Brandon C. Cox

Spontaneous hair cell regeneration in the neonatal mouse cochlea in vivo.

Loss of cochlear hair cells in mammals is currently believed to be permanent, resulting in hearing impairment that affects more than 10% of the population. Here, we developed two genetic strategies to ablate neonatal mouse cochlear hair cells in vivo. Both Pou4f3DTR/+ and Atoh1-CreER™; ROSA26DTA/+ alleles allowed selective and inducible hair cell ablation. After hair cell loss was induced at birth, we observed spontaneous regeneration of hair cells. Fate-mapping experiments demonstrated that neighboring supporting cells acquired a hair cell fate, which increased in a basal to apical gradient, averaging over 120 regenerated hair cells per cochlea. The normally mitotically quiescent supporting cells proliferated after hair cell ablation. Concurrent fate mapping and labeling with mitotic tracers showed that regenerated hair cells were derived by both mitotic regeneration and direct transdifferentiation. Over time, regenerated hair cells followed a similar pattern of maturation to normal hair cell development, including the expression of prestin, a terminal differentiation marker of outer hair cells, although many new hair cells eventually died. Hair cell regeneration did not occur when ablation was induced at one week of age. Our findings demonstrate that the neonatal mouse cochlea is capable of spontaneous hair cell regeneration after damage in vivo. Thus, future studies on the neonatal cochlea might shed light on the competence of supporting cells to regenerate hair cells and on the factors that promote the survival of newly regenerated hair cells.

In vivo proliferative regeneration of balance hair cells in newborn mice

The regeneration of mechanoreceptive hair cells occurs throughout life in non-mammalian vertebrates and allows them to recover from hearing and balance deficits that affect humans and other mammals permanently. The irreversibility of comparable deficits in mammals remains unexplained, but often has been attributed to steep embryonic declines in cellular production. However, recent results suggest that gravity-sensing hair cells in murine utricles may increase in number during neonatal development, raising the possibility that young mice might retain sufficient cellular plasticity for mitotic hair cell regeneration. To test for this we used neomycin to kill hair cells in utricles cultured from mice of different ages and found that proliferation increased tenfold in damaged utricles from the youngest neonates. To kill hair cells in vivo, we generated a novel mouse model that uses an inducible, hair cell-specific CreER allele to drive expression of diphtheria toxin fragment A (DTA). In newborns, induction of DTA expression killed hair cells and resulted in significant, mitotic hair cell replacement in vivo, which occurred days after the normal cessation of developmental mitoses that produce hair cells. DTA expression induced in 5-d-old mice also caused hair cell loss, but no longer evoked mitotic hair cell replacement. These findings show that regeneration limits arise in vivo during the postnatal period when the mammalian balance epitheliums supporting cells differentiate unique cytological characteristics and lose plasticity, and they support the notion that the differentiation of those cells may directly inhibit regeneration or eliminate an essential, but as yet unidentified pool of stem cells.

Regulation of p27Kip1 by Sox2 maintains quiescence of inner pillar cells in the murine auditory sensory epithelium

Sox2 plays critical roles in cell fate specification during development and in stem cell formation; however, its role in postmitotic cells is largely unknown. Sox2 is highly expressed in supporting cells (SCs) of the postnatal mammalian auditory sensory epithelium, which unlike non-mammalian vertebrates remains quiescent even after sensory hair cell damage. Here, we induced the ablation of Sox2, specifically in SCs at three different postnatal ages (neonatal, juvenile and adult) in mice. In neonatal mice, Sox2-null inner pillar cells (IPCs, a subtype of SCs) proliferated and generated daughter cells, while other SC subtypes remained quiescent. Furthermore, p27Kip1, a cell cycle inhibitor, was absent in Sox2-null IPCs. Similarly, upon direct deletion of p27Kip1, p27Kip1-null IPCs also proliferated but retained Sox2 expression. Interestingly, cell cycle control of IPCs by Sox2-mediated expression of p27Kip1 gradually declined with age. In addition, deletion of Sox2 or p27Kip1 did not cause a cell fate change. Finally, chromatin immunoprecipitation with Sox2 antibodies and luciferase reporter assays with the p27Kip1 promoter support that Sox2 directly activates p27Kip1 transcription in postmitotic IPCs. Hence, in contrast to the well known activity of Sox2 in promoting proliferation and cell fate determination, our data demonstrate that Sox2 plays a novel role as a key upstream regulator of p27Kip1 to maintain the quiescent state of postmitotic IPCs. Our studies suggest that manipulating Sox2 or p27Kip1 expression is an effective approach to inducing proliferation of neonatal auditory IPCs, an initial but necessary step toward restoring hearing in mammals.

Conditional Gene Expression in the Mouse Inner Ear Using Cre-loxP

In recent years, there has been significant progress in the use of Cre-loxP technology for conditional gene expression in the inner ear. Here, we introduce the basic concepts of this powerful technology, emphasizing the differences between Cre and CreER. We describe the creation and Cre expression pattern of each Cre and CreER mouse line that has been reported to have expression in auditory and vestibular organs. We compare the Cre expression patterns between Atoh1-CreERTM and Atoh1-CreERT2 and report a new line, Fgfr3-iCreERT2, which displays inducible Cre activity in cochlear supporting cells. We also explain how results can vary when transgenic vs. knock-in Cre/CreER alleles are used to alter gene expression. We discuss practical issues that arise when using the Cre-loxP system, such as the use of proper controls, Cre efficiency, reporter expression efficiency, and Cre leakiness. Finally, we introduce other methods for conditional gene expression, including Flp recombinase and the tetracycline-inducible system, which can be combined with Cre-loxP mouse models to investigate conditional expression of more than one gene.

