Hair Cell Generation by Notch Inhibition in the Adult Mammalian Cristae

Abstract

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)-Lalanyl]-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.

Sensory hair cell development and regeneration: similarities and differences

“…However, after hair cell loss, the inhibition of Notch signaling via either inhibition of the target gene Hes5 or pharmacological inhibition of γ-secretase was able to significantly increase Atoh1 expression, with a subset of supporting cells maturing to myosin VIIa-expressing hair cells (Burns et al, 2012;Jung et al, 2013;Lin et al, 2011). Similarly, more hair cells were detected following Notch inhibition in the damaged crista ampullaris (Slowik and Bermingham-McDonogh, 2013).…”

Severe streptomycin ototoxicity in the mouse utricle leads to a flat epithelium but the peripheral neural degeneration is delayed

“…The vestibular epithelium of mammals in vivo has spontaneous HC regeneration after HCs are lost (Forge et al, 1998; Forge et al, 1993; Golub et al, 2012; Kawamoto et al, 2009; Lopez et al, 1998; Rubel et al, 1995; Slowik et al, 2013). Both in vivo and in cultured explants, the extent of regeneration in the vestibular epithelium is limited and depends to a large extent on trans-differentiation of SCs (Forge et al, 1998; Forge et al, 1993; Golub et al, 2012; Kawamoto et al, 2009; Slowik et al, 2013; Tanyeri et al, 1995; Wang et al, 2010; Wang et al, 2015; Warchol et al, 1993).…”

“…Both in vivo and in cultured explants, the extent of regeneration in the vestibular epithelium is limited and depends to a large extent on trans-differentiation of SCs (Forge et al, 1998; Forge et al, 1993; Golub et al, 2012; Kawamoto et al, 2009; Slowik et al, 2013; Tanyeri et al, 1995; Wang et al, 2010; Wang et al, 2015; Warchol et al, 1993). In the current study, a small number of Myosin VIIa-positive cells are sparsely detected in the vestibular FE.…”

“…The mammalian vestibular sensory epithelium undergoes a limited amount of spontaneous hair cell (HC) regeneration after damage (Burns et al, 2012; Forge et al, 1998; Forge et al, 1993; Golub et al, 2012; Kawamoto et al, 2009; Slowik et al, 2013; Tanyeri et al, 1995; Wang et al, 2015; Warchol et al, 1993). The regenerated HCs are largely derived by transdifferentiation of surviving supporting cells (SCs) (Lin et al, 2011; Slowik et al, 2013; Wang et al, 2015).…”

“…The regenerated HCs are largely derived by transdifferentiation of surviving supporting cells (SCs) (Lin et al, 2011; Slowik et al, 2013; Wang et al, 2015). Therefore, the surviving SCs are potential targets for treatments to enhance the spontaneous HC regeneration, which is otherwise incomplete.…”