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

Abstract

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.

Keywords: Lgr5+ progenitors; Serpine2; hair cells; regeneration.

Figure 1

Expression pattern of Serpine2 in the mouse cochlea and conditional overexpression of Serpine2 in Lgr5+ progenitors. A) Flow chart of the Serpine2 expression pattern study in wild‐type mouse cochlea. B,C) Serpine2 mRNA and protein expression were assessed by RT‐qPCR (B) and western blotting (C). D) Construction of Serpine2 ^loxp/+ mice. A vector containing the Serpine2 gene sequence and a 3 × HA tag was inserted by CRISPR/Cas9 technology at the Hipp11 (H11) site. E) Genotyping of Serpine2 ^loxp/+ mice. The sizes of the DNA bands amplified by the wild‐type and mutant alleles were 360 and 434 bp, respectively. F) Flow chart of the Serpine2 expression pattern study in Serpine2 cOE mouse cochlea. (G,H) The Serpine2 expression in Serpine2 cOE mice and control mice was detected by RT‐qPCR (G) and immunofluorescence staining (H). Serpine2 expression was detected by HA tag staining (red). Myosin7a (green) was used as the HC marker. “n” refers to the number of mice used. Scale bars are 20 µm in (H). *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 2

Increased ectopic IHCs in heterozygous Serpine2 cOE mice. A) Flow chart of tamoxifen i.p. injection (0.075 mg g⁻¹ body weight) and cochlear imaging and quantification at P7. B) Ectopic IHCs (indicated by arrows) and OHCs (indicated by square brackets) were observed in heterozygous Serpine2 cOE mouse cochleae. Lgr5 ^{EGFP‐CreER/+} mice and Serpine2 ^loxp/+ mice were used as control groups. Myosin7a was used as the HC marker. Scale bars on the left and right sides in (B) are 200 and 20 µm, respectively. C–F) Quantification of the ectopic IHCs in each turn (C), total ectopic IHCs (D), ectopic OHCs in each turn (E), and total ectopic OHCs (F). “n” refers to the number of mice used. *p < 0.05, **p < 0.01, n.s., not significant.

Figure 3

Significantly increased ectopic HCs in homozygous Serpine2 cOE mice. A) Flow chart of tamoxifen i.p. injection (0.075 mg g⁻¹ body weight) and cochlear imaging and quantification at P7. B) Ectopic IHCs (arrows) and OHCs (square brackets) were observed in homozygous Serpine2 cOE cochleae. Lgr5 ^{EGFP‐CreER/+} mice and Serpine2 ^loxp/loxp mice were used as control groups. Myosin7a was used as the HC marker. Scale bars on the left and right sides in (B) are 200 and 20 µm, respectively. C–F) The quantification of ectopic IHCs in each turn (C), total ectopic IHCs (D), ectopic OHCs in each turn (E), and total ectopic OHCs (F). G–J) Quantification of the ectopic IHCs per turn (G), the total ectopic IHCs per cochlea (H), and quantification of the ectopic OHCs per turn (I), the total ectopic OHCs per cochlea (J) of Lgr5 ^{EGFP‐CreER/+} mice, Serpine2 ^loxp/+ mice, Lgr5 ^{EGFP‐CreER/+} Serpine2 ^loxp/+ mice, and Lgr5 ^{EGFP‐CreER/+} Serpine2 ^loxp/loxp mice (G). “n” refers to the number of mice in the experiment. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n.s., not significant.

Figure 4

EdU assay in the cochleae of Serpine2 cOE mice. A) Flow chart of the EdU assay. EdU (0.05 mg g⁻¹ body weight) was i.p. injected from P3 to P5 to label proliferating cells. B–D) EdU was stained (green) in the cochleae of Lgr5 ^{EGFP‐CreER/+} mice (B), heterozygous Serpine2 cOE mice (C), and homozygous Serpine2 cOE mice (D). Sox2 (red) was used as the SC marker, and DAPI (white) was used to label the nucleus. Scale bars are 20 µm in (B–D). E) Quantification of EdU+ SCs in Serpine2 cOE cochleae. “n” refers to the number of mice used.

