scholarly journals Lineage-tracing and translatomic analysis of damage-inducible mitotic cochlear progenitors identifies candidate genes regulating regeneration

PLoS Biology ◽  
2021 ◽  
Vol 19 (11) ◽  
pp. e3001445
Author(s):  
Tomokatsu Udagawa ◽  
Patrick J. Atkinson ◽  
Beatrice Milon ◽  
Julia M. Abitbol ◽  
Yang Song ◽  
...  
Keyword(s):  
Clustering Algorithms ◽  
Organ Of Corti ◽  
Gene Clusters ◽  
Lineage Tracing ◽  
Calcium Transients ◽  
Gene Clustering ◽  
Supporting Cells ◽  
Hearing Function ◽  
Cochlear Supporting Cells

Cochlear supporting cells (SCs) are glia-like cells critical for hearing function. In the neonatal cochlea, the greater epithelial ridge (GER) is a mitotically quiescent and transient organ, which has been shown to nonmitotically regenerate SCs. Here, we ablated Lgr5+ SCs using Lgr5-DTR mice and found mitotic regeneration of SCs by GER cells in vivo. With lineage tracing, we show that the GER houses progenitor cells that robustly divide and migrate into the organ of Corti to replenish ablated SCs. Regenerated SCs display coordinated calcium transients, markers of the SC subtype inner phalangeal cells, and survive in the mature cochlea. Via RiboTag, RNA-sequencing, and gene clustering algorithms, we reveal 11 distinct gene clusters comprising markers of the quiescent and damaged GER, and damage-responsive genes driving cell migration and mitotic regeneration. Together, our study characterizes GER cells as mitotic progenitors with regenerative potential and unveils their quiescent and damaged translatomes.

scholarly journals In vivo optogenetics reveals homeostatic control of cochlear sensitivity by supporting cells

2020 ◽  
Author(s):  
Victoria Lukashkina ◽  
Snezana Levic ◽  
Patricio Simões ◽  
Zhenhang Xu ◽  
Joseph DiGuiseppi ◽  
...  
Keyword(s):  
Hair Cells ◽  
Dynamic Range ◽  
Organ Of Corti ◽  
Feedback System ◽  
Supporting Cell ◽  
Outer Hair Cells ◽  
Supporting Cells ◽  
Cochlear Supporting Cells

Abstract We used optogenetics to investigate the control of auditory sensitivity by cochlear supporting cells that scaffold outer hair cells, which transduce and amplify cochlear responses to sound. In vivo and in vitro measurements of sound-induced cochlear mechanical and electrical responses were made from mice that conditionally expressed nonselective cationic channelrhodopsins in Deiters’ and outer pillar supporting cells in the organ of Corti. We demonstrated that cochlear light-stimulation and subsequent activation of channelrhodopsins depolarized the supporting cells, changed their extracellular electrical environment, and sensitized insensitive and desensitized sensitive cochlear responses to sound. We concluded that outer hair cells, Deiters’ cells and outer pillar cells interact through feedback which regulates their immediate ionic and electrical environment and controls energy flow in the mammalian cochlea to optimize its performance over its entire dynamic range. Activation of the supporting cell channelrhodopsins shunts this feedback system and restores cochlear sensitivity to a set level.

scholarly journals Vibration of the organ of Corti within the cochlear apex in mice

2014 ◽  
Vol 112 (5) ◽  
pp. 1192-1204 ◽  
Author(s):  
Simon S. Gao ◽  
Rosalie Wang ◽  
Patrick D. Raphael ◽  
Yalda Moayedi ◽  
Andrew K. Groves ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Phase Difference ◽  
Basilar Membrane ◽  
Organ Of Corti ◽  
Lateral Compartment ◽  
Outer Hair Cells ◽  
Supporting Cells ◽  
Tuning Curves ◽  
Cochlear Amplification

