| Background and objectiveThe development,repair,and regeneration of organs require the coordinated action of tissue stem cells in the epithelium and mesenchyme.These tissue stem cells reside in their own microenvironment(niche),which sends signals to regulate their fate and behaviour.Stem cells in the epithelium have been extensively studied in many organs,revealing a common principle that epithelial stem cells(epiSCs)express high levels of integrins and adhere to their underlying basement membrane extracellular matrix,an interface between the epithelium and the mesenchyme.On the other hand,although mesenchymal stem and progenitor cells have been isolated in many adult organs,their in vivo identities,characteristics,and niches remain unclear.Hair follicles are mini-organs covering almost the entire skin surface of mammals.They periodically regenerate hairs and the lower cycling portion of hair follicles throughout adult life,thus serving as an excellent model to study the biology of organ regeneration.Mammalian epiSC populations in hair follicles exist in the epithelial basal layer and adhere to their underlying basement membrane.The basement membrane plays a central role in controlling the fate and behaviour of skin epiSCs.Recent studies have reported the presence of multiple epiSC populations in and around the hair follicle bulge,as well as a primed population in the hair germ just below the bulge.The tissue architecture of the hair follicle mesenchyme also dynamically changes during the hair follicle regeneration cycle,suggesting the existence of tissue stem cells regenerating the mesenchymal portion of the hair follicle.However,the identity of the tissue stem cells in the hair follicle mesenchyme has long been unclear.In this study,we generate Itga8-eGFP-IRES-CreERT2 and Itga8-T2A-eGFP knock-in mouse lines to identify Itga8-expressing cells in skin mesenchyme and evaluate their lineage behaviour in the hair follicle regeneration process.We show that,in mouse hair follicles,?8 integrin(Itga8)expression marks thin mesenchymal cells that reside on the mesenchymal side of the epithelial major ligand.Itga8+ cells physically separate the epiSCs/progenitors and the dermal papilla of the mesenchymal signalling centre in quiescent hair follicles.Itga8+cells primarily give rise to the dermal sheath and dermal cup and are maintained over several regeneration cycles.Nephronectin deletion in epiSCs/progenitors depletes mesenchymal ?8 integrin from the basement membrane,ectopically activating hair follicle regeneration.Thus,Itga8+cells constitute a reservoir of mesenchymal stem cells(MSCs)residing on and regulated by the basement membrane of epiSCs/progenitors;these MSCs are involved in the quiescence of the hair follicle regeneration programme.Methods1.We employed a microhomology-mediated end-joining(MMEJ)-assisted gene knock-in using CRISPR/Cas9 with the precise integration into target chromosome(PITCh)system,and we generated Itga8-eGFP-IRES-CreERT2 and Itga8-T2A-eGFP knock-in mice expressing the eGFP and CreERT2 genes under the control of the Itga8 promoter.We confirmed that the knock-in cassette was precisely knocked-in with genomic PCR and sequencing.To examine the expression of transgenes in knock-in mice,we immunostained ?8 integrin and eGFP in adult telogen skin.2.To genetically label Itga8+cells,Itga8-eGFP-IRES-CreERT2 mice were crossed with R26R-Confetti mice.We administered 4-hydroxytamoxifen(4-OHT)topically to the dorsal skin of 8-week-old Itga8-eGFP-IRES-CreERT2;Confetti mice for 3 consecutive days.Confetti protein expression was examined by immunostaining.To further confirm the tissue localisation of Itga8-expressing cells,the eGFP+cells in adult skin of Itga8-T2A-eGFP mice were checked.3.To investigate the involvement of epiSC/progenitor basement membrane on the regulation of Itga8+MSCs and the hair follicle regeneration cycle,we first examined the tissue localisation of ?8 integrin in the absence of epiSC/progenitor-derived nephronectin.To this end,we generated mice lacking the nephronectin gene(Npnt)in skin basal epithelia by crossing Npnt-floxed mice with K5-Cre mice(K5-Cre;Npnt fl/fl).We then examined the effects of nephronectin deletion on the hair follicle regeneration process.Results1.We generated Itga8-eGFP-IRES-CreERT2 and Itga8-T2A-eGFP knock-in mice,and we identified that Itga8-eGFP-IRES-CreERT2 and Itga8-T2A-eGFP knock-in mice can serve as a useful tool for detection and lineage-tracing of Itga8+cells in adult skin.2.Itga8+ cells are a subset of dermal fibroblasts that are distinct from dermal papilla cells and are located at the interface between hair germ epiSCs and the dermal papilla,as well as in the periphery of the dermal papilla of telogen hair follicles.Our results also suggest that the dermal papilla cell cluster is segmented by Itga8+ cells and has limited contact with the hair germ basement membrane in the telogen phase.3.Itga8+ stem cells were quiescent in the telogen phase and became proliferative in the anagen phase,giving rise primarily to the dermal sheath and dermal cup.4.Through interaction with ?8 integrin-epithelial stem/progenitor cell-derived nephronectin is involved in the tissue localisation of Itga8+MSCs and in the quiescence of the hair follicle regeneration cycle.Deletion of epiSC-derived nephronectin depletes mesenchymal ?8 integrin and ectopically induces hair follicle regeneration.ConclusionsIn this study,we show that ?8 integrin defines a distinct hair follicle MSC population that resides on the mesenchymal surface of the epithelial stem/progenitor cell basement membrane.We also found that epithelial stem/progenitor cells specialise the molecular composition of the basement membrane to regulate the location of Itga8+ MSCs,as well as the quiescence of the hair follicle regeneration programme.Thus,our study elucidates the molecular and cellular underpinnings of the crosstalk between epithelial and mesenchymal stem/progenitor cells in organ regeneration via their shared basement membrane. |
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