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

Neonatal cochlear Lgr5+ progenitors can regenerate hair cells (HCs), and this process is regulated by several genes and signaling pathways. However, this regeneration ability is limited, and whether additional genes are involved in this process remains unknown. Serpine2 was shown to participate in regulating proliferation and differentiation of cochlear Lgr5+ progenitors in our previous in vitro study. Here, we explored the expression pattern and in vivo roles of Serpine2 in HC regeneration by constructing transgenic mice in which Serpine2 is conditionally overexpressed in cochlear Lgr5+ progenitors. We found that Serpine2 is expressed in the mouse cochlea after birth with a downward trend as the mice aged. In addition, Serpine2 conditional overexpression in vivo in Lgr5+ progenitors of neonatal mice cochlea resulted in an increase number of ectopic HCs in a dose-dependent manner. And Serpine2 knockdown ex vivo and in vivo can inhibit HC regeneration. EdU assay and lineage tracing assay demonstrated that these ectopic HCs likely originated from Lgr5+ progenitors through direct trans-differentiation rather than through mitotic regeneration. Moreover, snRNA-seq analysis and RT-qPCR results revealed that Serpine2 cOE likely induces HC regeneration via inhibiting sonic hedgehog (SHH) signal pathway and inducing Atoh1 and Pou4f3 transcription factor. In brief, our 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. snRNA-seq was performed using 19 cochleae from Serpine2loxp/loxp (control) and 20 cochleae from Lgr5EGFP-CreER/+Serpine2loxp/loxp (Serpine2 cOE) mice at P7, after tamoxifen injection at P1, with cochlear basement membrane pooled per group. Purified nuclei were diluted to 700–1200 nuclei/μl for 10× Genomics Chromium. Library preparation followed the 10× Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 protocol, and sequencing was performed on the Illumina NovaSeq 6000 system. Gene expression was aligned using the GRCm39 reference genome and quantified with the 10× Cell Ranger v3 pipeline (version 8.0.1).