Conditional Overexpression of <i>Serpine2</i> Promotes Hair Cell Regeneration from Lgr5+ Progenitors in the Neonatal Mouse Cochlea

Neonatal cochlear Lgr5+ progenitors retain limited hair cells (HCs) regenerative capacity, but the regulatory network remains incompletely defined. Serpin family E member 2 (Serpine2) is shown to participate in regulating proliferation and differentiation of cochlear Lgr5+ progenitors in the previous in vitro study. Here, the expression pattern and in vivo roles of Serpine2 in HC regeneration are explored by transgenic mice. It is found that Serpine2 is expressed in the mouse cochlea after birth with a downward trend as the mice age. In addition, Serpine2 conditional overexpression in vivo in Lgr5+ progenitors of neonatal mice cochlea results in an increased number of ectopic HCs in a dose-dependent manner. Serpine2 knockdown ex vivo and in vivo can inhibit HC regeneration. EdU assay and lineage tracing assay demonstrate these ectopic HCs likely originate from Lgr5+ progenitors through direct transdifferentiation rather than through mitotic regeneration. Moreover, single-nucleus RNA sequencing analysis and mRNA level validation reveal that conditionally overexpressed Serpine2 likely induces HC regeneration via inhibiting sonic hedgehog (SHH) signal pathway and inducing Atoh1 and Pou4f3 transcription factor. In brief, these data indicate that Serpine2 plays a pivotal role in HC regeneration from Lgr5+ progenitors in the neonatal mouse cochlea, and this suggests a new avenue for future research into HC regeneration.