Conditional Overexpression of Net1 Enhances the Trans-Differentiation of Lgr5+ Progenitors into Hair Cells in the Neonatal Mouse Cochlea

Abstract

Sensorineural hearing loss is mainly caused by damage to hair cells (HC), which cannot be regenerated spontaneously in adult mammals once damaged. Cochlear Lgr5⁺ progenitors are characterised by HC regeneration capacity in neonatal mice, and we previously screened several new genes that might induce HC regeneration from Lgr5⁺ progenitors. Net1, a guanine nucleotide exchange factor, is one of the screened new genes and is particularly active in cancer cells and is involved in cell proliferation and differentiation. Here, to explore in vivo roles of Net1 in HC regeneration, Net1 ^loxp/loxp mice were constructed and crossed with Lgr5 ^CreER/+ mice to conditionally overexpress (cOE) Net1 in cochlear Lgr5⁺ progenitors. We observed a large number of ectopic HCs in Lgr5 ^CreER/+ Net1 ^loxp/loxp mouse cochlea, which showed a dose-dependent effect. Moreover, the EdU assay was unable to detect any EdU⁺/Sox2⁺ supporting cells, while lineage tracing showed significantly more regenerated tdTomato⁺ HCs in Lgr5 ^CreER/+ Net1 ^loxp/loxp tdTomato mice, which indicated that Net1 cOE enhanced HC regeneration by inducing the direct trans-differentiation of Lgr5⁺ progenitors rather than mitotic HC regeneration. Additionally, qPCR results showed that the transcription factors related to HC regeneration, including Atoh1, Gfi1 and Pou4f3, were significantly upregulated and are probably the mechanism behind the HC regeneration induced by Net1. In conclusion, our study provides new evidence for the role of Net1 in enhancing HC regeneration in the neonatal mouse cochlea.

Keywords: Net1; HC regeneration; Lgr5+ progenitors; proliferation; trans‐differentiation.

FIGURE 1

Conditional overexpression of Net1 in Lgr5⁺ progenitors. (A, B) Net1 mRNA and protein expression from P3 to P30 in the mouse cochlea were detected by real‐time qPCR (A) and Western blotting (B). (C) Schematic of the construction of C57BL/6J‐Hipp11‐loxp‐STOP‐loxp‐Net1 (Net1 ^loxp/+ ) transgenic mice. The Net1 gene was expressed as a fusion with a 3× HA tag. (D, E) Breeding (E) and genotyping (D) of Lgr5 ^CreER/+ Net1 ^loxp/loxp mice. (F) P0‐P1 mice were I.P. injected with tamoxifen (0.075 mg/g body weight) and the cochleae were harvested at P7 to determine the efficiency of Net1 overexpression. (G) Schematic of cell types in the cochlea. OHCs, outer hair cells; IHCs, inner hair cells; IBCs, inner border cells; IPhCs, inner phalangeal cells; IPCs, inner pillar cells; OPCs, outer pillar cells; DCs, Deiters' cells; HECs, Hensen's cells; CCs, Claudius cells. (H, I) The Net1 overexpression in Net1 ^loxp/+ mice, Lgr5 ^CreER/+ Net1 ^loxp/+ mice and Lgr5 ^CreER/+ Net1 ^loxp/loxp mice was verified by real‐time qPCR (H). The 3× HA tag was stained (red) to identify overexpressed Net1 proteins in Lgr5⁺ progenitors of Lgr5 ^CreER/+ Net1 ^loxp/loxp mice, and SCs types are marked in the lateral view (I). Myo7a (green) was used to label HCs, and DAPI (blue) was used to label nuclei. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar, 20 μm.

