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BMP4 cocktail promotes utricular progenitor reprogramming and vestibular functional recovery in adult mice
Dan You1, Yunzhong Zhang1, Kunkun Wang1
1ENT Institute and Otorhinolaryngology, Department of Affiliated Eye and ENT Hospital, Key Laboratory of Hearing Medicine of NHFPC, Shanghai Key Laboratory of Gene Editing and Cell Therapy for Rare Diseases, State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200031, China.
Introduction:
Vestibular hair cells (HCs) are essential for maintaining balance and detecting head movements. In mammals, following vestibular damage HC regeneration derives from epithelial non-hair cells (ENHCs), which possess limited capacity for proliferation and reprogramming.
Objectives:
To examine the role of BMP4 in reprogramming utricular HCs following ototoxic injury in postnatal mice.
Methods:
The study utilized both wild-type mice and transgenic strains on a C57BL/6J background, including Notch1flox/flox, Pou4f3+/DTR, ROSA26tdTomato, Atoh1-eGFP; Sox9-CreERT2, and Fos-CreERT2, to investigate and lineage-trace the reprogramming of new HCs initiated by BMP4 protein. Advanced sequencing techniques, including single-cell RNA sequencing, bulk RNA sequencing, and CUT&Tag sequencing, were employed for transcriptomic and epigenomic analyses. To induce vestibular dysfunction, intraperitoneal injections of IDPN were administered to postnatal day 30 (P30) mice. Vestibular function was assessed through behavioral tests, including vestibulo-ocular reflex, off-vertical axis rotation, and gait analysis, to evaluate the functional outcomes of HC regeneration. For cellular studies, sphere or explant tissues from P2 mice, with gentamicin or small molecule cocktail in vitro, were used to evaluate proliferation and differentiation of ENHCs through EdU labelling or tdTomato lineage tracing, histological analyses, immunofluorescence staining, and Western blot analysis.
Results:
We found that increased BMP4 expression enhances ENHC reprogramming, accompanied by elevated levels of key HC transcription factors, including Gfi1, Pou4f3, and Atoh1, via c-Fos activation. Moreover, exogenous BMP4 further sensitized ENHCs to Notch inhibition and Wnt pathway activation, thus amplifying the regenerative outcomes. Conversely, inhibition of c-Fos or BMP4 diminished these effects, demonstrating that BMP4 is essential for both Notch inhibition and Wnt activation. Notably, the use of a combination of small molecules targeting these pathways successfully restored vestibular function and promoted HC regeneration in adult mice.
Conclusion:
Our findings suggest that BMP4 and its associated signaling pathways represent promising therapeutic targets for the restoration of hearing and balance.
