Hair Cell Regeneration in the Crista Ampullaris of the Mammalian Vestibular System
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The sensory modalities of hearing and balance are mediated by the six sensory organs of the inner ear that are each comprised of the same two main cell types, support cells and mechanosensory hair cells. Loss of the sensory hair cells from these organs causes permanent hearing loss and/or balance disorders, as there is currently no therapeutic treatment for hair cell loss. In developing organs, hair cells can be generated through the transdifferentiation of support cells caused by inhibition of the Notch signaling pathway, which is normally required to determine and maintain the precise ratio of hair cells and support cells through lateral inhibition. Although the efficacy of this method declines as the organs mature and Notch signaling is downregulated, previous research has shown that the Notch downstream effector, Hes5, is present in the adult cristae, suggesting that Notch signaling may be active and that the cristae of the adult mouse may retain some regenerative ability. In this dissertation, I tested this hypothesis and showed that Notch signaling is active in the peripheral region of the adult cristae and, using hair cell counts and lineage tracing, that supernumerary hair cells can be generated through inhibition of Notch signaling in vitro. Further, through an analysis of the spatial distribution of hair cell birth in the developing cristae, I showed that there is a correlation between the regions that maintain regenerative competence in the adult and the last regions to exit the cell cycle. In addition, to aid future regenerative studies, I identified a new support cell marker that can be used to lineage trace support cells and have used this marker to characterize spontaneous hair cell regeneration in the adult cristae in vivo. I also created standard protocols for lesioning hair cells in vivo in two common mouse strains using the known ototoxin 3,3’-Iminodipropionitrile (IDPN) and for quantifiably assaying vestibular behavior in mice with varying degrees of hair cell lesion. Together, this work establishes the previously uncharacterized mouse cristae as an additional model for studying the mechanisms of hair cell regeneration and provides some of the tools necessary for future studies. Supplemental Movie 1.1 The inner ear contains six distinct sensory organs: The cochlea, utricle, saccule, posterior cristae, horizontal cristae, and anterior cristae. These organs be seen in an intact E15.5 inner ear labeled for the sensory regions with Sox2 (white) and in a color coded model of the position of the Sox2-labeled sensory organs created by 3-dimensionally rendering tracings of the Sox2 regions in the individual confocal slices. Supplemental Movie 2.1 Cristae are highly three-dimensional, composed of two saddle-shaped hemicristae separated by the eminentia cruciatum. Sox9 (red) labels support cells as well as non-sensory 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 μm (w x h x d) - 544.9 x 272.5 x 75.5. Supplemental Movie 2.2 An example of a lineage traced transitional cell from the mTmG mouse (see Figure7B-B’’). The GFP+ cell expressed Gfi1, but had an elongated body similar to a support cell. The nucleus was lifting off of the basement membrane and the apical part of the cell had an unusual appearance unlike a normal hair cell or support cell. There was also another GFP+ support cell that spans the sensory epithelium as well as several non-sensory cells in view. Dimensions in μm (w x h x d) – 36.4 x 61.2 x 6.9. Supplemental Movie 2.3 An example of a lineage traced transitional cell from the mTmG mouse (see Fig. 7CC’’). The GFP+ cell expresses Gfi1 and overall has a normal appearance for a hair cell, except for a thin foot-like projection that extends to the basement membrane. Also in view are two support cells, one of which is directly next to the hair cell. Dimensions in μm (w x h x d) – 43.3 x 52.6 x 13.0. Supplemental Movie 2.4 An example of a lineage traced hair cell with a kinocilium from the mTmG mouse (see Fig. 7D-D’’). The GFP+ cell expressed Gfi1 (red) and had a flask shape with a rounded bottom and a thin neck. A long kinocilium extended up from the apical surface. Nuclei are labeled with Hoechst 33342 (white) and had prominent nucleoli at this fluorescent intensity. Also in view were a couple of GFP+ support cells and a non-sensory cell. Dimensions in μm (w x h x d) – 28.7 x 64.2 x 13.5.