Auditory hair cell-specific deletion of p27Kip1 in postnatal mice promotes cell-autonomous generation of new hair cells and normal hearing.

Hearing in mammals relies upon the transduction of sound by hair cells (HCs) in the organ of Corti within the cochlea of the inner ear. Sensorineural hearing loss is a widespread and permanent disability due largely to a lack of HC regeneration in mammals. Recent studies suggest that targeting the retinoblastoma (Rb)/E2F pathway can elicit proliferation of auditory HCs. However, previous attempts to induce HC proliferation in this manner have resulted in abnormal cochlear morphology, HC death, and hearing loss. Here we show that cochlear HCs readily proliferate and survive following neonatal, HC-specific, conditional knock-out of p27Kip1 (p27CKO), a tumor suppressor upstream of Rb. Indeed, HC-specific p27CKO results in proliferation of these cells without the upregulation of the supporting cell or progenitor cell proteins, Prox1 or Sox2, suggesting that they remain HCs. Furthermore, p27CKO leads to a significant addition of postnatally derived HCs that express characteristic synaptic and stereociliary markers and survive to adulthood, although a portion of the newly derived inner HCs exhibit cytocauds and lack VGlut3 expression. Despite this, p27CKO mice exhibit normal hearing as measured by evoked auditory brainstem responses, which suggests that the newly generated HCs may contribute to, or at least do not greatly detract from, function. These results show that p27Kip1 actively maintains HC quiescence in postnatal mice, and suggest that inhibition of p27Kip1 in residual HCs represents a potential strategy for cell-autonomous auditory HC regeneration.

Supporting Cells Remove and Replace Sensory Receptor Hair Cells in a Balance Organ of Adult Mice

Vestibular hair cells in the inner ear encode head movements and mediate the sense of balance. These cells undergo cell death and replacement (turnover) throughout life in non-mammalian vertebrates. However, there is no definitive evidence that this process occurs in mammals. We used fate-mapping and other methods to demonstrate that utricular type II vestibular hair cells undergo turnover in adult mice under normal conditions. We found that supporting cells phagocytose both type I and II hair cells. Plp1-CreERT2-expressing supporting cells replace type II hair cells. Type I hair cells are not restored by Plp1-CreERT2-expressing supporting cells or by Atoh1-CreERTM-expressing type II hair cells. Destruction of hair cells causes supporting cells to generate 6 times as many type II hair cells compared to normal conditions. These findings expand our understanding of sensorineural plasticity in adult vestibular organs and further elucidate the roles that supporting cells serve during homeostasis and after injury. DOI: http://dx.doi.org/10.7554/eLife.18128.001

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

Generation of Atoh1-rtTA transgenic mice: a tool for inducible gene expression in hair cells of the inner ear

Atoh1 is a basic helix-loop-helix transcription factor that controls differentiation of hair cells (HCs) in the inner ear and its enhancer region has been used to create several HC-specific mouse lines. We generated a transgenic tetracycline-inducible mouse line (called Atoh1-rtTA) using the Atoh1 enhancer to drive expression of the reverse tetracycline transactivator (rtTA) protein and human placental alkaline phosphatase. Presence of the transgene was confirmed by alkaline phosphatase staining and rtTA activity was measured using two tetracycline operator (TetO) reporter alleles with doxycycline administered between postnatal days 0–3. This characterization of five founder lines demonstrated that Atoh1-rtTA is expressed in the majority of cochlear and utricular HCs. Although the tetracycline-inducible system is thought to produce transient changes in gene expression, reporter positive HCs were still observed at 6 weeks of age. To confirm that Atoh1-rtTA activity was specific to Atoh1-expressing cells, we also analyzed the cerebellum and found rtTA-driven reporter expression in cerebellar granule neuron precursor cells. The Atoh1-rtTA mouse line provides a powerful tool for the field and can be used in combination with other existing Cre recombinase mouse lines to manipulate expression of multiple genes at different times in the same animal.

Impact of ageing on postsynaptic neuronal nicotinic neurotransmission in auditory thalamus

Neuronal nicotinic acetylcholine receptors (nAChRs) play a fundamental role in the attentional circuitry throughout the mammalian CNS. In the present study, we report a novel finding that ageing negatively impacts nAChR efficacy in auditory thalamus, and this is probably the result of a loss of nAChR density (Bmax) and changes in the subunit composition of nAChRs. Our data support the hypothesis that age‐related maladaptive changes involving nAChRs within thalamocortical circuits partially underpin the difficulty that elderly adults experience with respect to attending to speech and other salient acoustic signals.

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

ABSTRACTFour 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, ROSA26tdTomato and CAG-eGFP, to achieve the same goal. Our results confirm previous reports that Sox2CreERT2 and Fgfr3-iCreERT2 are not only expressed in neonatal SCs but also in HCs. Decreasing the tamoxifen dose did not reduce HC expression for Sox2CreERT2, 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-iCreERT2 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-CreERT2 was paired with the CAG-eGFP reporter; however, the total number of labeled cells decreased. Changes to the tamoxifen dose or reporter line with Prox1CreERT2 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 ROSA26tdTomato 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.