Hair Cell Generation by Notch Inhibition in the Adult Mammalian Cristae
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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 γ-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.
Keywordsinner ear regeneration vestibular DAPT
This work was supported by the following grants: PHS R21 DC010862 from NIDCD/NIH, PHS NRSA T32 GM07270 from NIGMS/NIH, and PHS P30 DC004661 from NIDCD/NIH. We thank Dr. Byron Hartman for his significant contribution to the development of this work; Dr. Verdon Taylor for the Hes5-GFP mice; Dr. Hugo Bellen for the Gfi1 antibody; Dr. Vidhya Munnamalai for the schematic of the inner ear; Catherine Ray and Katena Koemmpel for technical support; past and present members of the Bermingham-McDonogh, Reh, and Chao labs for helpful discussions; Drs. Thomas Reh, David Raible, Ajay Dhaka, Anna La Torre, and Yumi Ueki for critical comments on the manuscript; the Biology of the Inner Ear Course at the Marine Biological Laboratory for helpful instruction; Dr. Ronald Seifert for help with microscopy; and the Lynn and Mike Garvey Cell Imaging Lab.
Cristae are highly three-dimensional, composed of two saddle-shaped hemicristae separated by the eminentia cruciatum. Sox9 (red) labels support cells as well as nonsensory cells in the eminentia cruciatum and throughout the ampulla and semicircular canals. Gfi1 (white) labels all hair cells in the sensory epithelium. Hes5-GFP is expressed in a subset of support cells in the Calretinin-negative peripheral zone. Note that while the overall structure of the sensory epithelium was preserved, the normally dome-like Sox9+ ampulla flattened onto the sensory epithelium. Dimensions in micrometers (w × h × d) − 544.9 × 272.5 × 75.5 (MOV 17,420 kb)
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