fiber diameter and wool yield compared with Australian Merino sheep.The hair follicle is the main organ that controls the type of wool fiber.Secondary hair follicle morphogenesis is the key to point affecting the quality of wool.However,the process and molecular mechanism of hair follicle morphogenesis in Ordos fine wool sheep is still unclear.In summary,it is of great significance to analyze the molecular mechanism of hair follicle morphogenesis for improving the wool fiber diameter and wool yield of Ordos fine-wool sheep.In this study,single-cell sequencing and fluorescence immunohistochemistry were used to study the process of secondary hair follicle morphogenesis in Ordos fine-wool sheep and to reveal the cell heterogeneity and the differentiation mechanism of dermal and epidermal lineage cells in the process of secondary hair follicle morphogenesis in Ordos fine wool sheep.1.On the 93 rd day of pregnancy(E93),Ordos fine-wool sheep had differentiated obvious mature primary follicles,but no secondary follicles appeared.On the 110 th day of pregnancy(E110),Ordos fine-wool sheep had differentiated into obvious primary hair follicles and secondary hair follicles.The above two periods can be applied to the study of molecular characteristics of secondary hair follicles in finewool sheep.2.The transcriptional profiles of Ordos fine hair sheep at E93 and E110 were constructed at the single cell level.Based on cell fractionation,a total of 12 cell types including dermis,epidermis,fibroblasts,DP,funiculus cells,keratinocytes,sebaceous glands,melanocytes,periderm,immune cells and endothelial cells were successfully identified,and the above cell types basically contained the key cell types in the morphogenesis of hair follicles.And the molecular characteristics of each cell group were further described.3.Based on pseudo-timing analysis,the differentiation trajectory of dermal lineage cells was successfully constructed.By visualizing the dynamic expression of genes such as COL1A1,LFE1,PRDM1,and RSPO3 during this process,enriched signal pathways such as cell adhesion,cell migration,BMP,Notch,and Wnt / β-catenin signaling.The process of dermal lineage cells differentiating into dermal papilla through skin coagulation cells was analyzed.Furthermore,through the heterogeneity analysis of dermal papilla,the differential genes APOD,POSTN,KRT5 and KRT15 of dermal papilla related to primary hair follicles and secondary hair follicles were explored,and different molecular characteristics such as cell adhesion and osteoblast differentiation were enriched.4.Based on pseudo-temporal analysis,the differentiation trajectory of epidermal lineage cells was successfully constructed.By visualizing the dynamic expression of genes such as CTNNB1,KRT25,LUM,POSTN and SBSN during this process,enriched cell adhesion,cell migration,fibrous tissue development,WNT / β-CATENIN and BMP signaling pathways.The differentiation process of the primary hair follicle of the epidermal lineage cells,the differentiation process of the primary hair follicle and the secondary hair follicle-related keratinocytes,the differentiation process of the hair shaft,and the differentiation process of the muscle cells in the epidermis such as the primary hair follicle were analyzed.And through the analysis of protein interaction between primary hair follicles and secondary hair follicles in two different stages of keratinocyte differentiation,the differentiation of keratinocytes related to primary hair follicles is closely related to BMP signaling,translation and cell adhesion.Keratinocyte differentiation related to secondary hair follicles and WNT / β-CATENIN signaling are related to cell morphological changes but not to transcription-related pathways,further indicating that the molecular characteristics of keratinocytes at different differentiation stages are different.The results of this study can deepen the understanding of the morphogenesis of secondary hair follicles in fine wool sheep,and provide theoretical and technical support for breeding research of Ordos fine wool sheep.