Figure 5

Lineage tracing of Lgr5+ progenitors in Serpine2 cOE mice. A) Flow chart of the lineage tracing assay. B–D) Lineage tracing images of Lgr5 ^{EGFP‐CreER/+}Rosa26‐tdTomato mice (B), Lgr5 ^{EGFP‐CreER/+} Serpine2 ^loxp/+Rosa26‐tdTomato mice (C), and Lgr5 ^{EGFP‐CreER/+} Serpine2 ^loxp/loxpRosa26‐tdTomato mice (D). tdTomato+ IHCs and tdTomato+ OHCs are indicated by arrows and arrowheads, respectively. Scale bars are 20 µm in (B–D). E–H) Quantification of tdTomato+ IHCs (E) and OHCs (F) in each turn and the total number of tdTomato+ IHCs (G) and OHCs (H) per cochlea. “n” refers to the number of mice used. *p < 0.05, **p < 0.01, n.s., not significant.

Figure 6

Lineage tracing of Lgr5+ progenitors after Serpine2 knockdown ex vivo and in vivo. A) Flow chart of the lineage tracing assay after Serpine2 knockdown by siRNA ex vivo. B) Lineage tracing images of Lgr5 ^{EGFP‐CreER/+}Rosa26‐tdTomato mice treated with siRNA‐NC and siRNA‐Ser. Arrows indicate tdTomato+ HCs. C,D) Quantification of tdTomato+ HCs in each turn (C) and the total number of tdTomato+ HCs per cochlea (D) after Serpine2 knockdown ex vivo. E) Flow chart of the lineage tracing assay after Serpine2 knockdown by AAV in vivo. F) Lineage tracing images of Lgr5 ^{EGFP‐CreER/+}Rosa26‐tdTomato mice injected with AAV‐Sh‐Ser and AAV‐NC. Arrows indicate tdTomato+ HCs. Scale bars are 20 µm in (B) and (F). G,H) Quantification of tdTomato+ HCs in each turn (G) and the total number of tdTomato+ HCs per cochlea after Serpine2 knockdown in vivo (H). *p < 0.05, n.s., not significant.

Figure 7

Single‐nucleus transcriptomic analysis of Serpine2 cOE mice cochlea. A,B) UMAP plot (A) and Dotplot (B) show 11 distinct cell types in the control Serpine2 ^loxp/loxp mouse cochleae. C) The trajectory manifold of HCs from SCs of Serpine2 ^loxp/loxp mouse using the Monocle 2 R package. D,E) UMAP plot (D) and Dotplot (E) show 12 distinct cell types in the Lgr5 ^CreER/+ Serpine2 ^loxp/loxp mouse cochleae. F) The trajectory manifold of HCs from SCs of Lgr5 ^CreER/+ Serpine2 ^loxp/loxp mouse using the Monocle 2 R package. G) Serpine2 expression level in snRNA‐seq data of Serpine2 cOE mice cochlea and control mice cochlea. H–M) AUC pathway scoring of SHH (H), Wnt (I), cell cycle (J), Hippo (K), Notch (L), and TGF‐β (M) signaling pathway activity in Serpine2 ^loxp/loxp and Lgr5 ^CreER/+ Serpine2 ^loxp/loxp mouse cochleae. N–P) RT‐qPCR quantification of the expression of transcription factors (N), SHH signaling pathway genes (O), and Wnt signaling pathway genes (P) in the BM of P7 Serpine2 ^loxp/loxp and Lgr5 ^CreER/+ Serpine2 ^loxp/loxp mouse cochleae. IPhC, inner phalangeal cell; IBC, inner border cell; IHC, inner hair cell; OHC, outer hair cell; KO, Kölliker's organ cell; HeC, Hensen's cell; TBC, tympanic border cell; IdC, interdental cell; DC, Deiters’ cell; OSC, outer sulcus cell; RMC, Reissner's membrane cell; PC, pillar cell; SC, supporting cell; iOHC, immature outer hair cell; iIHC, immature inner hair cell. n = 4, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant.