Hippo-YAP Signaling Pathway Regulates The Regeneration Of Hair Cells In The Postnatal Mouse Cochlea

Objective: We aimed to explore the role of Hippo-YAP signaling in regulating the proliferation ability of cochlear Lgr5+(The Wnt-responsive leucine-rich repeat-containing G protein-coupled receptor 5)progenitor cell.Methods: We tested the expression of Hippo-YAP signaling pathway in the mouse cochlea by RT-PCR,Western blot and Immunofluorescence staining.We used three molecule drug to regulate the Hippo signaling in the mouse cochleae,and detected the effects of these small molecule drug via Western blot,Semi-quantification of the western blot,and q PCR.Then,we explored the regulation of Hippo-YAP signaling on the proliferation ability of cochlear Lgr5+ progenitors through Sphere assay and lineage traced cochlear explant culture.Lastly,we detected the relative m RNA level of YAP target genes,ccnd1,and Wnt target genes to research the possible mechanism.Results: 1.RT-PCR,western blot,and immunohistochemistry results confirmed that the Hippo-YAP signaling exists in the mouse cochlea.2.Small molecule drugs can efficiently regulate the Hippo-YAP signaling in the mouse cochlea,which detected via Western blot,Semi-quantification of the western blot,and q PCR.3.YAP inhibition made Lgr5+ progenitors form significantly less spheres than control,and the average diameter of spheres was significantly smaller than in the control group,while YAP activator-treated group formed significantly more spheres and the size of spheres were also larger than control.4.The numbers of Tomato+/EDU+ cells,representing the proliferated Lgr5+ progenitors,was obviously decreased in all three turns of YAP inhibitor-treated cochleae than DMSO control.While,YAP activator-treated group had significantly more Tomato+/EDU+ cells in the apical,middle,and basal turns of cochleae.5.Verteporfin and dobutamine significantly inhibited the YAP target genes(ctgf,cyr61 and ankrd1),cell cycling gene ccnd1 and the Wnt target genes.While XMU-MP-1 treatment significantly up-regulated these genes.Conclusions: we reported that the Hippo-YAP signaling is highly expressed in mouse cochlea.YAP activation significantly promoted the proliferation of the Lgr5+ progenitor cells both in isolated Lgr5+ cells and cultured whole organ explants,which may be related to the regulation of YAP in YAP target genes,cell cycle and β-catenin transcriptional capacity.Our findings may be useful in exploring the mechanisms regulating mammalian inner ear HC generation,and may provide a new strategy to restore hearing in clinic in the future.