The tonotopic map of the mammalian cochlea is commonly thought to be determined by the passive mechanical properties of the basilar membrane. The other tissues and cells that make up the organ of Corti also have passive mechanical properties; however, their roles are less well understood. In addition, active forces produced by outer hair cells (OHCs) enhance the vibration of the basilar membrane, termed cochlear amplification. Here, we studied how these biomechanical components interact using optical coherence tomography, which permits vibratory measurements within tissue. We measured not only classical basilar membrane tuning curves, but also vibratory responses from the rest of the organ of Corti within the mouse cochlear apex in vivo. As expected, basilar membrane tuning was sharp in live mice and broad in dead mice. Interestingly, the vibratory response of the region lateral to the OHCs, the “lateral compartment,” demonstrated frequency-dependent phase differences relative to the basilar membrane. This was sharply tuned in both live and dead mice. We then measured basilar membrane and lateral compartment vibration in transgenic mice with targeted alterations in cochlear mechanics. Prestin499/499, Prestin−/−, and TectaC1509G/C1509G mice demonstrated no cochlear amplification but maintained the lateral compartment phase difference. In contrast, SfswapTg/Tg mice maintained cochlear amplification but did not demonstrate the lateral compartment phase difference. These data indicate that the organ of Corti has complex micromechanical vibratory characteristics, with passive, yet sharply tuned, vibratory characteristics associated with the supporting cells. These characteristics may tune OHC force generation to produce the sharp frequency selectivity of mammalian hearing.

scholarly journals In vivo optogenetics reveals control of cochlear sensitivity by supporting cells

2021 ◽  
Author(s):  
Victoria Lukashkina ◽  
Snezana Levic ◽  
Patricio Simões ◽  
Zhenhang Xu ◽  
Joseph DiGuiseppi ◽  
...  
Keyword(s):  
Basilar Membrane ◽  
Ex Vivo ◽  
Organ Of Corti ◽  
Outer Hair Cells ◽  
Supporting Cells ◽  
Acoustic Environment ◽  
Cation Conductance ◽  
Fine Control ◽  
Electrical Responses

Abstract Cochlear sensitivity, essential for communication and exploiting the acoustic environment, is due to the sensory-motor outer hair cells (OHCs) that operate in the structural scaffold of supporting cells and extracellular spaces in the cochlear organ of Corti (OoC). It is unclear whether supporting cells (e.g., Deiters cells [DCs] and outer pillar cells [OPCs]) control cochlear sensitivity in vivo. Here we employed optogenetics to measure in vivo sound-induced cochlear mechanical and electrical responses, and ex vivo light-induced DC electrical responses in the OoC of mice that conditionally expressed channelrhodopsins (ChR2) specifically in DCs and OPCs. Illumination activated a nonselective ChR2 cation conductance and depolarized the DCs. This transient action reversibly blocked continuous, normally occurring, minor adjustments of tone-evoked basilar membrane displacements, and OHC voltage responses to tones at and close to their characteristic frequency, and speeded recovery from temporary acoustic desensitization. This is the first direct evidence for the interdependency of the structural, mechanical, and electrochemical arrangement of OHCs and OoC supporting cells which together fine control cochlear sensitivity.

scholarly journals Notch inhibition induces mitotically generated hair cells in mammalian cochleae via activating the Wnt pathway

2014 ◽  
Vol 112 (1) ◽  
pp. 166-171 ◽  
Author(s):  
Wenyan Li ◽  
Jingfang Wu ◽  
Jianming Yang ◽  
Shan Sun ◽  
Renjie Chai ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Lineage Tracing ◽  
Supporting Cells ◽  
Hearing Recovery ◽  
Wnt Pathways ◽  
Notch Inhibition ◽  
Canonical Wnt ◽  
G Protein Coupled

The activation of cochlear progenitor cells is a promising approach for hair cell (HC) regeneration and hearing recovery. The mechanisms underlying the initiation of proliferation of postnatal cochlear progenitor cells and their transdifferentiation to HCs remain to be determined. We show that Notch inhibition initiates proliferation of supporting cells (SCs) and mitotic regeneration of HCs in neonatal mouse cochlea in vivo and in vitro. Through lineage tracing, we identify that a majority of the proliferating SCs and mitotic-generated HCs induced by Notch inhibition are derived from the Wnt-responsive leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5+) progenitor cells. We demonstrate that Notch inhibition removes the brakes on the canonical Wnt signaling and promotes Lgr5+ progenitor cells to mitotically generate new HCs. Our study reveals a new function of Notch signaling in limiting proliferation and regeneration potential of postnatal cochlear progenitor cells, and provides a new route to regenerate HCs from progenitor cells by interrupting the interaction between the Notch and Wnt pathways.

scholarly journals Dual expression of Atoh1 and Ikzf2 promotes transformation of adult cochlear supporting cells into outer hair cells

2021 ◽  
Author(s):  
Suhong Sun ◽  
Shuting Li ◽  
Zhengnan Luo ◽  
Minhui Ren ◽  
Shunji He ◽  
...  
Keyword(s):  
Single Cell ◽  
Hearing Impairment ◽  
Hair Cells ◽  
Outer Hair Cells ◽  
Supporting Cells ◽  
Differentiation Status ◽  
Cochlear Supporting Cells ◽  
Simultaneous Expression ◽  
Dual Expression

ABSTRACTMammalian cochlear outer hair cells (OHCs) are essential for hearing. OHC degeneration causes severe hearing impairment. Previous attempts of regenerating new OHCs from cochlear supporting cells (SCs) had yielded cells lacking Prestin, a key motor protein for OHC function. Thus, regeneration of Prestin+ OHCs remains a challenge for repairing OHC damage in vivo. Here, we reported that successful in vivo conversion of adult cochlear SCs into Prestin+ OHC-like cells could be achieved by simultaneous expression of Atoh1 and Ikzf2, two key transcriptional factors necessary for OHC development. New OHC-like cells exhibited upregulation of hundreds of OHC genes and downregulation of SC genes. Single cell transcriptomic analysis demonstrated that the differentiation status of these OHC-like cells was much more advanced than previously achieved. Thus, we have established an efficient approach to promote regeneration of Prestin+ OHCs and paved the way for repairing damaged cochlea in vivo via transdifferentiation of SCs.

scholarly journals Dual expression of Atoh1 and Ikzf2 promotes transformation of adult cochlear supporting cells into outer hair cells

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Suhong Sun ◽  
Shuting Li ◽  
Zhengnan Luo ◽  
Minhui Ren ◽  
Shunji He ◽  
...  
Keyword(s):  
Hair Cells ◽  
Adult Mouse ◽  
Outer Hair Cells ◽  
Auditory Function ◽  
Supporting Cells ◽  
Single Cell Rna Sequencing ◽  
Differentiation Status ◽  
Cochlear Supporting Cells ◽  
Dual Expression

Mammalian cochlear outer hair cells (OHCs) are essential for hearing. Severe hearing impairment follows OHC degeneration. Previous attempts at regenerating new OHCs from cochlear supporting cells (SCs) have been unsuccessful, notably lacking expression of the key OHC motor protein, Prestin. Thus, regeneration of Prestin+ OHCs represents a barrier to restore auditory function in vivo. Here, we reported the successful in vivo conversion of adult mouse cochlear SCs into Prestin+ OHC-like cells through the concurrent induction of two key transcriptional factors known to be necessary for OHC development: Atoh1 and Ikzf2. Single-cell RNA sequencing revealed the upregulation of 729 OHC genes and downregulation of 331 SC genes in OHC-like cells. The resulting differentiation status of these OHC-like cells was much more advanced than previously achieved. This study thus established an efficient approach to induce the regeneration of Prestin+ OHCs, paving the way for in vivo cochlear repair via SC transdifferentiation.

scholarly journals In Vivo Proliferation of Postmitotic Cochlear Supporting Cells by Acute Ablation of the Retinoblastoma Protein in Neonatal Mice

2010 ◽  
Vol 30 (17) ◽  
pp. 5927-5936 ◽  
Author(s):  
Y. Yu ◽  
T. Weber ◽  
T. Yamashita ◽  
Z. Liu ◽  
M. B. Valentine ◽  
...  
Keyword(s):  
Retinoblastoma Protein ◽  
Supporting Cells ◽  
Neonatal Mice ◽  
Cochlear Supporting Cells

