Research paper
Response of the flat cochlear epithelium to forced expression of Atoh1

https://doi.org/10.1016/j.heares.2008.02.007Get rights and content

Abstract

Following hair cell elimination in severely traumatized cochleae, differentiated supporting cells are often replaced by a simple epithelium with cuboidal or flat appearance. Atoh1 (previously Math1) is a basic helix–loop–helix transcription factor critical to hair cell differentiation during mammalian embryogenesis. Forced expression of Atoh1 in the differentiated supporting cell population can induce transdifferentiation leading to hair cell regeneration. Here, we examined the outcome of adenovirus mediated over-expression of Atoh1 in the non-sensory cells of the flat epithelium. We determined that seven days after unilateral elimination of hair cells with neomycin, differentiated supporting cells are absent, replaced by a flat epithelium. Nerve processes were also missing from the auditory epithelium, with the exception of infrequent looping nerve processes above the habenula perforata. We then inoculated an adenovirus vector with Atoh1 insert into the scala media of the deafened cochlea. The inoculation resulted in upregulation of Atoh1 in the flat epithelium. However, two months after the inoculation, Atoh1-treated ears did not exhibit clear signs of hair cell regeneration. Combined with previous data on induction of supporting cell to hair cell transdifferentiation by forced expression of Atoh1, these results suggest that the presence of differentiated supporting cells in the organ of Corti is necessary for transdifferentiation to occur.

Introduction

The cochlear sensory epithelium contains two types of differentiated epithelial cells: hair cells and supporting cells. When hair cells degenerate, supporting cells expand and replace them to maintain a confluent layer of cells lining the scala media and separating endolymph from perilymph. In some cases, supporting cells in lesioned ears remain differentiated and the organ of Corti maintains its tall appearance despite the lack of hair cells. However, in many cases the supporting cells that remain after hair cell loss do not maintain their differentiated state. As a result, the area of the organ of Corti becomes a flat or cuboidal simple epithelium with no patterned organization (Forge et al., 1998, Kim and Raphael, 2007). The condition of supporting cells in deaf ears will dictate the choice of therapy, once therapies such as hair cell regeneration or stem cell implantation become a reality.

The flat epithelium has been described after several types of trauma. For instance, ears that receive cochlear implants often exhibit a flat epithelium in both human and animal models (Nadol et al., 1994). A variety of etiologies may lead to degeneration of the auditory epithelium to the flat state, including severe presbycusis (Bhatt et al., 2001), extremely severe ototoxic injury (Coco et al., 2007, Forge et al., 1998, Kim and Raphael, 2007) or hereditary cochlear pathologies (Webster, 1992). In many cases, the loss of hair cells does not initially involve supporting cell degeneration, but over time the non-sensory auditory epithelium is replaced by a flat epithelium. Because of the prevalence of this pathology in humans, the flat epithelium constitutes the substrate for potential future therapy in many clinical cases. It is therefore important to characterize the flat epithelium and determine how it responds to therapeutic manipulations.

In the present study, we have used the neomycin model to eliminate hair cells and induce transformation of supporting cells into the flat epithelium state. We tested the ability of the flat epithelium to be transduced with an adenovirus and whether forced expression of a developmental gene, Atoh1, in the flat epithelium can induce transdifferentiation of these cells into new hair cells. Atoh1 is the mouse homolog of the Drosophila gene atonal, a basic helix–loop–helix transcription factor that acts as a ‘pro-hair cell gene’ (Jones et al., 2006). Forced expression of Atoh1 in deaf ears with differentiated supporting cells can induce transdifferentiation of these supporting cells to new hair cells (Izumikawa et al., 2005, Shou et al., 2003).

We found that the adenovirus-mediated expression of a reporter gene in the flat epithelium was robust. However, forced expression of Atoh1 did not induce noticeable changes in the morphology of the flat epithelium. The results point to the importance of designing ways to prevent supporting cell degeneration and indicate that once the auditory epithelium is flat, therapies other than Atoh1 over-expression should be considered.

Section snippets

Animals

All animal experiments were approved by the University of Michigan Institutional Committee on Care and Use of Animals (UCUCA) and performed using accepted veterinary standards. We used 72 young adult guinea pigs (Elm Hills Breeding Laboratory). At the beginning of the experiments, animals weighed 250–400 g and displayed normal Preyer’s reflex. All animals were deafened unilaterally with neomycin (see below) and received one of the following treatments: Ad.Atoh1 (n = 33), Ad.Atoh1-GFP (n = 21), Ad.GFP

Results

Morphological analysis reported here is based on observations in the first three turns of the cochlea. Little variation was seen among individuals. In normal ears that were not deafened, the combined staining with phalloidin and neurofilament shows presence of hair cells and nerves extending in the direction of the hair cells (Fig. 1a). In contrast, in animals sacrificed at six days after the deafening procedure, whole-mounts of the organ of Corti stained with phalloidin show that hair cells

Discussion

The data show that six days after placing neomycin in the perilymph neither hair cells nor differentiated supporting cells can be found. The rapid and devastating effect of this ototoxic regimen on hair cells has been described in the past (Jyung et al., 1989, Zappia and Altschuler, 1989). In this study, we show an equally devastating effect on supporting cells, such that the epithelium replacing the organ of Corti is flat and neuronal fibers are absent. We also show that this transformation

Acknowledgments

We thank Lisa Beyer for technical assistance. Viral vectors were kindly provided by Doug Brough (GenVec). Work was supported by the Taubman Institute, a gift from Berte and Alan Hirschfield, the R. Jamison and Betty Williams Professorship, a Research Grant from Kansai Medical University, and NIH/NIDCD Grants R01-DC01634, R01-DC05401, R01-DC03685, T32-DC00011 and P30-DC05188.

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