Atoh1 Overexpression Research Articles

Precise genetic control of ATOH1 enhances maturation of regenerated hair cells in the mature mouse utricle

Vestibular hair cells are mechanoreceptors critical for detecting head position and motion. In mammals, hair cell loss causes vestibular dysfunction as spontaneous regeneration is nearly absent. Constitutive expression of exogenous ATOH1, a hair cell transcription factor, increases hair cell regeneration, however, these cells fail to fully mature. Here, we profiled mouse utricles at 14 time points, and defined transcriptomes of developing and mature vestibular hair cells. To mimic native hair cells which downregulate endogenous ATOH1 as they mature, we engineered viral vectors carrying the supporting cell promoters GFAP and RLBP1. In utricles damaged ex vivo, both CMV-ATOH1 and GFAP-ATOH1 increased regeneration more effectively than RLBP1-ATOH1, while GFAP-ATOH1 and RLBP1-ATOH1 induced hair cells with more mature transcriptomes. In utricles damaged in vivo, GFAP-ATOH1 induced regeneration of hair cells expressing genes indicative of maturing type II hair cells, and more hair cells with bundles and synapses than untreated organs. Together our results demonstrate the efficacy of spatiotemporal control of ATOH1 overexpression in inner ear hair cell regeneration.

Atoh1 overexpression promotes Guinea pig bone marrow mesenchymal stem cells to differentiate into neural stem cell

Sensorineural hearing loss (SNHL) is a prevalent condition in otolaryngology. A key obstacle is finding effective strategies for regenerating damaged cochlear hair cells in adult animals. A practical and reliable approach has been developed to create a superior cell source for stem cell transplantation in the inner ear to treat SNHL. Atoh1 is involved in the differentiation of neurons, intestinal secretory cells, and mechanoreceptors including auditory hair cells, and thus plays an important role in neurogenesis. Lentivirus-mediated transfection of bone marrow mesenchymal stem cells (BMSCs) was utilized to achieve stable expression of the essential transcription factor Atoh1, which is crucial for developing auditory hair cells without compromising cell survival. By manipulating the induction conditions through altering the cell growth environment using anti-adherent culture, the synergistic impact of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) was effectively applied to significantly improve the differentiation efficiency of bone marrow-derived mesenchymal stem cells (BMSC) into neural stem cells (NSCs) following Atoh1 transfection, thereby reducing the induction time. The study indicated that the newly proposed transdifferentiation method effectively transformed BMSCs into NSCs in a controlled environment, presenting a potential approach for stem cell transplantation to promote hair cell regeneration.

The proper timing of Atoh1 expression is pivotal for hair cell subtype differentiation and the establishment of inner ear function

Atoh1 overexpression is essential for hair cell (HC) regeneration in the sensory epithelium of mammalian auditory and vestibular organs. However, Atoh1 overexpression alone cannot induce fully mature and functional HCs in the mammalian inner ear. In the current study, we investigated the effect of Atoh1 constitutive overexpression in native HCs by manipulating Atoh1 expression at different developmental stages. We demonstrated that constitutive overexpression of Atoh1 in native vestibular HCs did not affect cell survival but did impair vestibular function by interfering with the subtype differentiation of HCs and hair bundle development. In contrast, Atoh1 overexpression in cochlear HCs impeded their maturation, eventually leading to gradual HC loss in the cochlea and hearing dysfunction. Our study suggests that time-restricted Atoh1 expression is essential for the differentiation and survival of HCs in the inner ear, and this is pivotal for both hearing and vestibular function re-establishment through Atoh1 overexpression-induced HC regeneration strategies.

The role of Espin in the stereocilia regeneration and protection in Atoh1-overexpressed cochlear epithelium.

Hair cells (HCs) in mammals cannot spontaneously regenerate after damage. Atoh1 overexpression can promote HC regeneration in the postnatal cochlea, but the regenerated HCs do not possess the structural and functional characteristics of HCs in situ. The stereocilia on the apical surface of HCs are the first-level structure for sound conduction, and regeneration of functional stereocilia is the key basis for the reproduction of functional HCs. Espin, as an actin bundling protein, plays an important role in the development and structural maintenance of the stereocilia. Here, we found that the upregulation of Espin by AAV-ie was able to induced the aggregation of actin fibres in Atoh1-induced HCs in both cochlear organoids and explants. In addition, we found that persistent Atoh1 overexpression resulted in impaired stereocilia in both endogenous and newly formed HCs. In contrast, the forced expression of Espin in endogenous and regenerative HCs was able to eliminate the stereocilia damage caused by persistent Atoh1 overexpression. Our study shows that the enhanced expression of Espin can optimize the developmental process of stereocilia in Atoh1-induced HCs and can attenuate the damage to native HCs induced by Atoh1 overexpression. These results suggest an effective method to induce the maturation of stereocilia in regenerative HCs and pave the way for functional HC regeneration via supporting cell transdifferentiation.

A Novel in vitro Model Delineating Hair Cell Regeneration and Neural Reinnervation in Adult Mouse Cochlea.

