Combinatorial Atoh1, Gfi1, Pou4f3, and Six1 gene transfer induces hair cell regeneration in the flat epithelium of mature guinea pigs

Abstract

Flat epithelium (FE) is a condition characterized by the loss of both hair cells (HCs) and supporting cells and the transformation of the organ of Corti into a simple flat or cuboidal epithelium, which can occur after severe cochlear insults. The transcription factors Gfi1, Atoh1, Pou4f3, and Six1 (GAPS) play key roles in HC differentiation and survival in normal ears. Previous work using a single transcription factor, Atoh1, to induce HC regeneration in mature ears in vivo usually produced very few cells and failed to produce HCs in severely damaged organs of Corti, especially those with FE. Studies in vitro suggested combinations of transcription factors may be more effective than any single factor, thus the current study aims to examine the effect of co-overexpressing GAPS genes in deafened mature guinea pig cochleae with FE. Deafening was achieved through the infusion of neomycin into the perilymph, leading to the formation of FE and substantial degeneration of nerve fibers. Seven days post neomycin treatment, adenovirus vectors carrying GAPS were injected into the scala media and successfully expressed in the FE. One or two months following GAPS inoculation, cells expressing Myosin VIIa were observed in regions under the FE (located at the scala tympani side of the basilar membrane), rather than within the FE. The number of cells, which we define as induced HCs (iHCs), was not significantly different between one and two months, but the larger N at two months made it more apparent that there were significantly more iHCs in GAPS treated animals than in controls. Additionally, qualitative observations indicated that ears with GAPS gene expression in the FE had more nerve fibers than FE without the treatment. In summary, our results showed that co-overexpression of GAPS enhances the potential for HC regeneration in a severe lesion model of FE.

Keywords: Adenovirus; Cochlea; Deafness; Flat epithelium; Gene transfer; Hair cell regeneration.

Fig. 1.

Whole mounts of the auditory epithelium stained with markers for actin (green) and Myosin VIIa (red) and photographed with epifluorescence. (a) In the normal auditory epithelium, one array of inner hair cells (I), 3 arrays of outer hair cells (1, 2, 3, indicating the 1^st, 2^nd, and 3^rd rows), and pillar cells (P) are displayed. (b) Seven days after neomycin administration, the distribution of actin in adherens junctions between cells reveals a single layer FE of non-sensory cells with irregular apical outlines; whereas HCs and differentiated SCs are absent. All images at the same magnification; scale bar = 50 μm.

Fig. 2.

Whole-mounts of the neomycin-deafened auditory epithelium from the 4 cochlear turns of a guinea pig, from apex (a) to base (d), demonstrating FE without HCs or SCs throughout the cochlear duct. All images at the same magnification, scale bar = 50 μm.

Fig. 3.

Whole mounts of the FE stained with markers for actin (blue) and GFP (green) and photographed with epifluorescence. Seven days after Ad.EF1a-Venus delivery through a scala media infusion, gene expression was detected at the level of the FE, as seen in lower magnification (a, scale bar = 100 μm) and at higher magnification (b, scale bar = 20 μm). The approximate location of the habenula perforata is marked with a dashed line and the medial aspect of the epithelium is at the bottom of the images.

Fig. 4.

Whole mounts of the FE stained with markers for actin (green) and Myosin VIIa (red) and photographed with epifluorescence. (a) One month after Ad.GAPS-Venus delivery through scala media infusion, Myosin VIIa-positive iHCs are seen at a focal plane of the scala tympani side of the basilar membrane, under the FE. (b) Two months after Ad.GAPS-Venus delivery through scala media infusion, Myosin VIIa-positive cells are seen on the scala tympani side under the level of the FE. (c) Two months after Ad.GAPS-Venus delivery through scala media infusion, Myosin VIIa-positive iHCs are seen at the level of the FE. All images at the same magnification, scale bar = 50 μm.

Fig. 5.

Whole mounts of the FE stained with markers for Myosin VIIa (red), actin (green) and neurofilament (blue) and photographed with epifluorescence. (a) In a control cochlea that was only deafened, neural fibers were sparse and seldom extended far beyond the habenula perforata (dotted line). (b) Two months after Ad.GAPS-Venus delivery through scala media infusion, several fibers could be seen passing through regions with numerous Myosin VIIa-positive iHCs. (c) In rare cases, a nerve fiber appeared to terminate in the immediate vicinity of a Myosin VIIa-positive cell (arrow). All images at the same magnification, scale bar = 50 μm.