Hair Cell Generation by Notch Inhibition in the Adult Mammalian Cristae

Balance disorders caused by hair cell loss in the sensory organs of the vestibular system pose a significant health problem worldwide, particularly in the elderly. Currently, this hair cell loss is permanent as there is no effective treatment. This is in stark contrast to nonmammalian vertebrates who robustly regenerate hair cells after damage. This disparity in regenerative potential highlights the need for further manipulation in order to stimulate more robust hair cell regeneration in mammals. In the utricle, Notch signaling is required for maintaining the striolar support cell phenotype into the second postnatal week. Notch signaling has further been implicated in hair cell regeneration after damage in the mature utricle. Here, we investigate the role of Notch signaling in the mature mammalian cristae in order to characterize the Notch-mediated regenerative potential of these sensory organs. For these studies, we used the gamma-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), in conjunction with a method we developed to culture cristae in vitro. In postnatal and adult cristae, we found that 5 days of DAPT treatment resulted in a downregulation of the Notch effectors Hes1 and Hes5 and also an increase in the total number of Gfi1(+) hair cells. Hes5, as reported by Hes5-GFP, was downregulated specifically in peripheral support cells. Using lineage tracing with proteolipid protein (PLP)/CreER;mTmG mice, we found that these hair cells arose through transdifferentiation of support cells in cristae explanted from mice up to 10 weeks of age. These transdifferentiated cells arose without proliferation and were capable of taking on a hair cell morphology, migrating to the correct cell layer, and assembling what appears to be a stereocilia bundle with a long kinocilium. Overall, these data show that Notch signaling is active in the mature cristae and suggest that it may be important in maintaining the support cell fate in a subset of peripheral support cells.