A technique for protecting delicate specimens during processing

1989 ◽  
Vol 47 ◽  
pp. 982-983
Author(s):  
R.J. Mount ◽  
R.V. Harrison
Keyword(s):  
Basilar Membrane ◽  
Low Cost ◽  
Organ Of Corti ◽  
Region Of Interest ◽  
Sensory Epithelium ◽  
Physical Damage ◽  
Air Drying ◽  
Specimen Handling ◽  
Two Component

The sensory end organ of the ear, the organ of Corti, rests on a thin basilar membrane which lies between the bone of the central modiolus and the bony wall of the cochlea. In vivo, the organ of Corti is protected by the bony wall which totally surrounds it. In order to examine the sensory epithelium by scanning electron microscopy it is necessary to dissect away the protective bone and expose the region of interest (Fig. 1). This leaves the fragile organ of Corti susceptible to physical damage during subsequent handling. In our laboratory cochlear specimens, after dissection, are routinely prepared by the O-T- O-T-O technique, critical point dried and then lightly sputter coated with gold. This processing involves considerable specimen handling including several hours on a rotator during which the organ of Corti is at risk of being physically damaged. The following procedure uses low cost, readily available materials to hold the specimen during processing ,preventing physical damage while allowing an unhindered exchange of fluids.Following fixation, the cochlea is dehydrated to 70% ethanol then dissected under ethanol to prevent air drying. The holder is prepared by punching a hole in the flexible snap cap of a Wheaton vial with a paper hole punch. A small amount of two component epoxy putty is well mixed then pushed through the hole in the cap. The putty on the inner cap is formed into a “cup” to hold the specimen (Fig. 2), the putty on the outside is smoothed into a “button” to give good attachment even when the cap is flexed during handling (Fig. 3). The cap is submerged in the 70% ethanol, the bone at the base of the cochlea is seated into the cup and the sides of the cup squeezed with forceps to grip it (Fig.4). Several types of epoxy putty have been tried, most are either soluble in ethanol to some degree or do not set in ethanol. The only putty we find successful is “DUROtm MASTERMENDtm Epoxy Extra Strength Ribbon” (Loctite Corp., Cleveland, Ohio), this is a blue and yellow ribbon which is kneaded to form a green putty, it is available at many hardware stores.

Hair cell changes after cationic stimulation of corti's organ

1989 ◽  
Vol 47 ◽  
pp. 798-799
Author(s):  
Cesar D. Fermin ◽  
Hans-Peter Zenner
Keyword(s):  
Organ Of Corti ◽  
Cross Sectional ◽  
Surface Areas ◽  
High K ◽  
Anatomical Arrangement ◽  
Cell Cytosol ◽  
High Concentrations ◽  
K Concentration ◽  
Cell Changes

Contraction of outer and inner hair cells (OHC&IHC) in the Organ of Corti (OC) of the inner ear is necessary for sound transduction. Getting at HC in vivo preparations is difficult. Thus, isolated HCs have been used to study OHC properties. Even though viability has been shown in isolated (iOHC) preparations by good responses to current and cationic stimulation, the contribution of adjoining cells can not be explained with iOHC preparations. This study was undertaken to examine changes in the OHC after expossure of the OHC to high concentrations of potassium (K) and sodium (Na), by carefully immersing the OC in either artifical endolymph or perilymph. After K and Na exposure, OCs were fixed with 3% glutaraldehyde, post-fixed in osmium, separated into base, middle and apex and embedded in Araldite™. One μm thick sections were prepared for analysis with the light and E.M. Cross sectional areas were measured with Bioquant™ software.Potassium and sodium both cause isolated guinea pig OHC to contract. In vivo high K concentration may cause uncontrolled and sustained contractions that could contribute to Meniere's disease. The behavior of OHC in the vivo setting might be very different from that of iOHC. We show here changes of the cell cytosol and cisterns caused by K and Na to OHC in situs. The table below shows results from cross sectional area measurements of OHC from OC that were exposed to either K or Na. As one would expect, from the anatomical arrangement of the OC, OHC#l that are supported by rigid tissue would probably be displaced (move) less than those OHC located away from the pillar. Surprisingly, cells in the middle turn of the cochlea changed their surface areas more than those at either end of the cochlea. Moreover, changes in surface area do not seem to differ between K and Na treated OCs.

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