Whole Transcriptomic Profiling Of Dermal Papilla Cells From Cashmere Goats And Related Molecular Mechanisms Underlying Their Intercellular Interaction With Hair Matrix Cells

Hair is one of the most obvious features shared by mammals,which is produced by the miniorgan called hair follicle?HF?in skins.HF is mainly composed of mesenchymal and epithelia cells.Dermal papilla cells?DPCs?are the most important mesenchymal cells inside HF that control the proliferation and differentiation of epithelial cells through secreting a myriad of growth factors and cytokines.Hair matrix cells?HMCs?,which are the progenies of hair follicle stem cells,are the progenitors of hair-forming cells.Their rapid division and terminal differentiation are the basis of fast hair growth.Cashmere goats are the farm animals with exceptional status in China.Exploration of related mechanism underlying HF development lays solid foundation for improving the quality and quantity of hair fibers.Aims of our present study include: 1)isolation and cultivation of DPCs and dermal fibroblasts?DFs?,profiling their whole transcriptomic data and screening potential coding and non-coding candidates related to hair growth through comparing corresponding data;2)verification of the roles of CRABP1 and LEPR related all-trans retinoic acid?ATRA?and leptin on DPCs;3)establishment and optimization of culturing system of HMCs;4)probing the molecular mechanisms underlying the intercellular communication between DPCs and HMCs through cell coculture and RNA-seq.Our main results were listed as follows:1.Using microdissection and tissues culture methods,we successfully obtained DPCs and DFs from goat skins;they displayed distinct morphologies,DPCs are flatten with multiple projection and DFs are in spindle shape with smaller volume;through transcriptomic profiling,we totally identified 43766 mRNAs,2540 lncRNAs,759 TUCP,536 miRNAs and 3706 circRNAs.2.Through analysis,we showed that 2538 mRNAs were differentially expressed between DPCs and DFs,including 1286 mRNAs upregulated in DPCs,and 1252 downregulated in DPCs;71 lncRNAs showed differential expressions,comprising 18 upregulated and 53 downregulated in DPCs respectively;18 TUCP exhibited differential expressions,containing 3 upregulated and 15 downregulated in DPCs respectively;121 circRNAs exhibited differential expressions,including 76 upregulated and 45 downregulated in DPCs respectively;86 miRNAs exhibited differential expressions,including 42 upregulated and 44 downregulated in DPCs respectively.KEGG analysis of upregulated genes in DPCs and the target genes of non-coding RNAs displayed that Focal adhesion,ECM-receptor interaction and Signaling pathways regulating pluripotency of stem cells were enriched.Moreover,several hormones related pathways such as Estrogen signaling pathway,Adipocytokine signaling pathway and Thyroid hormone signaling pathway also appeared.3.Through overlapping with mouse data,we defined 25 core signatures of DPCs.Some of the are SPP1,LEPR,WNT5 A,PTGFR,RSPO1,HOXC8,MAGED2 and others.We chose two genes responsible for hair follicle stem cell activation for further analysis,and found that their expressions were under positive and negative regulations of miRNAs and lncRNAs such as chi-miR-144-5P,lnc₀00335 and lnc₀01710.Moreover,some lncRNAs and circRNAs such as XR₃10320.3 and chi_circ₀00573,served as competing endogenous RNAs to absorb miRNAs for relieving their inhibitory impacts on gene expressions.4.Two genes inside retinoic acid signaling,CRABP1 and RAR? were highly expressed in DPCs compared with DFs;treatment of DPCs with ATRA weakened their cellular activities,induced their apoptosis,increased their percentage at G1 phase and decreased quantities at G2 phase;ATRA stimulated the expressions of CRABP1 and RAR? in DPCs?P<0.05?,and inhibited the expression of FGF7?P<0.05?;ATRA impaired FGF7 expression at transcriptional level?P<0.05?and several RAR? binding sites existed in the promoter region of FGF7;mRNA and protein expressions of LEPR are relatively more in DPCs than DFs;treatment of recombinant leptin enhanced the cellular activities of DPCs,and the expressions of FGF7 and IGF-1?P<0.05?.5.We successfully cultivated HMCs from goat HF,and their primary and passaged cultures exhibited similar morphologies with keratinocytes;RPMI 1640 without calcium was the preferred medium,and Coating Matrix and Collagen Type IV were the preferred coating substances;cell doubling time of HMCs was 23.60 hours;specific markers for HMCs such as HOXC13 and SOX21 were highly expressed in HMCs than DPCs.6.Calcium stimulated the expressions of several keratinocyte differentiation associated genes including loricrin? involucrin and KRT1 in HMCs?P<0.05?,improved the ratio of proliferating cells of HMCs,but showed no effects on cell cycle distribution of HMCs;ATRA stimulated HMCs proliferation,decreased the percentage of HMCs at G1 stage,increased their percentage at G2 and S stage;ATRA stimulated the expressions of CRABP1 and RAR??P<0.05?,as well as the expression of involucrin?P<0.05?.7.Coculture changed cell cycle distribution of DPCs and HMCs.The percentage of DPCs at S phase were significantly higher when cocultured.Moreover,the percentage of HMCs at G1 stage decreased,and G2 and S increased when incubated with DPCs.8.Cell coculture significantly altered gene expression patterns of both cell populations.Compared with mono-cultured DPCs,3358 mRNAs including some well-noticed genes such as FGF7,FGF10,WNT5 A,and IL-6 were upregulated,and 2843 mRNAs were downregulated;3436 mRNAs were upregulated in cocultured HMCs,including a well-described gene IL-1,and 3059 mRNAs were downregulated including several genes encoding keratins and a regulatory gene HOXC13 for keratin expressions.Overall,we provided adequate information for the gene expressions inside DPCs,and screened potential coding and non-coding genes associated with hair growth.We also hinted the genes responsible for intercellular communication between DPCs and HMCs.Our study is informative for exploration of the rationales underlying hair growth in cashmere goats and other mammals.