Multiple supporting cell subtypes are capable of spontaneous hair cell regeneration in the neonatal mouse cochlea.

Supporting cells (SCs) are known to spontaneously regenerate hair cells (HCs) in the neonatal mouse cochlea, yet little is known about the relative contribution of distinct SC subtypes which differ in morphology and function. We have previously shown that HC regeneration is linked to Notch signaling, and some SC subtypes, but not others, lose expression of the Notch effector Hes5 Other work has demonstrated that Lgr5-positive SCs have an increased capacity to regenerate HCs; however, several SC subtypes express Lgr5. To further investigate the source for spontaneous HC regeneration, we used three CreER lines to fate-map distinct groups of SCs during regeneration. Fate-mapping either alone or combined with a mitotic tracer showed that pillar and Deiters' cells contributed more regenerated HCs overall. However, when normalized to the total fate-mapped population, pillar, Deiters', inner phalangeal and border cells had equal capacity to regenerate HCs, and all SC subtypes could divide after HC damage. Investigating the mechanisms that allow individual SC subtypes to regenerate HCs and the postnatal changes that occur in each group during maturation could lead to therapies for hearing loss.

The Notch Ligand Jagged1 Is Required for the Formation, Maintenance, and Survival of Hensen's Cells in the Mouse Cochlea.

During cochlear development, the Notch ligand JAGGED 1 (JAG1) plays an important role in the specification of the prosensory region, which gives rise to sound-sensing hair cells and neighboring supporting cells (SCs). While JAG1's expression is maintained in SCs through adulthood, the function of JAG1 in SC development is unknown. Here, we demonstrate that JAG1 is essential for the formation and maintenance of Hensen's cells, a highly specialized SC subtype located at the edge of the auditory epithelium. Using Sox2CreERT2/+::Jag1loxP/loxP mice of both genders, we show that Jag1 deletion at the onset of differentiation, at embryonic day 14.5, disrupted Hensen's cell formation. Similar loss of Hensen's cells was observed when Jag1 was deleted after Hensen's cell formation at postnatal day (P) 0/P1 and fate-mapping analysis revealed that in the absence of Jag1, some Hensen's cells die, but others convert into neighboring Claudius cells. In support of a role for JAG1 in cell survival, genes involved in mitochondrial function and protein synthesis were downregulated in the sensory epithelium of P0 cochlea lacking Jag1 Finally, using Fgfr3-iCreERT2 ::Jag1loxP/loxP mice to delete Jag1 at P0, we observed a similar loss of Hensen's cells and found that adult Jag1 mutant mice have hearing deficits at the low-frequency range.SIGNIFICANCE STATEMENT Hensen's cells play an essential role in the development and homeostasis of the cochlea. Defects in the biophysical or functional properties of Hensen's cells have been linked to auditory dysfunction and hearing loss. Despite their importance, surprisingly little is known about the molecular mechanisms that guide their development. Morphologic and fate-mapping analyses in our study revealed that, in the absence of the Notch ligand JAGGED1, Hensen's cells died or converted into Claudius cells, which are specialized epithelium-like cells outside the sensory epithelium. Confirming a link between JAGGED1 and cell survival, transcriptional profiling showed that JAGGED1 maintains genes critical for mitochondrial function and tissue homeostasis. Finally, auditory phenotyping revealed that JAGGED1's function in supporting cells is necessary for low-frequency hearing.

The FBN rat model of aging: investigation of ABR waveforms and ribbon synapse changes.

Age-related hearing loss is experienced by one-third of individuals aged 65 years and older and can be socially debilitating. Historically, there has been poor correlation between age-related threshold changes, loss of speech understanding, and loss of cochlear hair cells. We examined changes in ribbon synapse number at four different ages in Fisher Brown Norway rats, an extensively studied rat model of aging. In contrast to previous work in mice/Wistar rats, we found minimal ribbon synapse loss before 20 months, with significant differences in 24- and 28-month-old rats at 4 kHz. Significant outer HC loss was observed at 24 and 28 months in low- to mid-frequency regions. Age-related reductions in auditory brainstem response wave I amplitude and increases in threshold were strongly correlated with ribbon synapse loss. Wave V/I ratios increased across age for click, 2, 4, and 24 kHz. Together, we find that ribbon synapses in the Fisher Brown Norway rat cochlea show resistance to aging until ∼60% of their life span, suggesting species/strain differences may underpin decreased peripheral input into the aging central processor.

Quantitative Analysis of Supporting Cell Subtype Labeling Among CreER Lines in the Neonatal Mouse Cochlea.

Four CreER lines that are commonly used in the auditory field to label cochlear supporting cells (SCs) are expressed in multiple SC subtypes, with some lines also showing reporter expression in hair cells (HCs). We hypothesized that altering the tamoxifen dose would modify CreER expression and target subsets of SCs. We also used two different reporter lines, ROSA26 tdTomato and CAG-eGFP, to achieve the same goal. Our results confirm previous reports that Sox2 CreERT2 and Fgfr3-iCreER T2 are not only expressed in neonatal SCs but also in HCs. Decreasing the tamoxifen dose did not reduce HC expression for Sox2 CreERT2 , but changing to the CAG-eGFP reporter decreased reporter-positive HCs sevenfold. However, there was also a significant decrease in the number of reporter-positive SCs. In contrast, there was a large reduction in reporter-positive HCs in Fgfr3-iCreER T2 mice with the lowest tamoxifen dose tested yet only limited reduction in SC labeling. The targeting of reporter expression to inner phalangeal and border cells was increased when Plp-CreER T2 was paired with the CAG-eGFP reporter; however, the total number of labeled cells decreased. Changes to the tamoxifen dose or reporter line with Prox1 CreERT2 caused minimal changes. Our data demonstrate that modifications to the tamoxifen dose or the use of different reporter lines may be successful in narrowing the numbers and/or types of cells labeled, but each CreER line responded differently. When the ROSA26 tdTomato reporter was combined with any of the four CreER lines, there was no difference in the number of tdTomato-positive cells after one or two injections of tamoxifen given at birth. Thus, tamoxifen-mediated toxicity could be reduced by only giving one injection. While the CAG-eGFP reporter consistently labeled fewer cells, both reporter lines are valuable depending on the goal of the study.