THE ROLE OF NOTCH SIGNALING IN THE DIFFERENTIAL ABILITY OF SUPPORTING CELL SUBTYPES TO SPONTANEOUSLY REGENERATE HAIR CELLS IN THE NEONATAL MOUSE COCHLEA

Author

Melissa M. McGovern, Southern Illinois University CarbondaleFollow

Date of Award

12-1-2017

Degree Name

Doctor of Philosophy

Department

Pharmacology

First Advisor

Cox, Brandon

Abstract

One of the most common disabilities in the US, hearing loss, is reported by The National Institutes of Health to affect approximately 36 million Americans. One of the major contributing factors to this loss in hearing is the loss of the sensory hair cells (HCs) within the cochlea. Also in the mammalian cochlea, six major groups of supporting cell (SC) subtypes reside in close proximity to HCs and may have the potential to regenerate HCs after damage. These subtypes include cells of the greater epithelial ridge, inner phalangeal/border cells, inner and outer pillar cells, Deiters’ cells, Hensen cells, and Claudius cells. During embryonic development, progenitor cells differentiate into HCs or one of the SC subtypes by Notch-mediated lateral inhibition. In the neonatal mouse cochlea, many studies have shown that inhibition of Notch signaling allows SCs to convert into HCs in both normal undamaged cochleae, as well as in drug-damaged cochlear explants. This mechanism is also implicated during spontaneous HC regeneration that occurs in non-mammalian vertebrates. We and others have recently observed that spontaneous HC regeneration can also occur in the neonatal mouse cochlea. However, little is known about the molecular mechanism or the SC subtypes which act as the source of regenerated HCs. In the neonatal mouse cochlea, HCs were killed in vivo at birth using a genetically-modified mouse model to express a toxin in HCs. Subsequently, SCs formed new HCs by either direct transdifferentiation, where no cell division occurred, or by mitotic regeneration. My dissertation investigated the role of Notch signaling in the ability of SC subtypes to regenerate HCs after damage. My central hypothesis is that after HC ablation is induced at birth, Notch signaling is partially eliminated and therefore lateral inhibition is lost in neonatal SCs in a subtype specific manner, which allows some SCs, but not others, to differentiate into and regenerate HCs. Aim 1 focused on changes in the Notch signaling pathway in response to HC damage during the window of spontaneous HC regeneration. Changes in the expression of genes in the Notch pathway were measured using real time qPCR, immunostaining, and in situ hybridization. The Notch effector HeyL was increased in the apical one-third of the cochlea while other Notch players are decreased. The most notable example is the Notch effector Hes5, which is directly responsible for inhibiting HC fate, and was reduced in outer pillar cells and Deiters’ cells, but not in other SC subtypes. From this we conclude that Notch signaling is reduced differentially among SC subtypes. In Aim 2 we investigated whether inhibition of Notch signaling is required for spontaneous HC regeneration to occur by maintaining active Notch signaling in all SCs in the context of HC damage. We hypothesized that maintaining active Notch signaling after HC damage will prevent SC-to-HC conversion thus preventing HC regeneration. We found significantly fewer regenerated HCs while maintaining Notch expression compared to controls with HC damage and no manipulation of Notch signaling. Therefore we conclude loss of Notch mediated lateral inhibition is required for the majority of spontaneous HC regeneration. In Aim 3 we investigated the ability of different SC subtypes to regenerate HCs by fate-mapping SC subtypes during the HC regeneration process. Since fate-mapping creates a permanent label in targeted cells, we can track their potential change in cell fate or reentry in the cell cycle after HC damage. We hypothesized that pillar cells and Deiters’ cells are the source for spontaneously regenerated HC within the neonatal mouse cochlea based on our results from Aim 1. We used three CreER mouse lines to fate-map distinct groups of SC subtypes during the HC damage and regeneration process. More pillar and Deiters’ cells regenerated HCs after damage than other SC populations. We found that outer pillar cells and Deiters’ cells are capable of downregulating the cell cycle inhibitor, p27Kip1, after HC damage. Therefore we investigated the ability of SC subtypes to mitotically regenerate HCs by including a mitotic tracer along with fate-mapping. A larger proportion of mitotically regenerated HCs came from pillar and Deiters’ cells. From these experiments, we conclude that outer pillar and Deiters’ cells are the source for the majority of spontaneously regenerated HCs in vivo. This knowledge will allow targeted investigation into outer pillar cells and Deiters’ cells that maintain regenerative plasticity at postnatal ages. Understanding how these cells change with age will inform efforts to induce HC regeneration in more mature cochleae. Additionally, understanding how Notch signaling regulates this regenerative plasticity will lead to the development of potential targets for the treatment of hearing loss.