FIGURE 2

Ectopic HCs were significantly increased in Net1 homozygous cOE Lgr5 ^CreER/+ Net1 ^loxp/loxp mice. (A) Tamoxifen was I.P. injected into P0‐P1 Lgr5 ^CreER/+ Net1 ^loxp/loxp mice to activate Cre recombinase and subsequently overexpress Net1 in Lgr5⁺ progenitors. (B) Ectopic OHCs (white arrows and white brackets) and IHCs (yellow arrows) were seen in the apical, middle and basal turns of the P7 Lgr5 ^CreER/+ Net1 ^loxp/loxp mouse cochlea. Lgr5 ^CreER/+ and Net1 ^loxp/loxp mice were used as controls. Myo7a (green) was used as the HC marker. Scale bar, 50 μm. (C–F) Quantification of the ectopic OHCs (C) and IHCs (D) per turn and the total ectopic OHCs (E) and total ectopic IHCs (F) per cochlea. ‘n’ refers to the number of mice. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

Only ectopic IHCs were slightly but significantly increased in Net1 heterozygous cOE Lgr5 ^CreER/+ Net1 ^loxp/+ mice. (A) Lgr5 ^CreER/+ Net1 ^loxp/+ mice and Net1 ^loxp/+ mice were I.P. injected with tamoxifen at P0‐P1, and the mice were sacrificed at P7. Net1 ^loxp/+ mice were used as controls. (B) Ectopic OHCs (white arrows) and IHCs (yellow arrows) were seen in the apical, middle and basal turns of Lgr5 ^CreER/+ Net1 ^loxp/+ mice. Myo7a (green) was used as the HC marker. Scale bar, 50 μm. (C, D) Quantification of the ectopic OHCs per turn and the total ectopic OHCs per cochlea (C), and quantification of the ectopic IHCs per turn and the total ectopic IHCs per cochlea of Net1 ^loxp/+ mice and Lgr5 ^CreER/+ Net1 ^loxp/+ mice (D). ‘n’ refers to the number of cochleae. (E–G) Quantification of the ectopic OHCs per turn (E) and the ectopic IHCs per turn (F), and quantification of the total ectopic OHCs and IHCs per cochlea of Net1 ^loxp/+ mice, Lgr5 ^CreER/+ Net1 ^loxp/+ mice and Lgr5 ^CreER/+ Net1 ^loxp/loxp mice (G). ‘n’ refers to the number of cochleae. *p < 0.05. **p < 0.01, ***p < 0.001.

FIGURE 4

EdU assay and lineage tracing of Lgr5⁺ progenitors. (A) Tamoxifen was injected into Lgr5 ^CreER/+ Net1 ^loxp/loxp mice to conditionally overexpress Net1 in Lgr5⁺ progenitors. EdU (50 mg/kg body weight) was injected at P3‐P5 to label proliferating cells. (B) EdU was stained (red) in Net1 ^loxp/loxp mice, Lgr5 ^CreER/+ Net1 ^loxp/loxp mice and positive control cochlea. Sox2 (blue) was used as the SC marker. EdU⁺/Sox2⁺ SCs are indicated by yellow arrows. Scale bar, 20 μm. (C) Quantification of EdU⁺ SCs per cochlea. ‘n’ represents the number of mice. (D) Tamoxifen was injected into Lgr5 ^CreER/+ Net1 ^loxp/loxp tdTomato and Lgr5 ^CreER/+ tdTomato mice at P0‐P1 to lineage trace Lgr5⁺ progenitors at P7. (E, F) Lineage tracing images of tdTomato⁺ HCs in the cochlea of Lgr5 ^CreER/+ tdTomato mice (E) and Lgr5 ^CreER/+ Net1 ^loxp/loxp tdTomato mice (F). Myo7a (green) was used as the HC marker. tdTomato⁺ OHCs are indicated by white arrows, and tdTomato⁺ IHCs are indicated by blue arrows. Scale bar, 20 μm. (G–K) Quantification of tdTomato⁺ OHCs (G) and IHCs (H) per turn, total tdTomato⁺ OHCs (I) and IHCs (J) per cochlea, and total tdTomato⁺ HCs (K). ‘n’ represents the number of mice. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

Quantification of the expression of related genes and signalling pathways in Lgr5 ^CreER/+ Net1 ^loxp/loxp mice. (A) Lgr5 ^CreER/+ Net1 ^loxp/loxp mice and Net1 ^loxp/loxp mice were injected with tamoxifen at P0‐P1, and the RNA of the mouse cochlea was extracted at P7. Net1 ^loxp/loxp mice were used as controls. (B–F) Relative mRNA expression of transcription factors related to HC regeneration (B), cell cycle (C), Wnt signalling (D), Notch signalling (E) and TGFβ signalling pathways (F). Three independent qPCR experiments were performed. *p < 0.05, **p < 0.01, ***p < 0.001.