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Updated: Jun 6, 2026
Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation
Published on: January 12, 2015
Conditional knockout of Dkk3 drives Lgr5+ progenitor reprogramming into hair cells in the mouse cochlea
Hairong Xiao1,2, Xinlin Wang1, Zixuan Ye1
1State Key Laboratory of Digital Medical Engineering, Department of Otolaryngology Head and Neck Surgery, Zhongda Hospital, School of Life Sciences and Technology, Advanced Institute for Life and Health, Jiangsu Province High-Tech Key Laboratory for Bio-Medical Research, Southeast University, Nanjing, China.
Background:
Neonatal cochlear Lgr5+ progenitors possess a transient regenerative capacity that diminishes rapidly after birth, severely limiting the potential for hearing restoration. Identifying the molecular mechanisms that restrict this plasticity is critical for developing effective regenerative therapies to treat hearing loss (HL).
Methods:
To uncover regulators of cochlear progenitor plasticity, we performed an integrative transcriptomic analysis across spatial, injury, and lineage contexts. Following the identification of candidate regulators, we investigated the function of Dkk3, a canonical Wnt antagonist, using in vitro Lgr5+ sphere formation assays and in vivo conditional knockout (cKO) models in neonatal progenitors. We assessed hair cell (HC) generation and maturation using lineage tracing, histological analysis of stereocilia and synapses, and electrophysiological recordings. Furthermore, we employed single-nucleus transcriptomics (snRNA-seq) of the Dkk3 cKO cochlea to elucidate the underlying molecular signaling networks.
Results:
Our screen identified 14 candidate regulators, highlighting Dkk3 as a previously uncharacterized factor in the auditory epithelium that functions as a physiological gatekeeper of Lgr5+ progenitor plasticity. In vitro, Dkk3 knockdown significantly enhanced Lgr5+ sphere formation. In vivo, Dkk3 cKO induced the spontaneous generation of HCs through direct trans-differentiation. Crucially, these ectopic HCs achieved structural maturity, characterized by organized stereocilia and synaptic connections, displayed partial electrophysiological activity, and survived long-term into adulthood without disrupting native auditory function. Mechanistically, snRNA-seq analysis and RT-qPCR validation suggested that Dkk3 cKO may activate a pro-regenerative network involving the Wnt, Hedgehog, and mTOR signaling pathways.
Conclusions:
Our findings establish Dkk3 as a key molecular inhibitor of sensory fate reprogramming in the cochlea. These results suggest that targeting Dkk3 represents a promising therapeutic strategy for functional and durable HC reprogramming.
