Notch signaling and Hes labeling in the normal and drug-damaged organ of Corti

Abstract

During the development of the inner ear, the Notch cell signaling pathway is responsible for the specification of the pro-sensory domain and influences cell fate decisions. It is assumed that Notch signaling ends during maturity and cannot be reinitiated to alter the fate of new or existing cells in the organ of Corti. This is in contrast to non-mammalian species which reinitiate Delta 1-Notch1 signaling in response to trauma in the auditory epithelium, resulting in hair cell regeneration through transdifferentiation and/or mitosis. We report immunohistochemical data and Western protein analysis showing that in the aminoglycoside-damaged guinea pig organ of Corti, there is an increase in proteins involved in Notch activation occurring within 24h of a chemical hair cell lesion. The signaling response is characterized by the increased presence of Jagged1 ligand in pillar and Deiters cells, Notch1 signal in surviving supporting cell nuclei, and the absence of Jagged2 and Delta-like1. The pro-sensory bHLH protein Atoh1 was absent at all time points following an ototoxic lesion, while the repressor bHLH transcription factors Hes1 and Hes5 were detected in surviving supporting cell nuclei in the former inner and outer hair cell areas, respectively. Notch pathway proteins peaked at 2 weeks, decreased at 1 month, and nearly disappeared by 2 months. These results indicate that the mammalian auditory epithelium retains the ability to regulate Notch signaling and Notch-dependent Hes activity in response to cellular trauma and that the signaling is transient. Additionally, since Hes activity antagonizes the transcription of pro-sensory Atoh1, the presence of Hes after a lesion may prohibit the occurrence of transdifferentiation in the surviving supporting cells.

Figure 1. Confocal images of whole mounts of the normal guinea pig organ of Corti stained with fluorescent phalloidin (green), DAPI (blue), and antibodies to Notch signaling molecules (red: A–C, Notch1; D–E, Hes1; F–G, Hes5; H, Jagged1)

A, B and inset C: The region of the organ of Corti that contains nuclei is largely negative for the Notch1 receptor (A), punctate Notch1 positive areas are evident in non-nuclear locations such as the cell membrane and cytoplasm of Deiters cells more apical to the cell nuclei (B, and higher magnification inset C). D and E: Hes1 is found to be co-labeled in some supporting cell nuclei (D) in the inner hair cell area. An arrow labels one of several Hes1 positive nuclei (D is Hes1, phalloidin and DAPI, and E is Hes1 and phalloidin). F and G: Nuclei in the outer hair cell region (F) and inner hair cell region (G) are all negative for Hes5 although some cytoplasmic Hes5 signal was observed. H and H’: Low-level Jagged1 signal was seen in the pillar cells (H, shown focused on the area of positive outer pillar cell bodies in higher magnification in H’). Labels: ‘O’ refers to the outer hair cell area; ‘I’ refers to the inner hair cell area; ‘P’ refers to the apical surface of the inner and outer pillar cells. The lateral edge of the organ of Corti is oriented towards the top of each panel, and the medial edge is oriented towards the bottom. Scale bars represent 2μm.

Figure 2. Confocal images of guinea pig organ of Corti whole mounts one day following ototoxic drug administration, stained with fluorescent phalloidin (green), DAPI (blue), and antibodies to Notch signaling molecules (red: A–A’’, Notch1; B–B’’, Hes5; C–D’, Hes1; E–E’, Jagged1)

A, A’, and A’’: One day following drug administration, many DAPI-labeled nuclei (blue, in A and A’) were also positive for Notch1 (red in A’ and A’’, arrows point to examples of double-labeling). A’ and A’’ are higher magnification of the Deiters cell nuclei. Focus is at the level in the tissue where the nuclei of the remaining supporting cells were visible. Some diffuse membrane- or cytoplasmically-located Notch1 signal was still evident. B, B’, and B’’: Many DAPI-labeled nuclei in the remaining supporting cells in the outer hair cell area were co-labeled with Hes5 (B, higher magnification of DAPI and Hes5 signal in B’ and Hes5 signal alone in B’’). C, C’, D, D’: Many DAPI-labeled nuclei (blue in C and D) in the supporting cells of the inner hair cell area (C, C’) as well as the Henson cells (D, D’) were co-labeled with Hes1 signal. Cytoplasmic Hes1 signal was also observed in these same cell types to a lesser degree. E, E’: Both inner (E) and outer (E’, at a lower focal plane with the red channel alone) pillar cell bodies, and the Deiters cells were very brightly positive for Jagged1. Labels: ‘O’ refers to the outer hair cell area; ‘I’ refers to the inner hair cell area; ‘P’ refers to the apical surface of the inner and outer pillar cells. The lateral edge of the organ of Corti is oriented towards the top of each panel, and the medial edge is oriented towards the bottom. Scale bars represent 2μm.

Figure 3. Western blot analysis and quantification graph of Notch1, NICD, Hes1, and Hes5 in normal and drug-damaged organ of Corti

Notch1, NICD, Hes1, and Hes5 all appeared to rise after a lesion, with the first three peaking at 2 weeks and Hes5 appearing to peak at one month following ototoxicity. The Notch1 blot exhibited bands at ~280 kDa corresponding to the whole receptor, ~120 kDa corresponding to the cleaved portion (NICD), and one faint non-specific band at ~250kDa. The Hes1 blot exhibited the predicted band at ~30 kDa and no nonspecific bands. The blot to Hes5 exhibited the predicted band at ~20 kDa and one faint nonspecific band at ~40 kDa. GAPDH was used as a loading control for relative quantification of protein amounts with ImageJ (graph at right). N = normal, 1d = 1 day following the lesion, 14d = 14 days after the lesion, 30d = thirty days after the lesion, 60d = 60 days after the lesion.

Figure 4. Confocal images of guinea pig organ of Corti whole mounts two weeks following ototoxic drug administration, stained with fluorescent phalloidin (green), DAPI (blue), and antibodies to Notch signaling molecules (red: A–B, Notch1; C, Jagged1; D–D’, Hes1; E–E’, Hes5)

A, B: Nearly all DAPI-labeled nuclei (blue) of remaining supporting cells were also positive for Notch1 (red). In B’, DAPI signal is omitted to more clearly show Notch1. Arrows in B and B’ point to two double-labeled nuclei. Very little diffuse membrane- or cytoplasmically-located Notch1 signal was evident at this time point. C: All the inner and outer pillar cells and most of the Deiters cells were still brightly positive for Jagged1 signal (red). D, D’: Many supporting cell nuclei medial to the inner hair cell area (I) were co-labeled with DAPI and Hes1 (red). Additionally, there were high concentrations of non-nuclear Hes1 protein in some scars where inner hair cells had died (arrows in D, D’ which shows DAPI and Hes1 only). E, E’: Supporting cell nuclei in the outer hair cell area were co-labeled with DAPI and Hes5 signal, while all nuclei in the inner hair cell area were negative for Hes5. Labels: ‘O’ refers to the outer hair cell area; ‘I’ refers to the inner hair cell area; ‘P’ refers to the apical surface of the inner and outer pillar cells with ‘IP’ and ‘OP’ referring to inner and outer pillar cells, respectively. The lateral edge of the organ of Corti is oriented towards the top of each panel, and the medial edge is oriented towards the bottom. Scale bars represent 2μm.