Live imaging and multimodal profiling reveal transdifferentiation of a cochlear supporting cell subpopulation upon Notch inhibition

Lama Khalaily¹, Shahar Kasirer^3.4, Katherine Domb¹, Mi Zhou², Buwei Shao¹, Shahar Taiber¹, Ran Elkon¹, Litao Tao², David Sprinzak³, Karen B. Avraham<sup>1

1</sup>Department of Human Genetics and Computational Medicine, Gray Faculty of Medical and Health Sciences and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel
²Department of Biomedical Sciences, School of Medicine, Creighton University, Omaha, NE, USA
³School of Neurobiology, Biochemistry & Biophysics, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
⁴School of Physics and Astronomy, Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, Israel

Regeneration enables organisms to repair damaged tissues, yet this capacity is strikingly limited in the cochlear sensory epithelium, essential for sound detection. A major cause of hearing loss arises from the irreversible loss of sensory hair cells (HCs) in the cochlea. While supporting cells (SCs) have a latent ability to transdifferentiate into HCs, this regenerative potential is rapidly lost after development. Using live imaging and single-cell multi-omics of cochlear explants, we uncovered the cellular and molecular heterogeneity underlying the limited regenerative capacity of the neonatal mouse cochlea. Notch repression broadly silenced key SC genes, yet only a rare subpopulation of Deiters cells (DC), termed transdifferentiating DCs (tDCs), initiated the transdifferentiation into HC fate. These cells underwent coordinated transcriptional and enhancer remodeling, linking epigenetic priming with morphological plasticity, while other SCs remained refractory despite robust Notch targets downregulation. Our study provides a molecular definition of an early induced transitional DC to HC state, revealing Notch inhibition as a selective trigger that unmasks rare regenerative competence.

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