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Background:Tissue injury activates endogenous stem/progenitor cells to regenerate lost cells.The inner ear utricle requires mechanosensory hair cells (HCs) to detect linear acceleration.The utricle consists of two types of HCs (Type Ⅰ and Ⅱ);after damage,non-mammalian utricles mitotically regenerate HCs,and both types of HCs are replenished.In adult mammalian utricles,supporting cells (SCs) nonmitotically regenerate HCs,which are predominantly extrastriolar Type Ⅱ HCs.Recent studies have revealed that the neonatal utricles can mitotically regenerate HCs after damage.The current study aims to identify and characterize these damage-recruited progenitor cells.Methods:We used the Pou4f3-DTR mice,where the human diphtheria toxin receptor is driven by Pou4f3 expression,to ablate HCs.Diphtheria toxin was injected on postnatal day (P)1 and tissues were examined from P3 to P30.To detect Lgr5 expression,we used the Lgr5-EGFP-CreERT2 reporter mice.In parallel,we fate-mapped Lgr5+progenitors and Plp1+ SC in this neonatal HC damage model using Pou4f3-DTR;Lgr5-EGFP-CreERT2/Plp1-CreERT;ROSA26R-tdTomato mice.Results:After DT injection at P1,~27% of HCs were present in utricles from P15 Pou4f3-DTR mice in comparison toage-matched,wildtype controls.At P30,normalized HC number increased to 55%,suggesting HC regeneration.Without damage,Lgr5 is absent from the postnatal utricle.After HC ablation,Lgr5 expression is foundpredominantly in striolar supporting cells at P4.Lineage tracing of Lgr5+ cells revealed that they contributed to the striola,Myosin+ HCs at P30.These fate-mapped HCs displayed abnormal-appearing f-actin+,espin+stereocila and Ctbp2+,Shank1+ synapses.Quantification of Calbindin,Sox2,and Tuj1+ calyx expression in fate-mapped,Myosin7a+ HCs showed that ~50% regenerated HCs were Type Ⅰ.Prior studies have shown that tamoxifen preferentially activates Cre recombinase in extrastriolar SCs in Plp1-CreERT mice.Lineage tracing revealed that Plp1+ SCs contribute to 81.1±1.8% of Myo7a+ HCs in the extrastriolar region at P30 after HC ablation at P1.Without damage,no Edu+,Myo7a+ HCs,~1-2 Edu+,Sox2+ SCs (per 20,000 μm2) weredetected in the striola or extrastriolar of P30 utricles.After damage,~14 Edu+,Myo7a+ HCs,~96 Edu+,Sox2+ SCs were found in striolar and ~6 Edu+,Myo7a+ HCs,~28 Edu+,Sox2+ SCs were found in extrastriolar region.Conclusions:HC ablation induces Lgr5 expression in striolar SCs,which contribute to regeneration of Type Ⅰ and Ⅱ striolar HCs in the neonatal utricle.Lgr5+ SCs can re-enter cell cycle to mitotically regenerate HCs.Thus,Lgr5+ SCs act as facultative HC progenitors.