The study of an adult mammalian auditory system, such as regeneration, has been hampered by the lack of an in vitro system in which hypotheses can be tested efficiently. This is primarily due to the fact that the adult inner ear is encased in the toughest bone of the body, whereas its removal leads to the death of the sensory epithelium in culture. We hypothesized that we could take advantage of the integral cochlear structure to maintain the overall inner ear architecture and improve sensory epithelium survival in culture. We showed that by culturing adult mouse cochlea with the (surrounding) bone intact, the supporting cells (SCs) survived and almost all hair cells (HCs) degenerated. To evaluate the utility of the explant culture system, we demonstrated that the overexpression of Atoh1, an HC fate-determining factor, is sufficient to induce transdifferentiation of adult SCs to HC-like cells (HCLCs). Transdifferentiation-derived HCLCs resemble developmentally young HCs and are able to attract adult ganglion neurites. Furthermore, using a damage model, we showed that degenerated adult ganglions respond to regenerated HCLCs by directional neurite outgrowth that leads to HCLC-neuron contacts, strongly supporting the intrinsic properties of the HCLCs in establishing HCLC-neuron connections. The adult whole cochlear explant culture is suitable for diverse studies of the adult inner ear including regeneration, HC-neuron pathways, and inner ear drug screening.

Generation of mature and functional hair cells by co-expression of Gfi1, Pou4f3, and Atoh1 in the postnatal mouse cochlea.

The mammalian cochlea cannot regenerate functional hair cells (HCs) spontaneously. Atoh1 overexpression as well as other strategies are unable to generate functional HCs. Here, we simultaneously upregulated the expression of Gfi1, Pou4f3, and Atoh1 in postnatal cochlear supporting cells (SCs) invivo, which efficiently converted SCs into HCs. The newly regenerated HCs expressed HC markers Myo7a, Calbindin, Parvalbumin, and Ctbp2 and were innervated by neurites. Importantly, many new HCs expressed the mature and terminal marker Prestin or vesicular glutamate transporter 3 (vGlut3), depending on the subtypes of the source SCs. Finally, our patch-clamp analysis showed that the new HCs in the medial region acquired a large K+ current, fired spikes transiently, and exhibited signature refinement of ribbon synapse functions, in close resemblance to native wild-type inner HCs. We demonstrated that co-upregulating Gfi1, Pou4f3, and Atoh1 enhances the efficiency of HC generation and promotes the functional maturation of new HCs.

AAV8-mediated Atoh1 overexpression induces dose-dependent regeneration of vestibular hair cells in adult mice

Vestibular hair cells (HCs) are mechanoreceptors for the detection of head movement. Vestibular HCs of adult mammals never completely regenerate after damage, resulting in vestibular dysfunction. Overexpression of Atoh1 is effective for inducing HC regeneration. However, method of clinical feasibility and improvement of regenerative extent are both in need. Here we used an adeno-associated virus (AAV) serotype 8 vector of two different titers to overexpress Atoh1 in the injured utricles of adult mice. One month after virus inoculation, abundant myosin VIIa-positive cells and immature stereocilia were observed. Quantitative analyses revealed that Atoh1 overexpression replenished vestibular HCs in a dose-dependent manner. Vectors of a higher titer increased the number of myosin VIIa-positive cells compared to those of lower titer. Moreover, only Atoh1 overexpression in the higher titer group enhanced stereocilium regeneration, which is an important step in the maturation of regenerated HCs. Although the current treatment failed to initiate functional recovery of the animals, our results prompt further improvements in the recovery of vestibular dysfunction by AAV.

Simultaneous gentamicin-mediated damage and Atoh1 overexpression promotes hair cell regeneration in the neonatal mouse utricle

Loss of hair cells from vestibular epithelium results in balance dysfunction. The current therapeutic regimen for vestibular diseases is limited. Upon injury or Atoh1 overexpression, hair cell replacement occurs rapidly in the mammalian utricle, suggesting a promising approach to induce vestibular hair cell regeneration. In this study, we applied simultaneous gentamicin-mediated hair cell ablation and Atoh1 overexpression to induce neonatal utricular hair cell formation in vitro. We confirmed that type I hair cells were the primary targets of gentamicin. Furthermore, injury and Atoh1 overexpression promoted hair cell regeneration in a timely and efficient manner through robust viral transfection. Hair cells regenerated with type II characteristics in the striola and type I/II characteristics in non-sensory regions. Rare EdU+/myosin7a+ cells in sensory regions and robust EdU+/myosin7a+ signals in ectopic regions indicate that transdifferentiation of supporting cells in situ, and mitosis and differentiation of non-sensory epithelial cells in ectopic regions, are sources of regenerative hair cells. Distinct regeneration patterns in in situ and ectopic regions suggested robust plasticity of vestibular non-sensory epithelium, generating more developed hair cell subtypes and thus providing a promising stem cell-like source of hair cells. These findings suggest that simultaneously causing injury and overexpressing Atoh1 promotes hair cell regeneration efficacy and maturity, thus expanding the understanding of ectopic plasticity in neonatal vestibular organs.

Combinatorial Atoh1 and Gfi1 induction enhances hair cell regeneration in the adult cochlea

Mature mammalian cochlear hair cells (HCs) do not spontaneously regenerate once lost, leading to life-long hearing deficits. Attempts to induce HC regeneration in adult mammals have used over-expression of the HC-specific transcription factor Atoh1, but to date this approach has yielded low and variable efficiency of HC production. Gfi1 is a transcription factor important for HC development and survival. We evaluated the combinatorial effects of Atoh1 and Gfi1 over-expression on HC regeneration using gene transfer methods in neonatal cochlear explants, and in vivo in adult mice. Adenoviral over-expression of Atoh1 and Gfi1 in cultured neonatal cochlear explants resulted in numerous ectopic HC-like cells (HCLCs), with significantly more cells in Atoh1 + Gfi1 cultures than Atoh1 alone. In vitro, ectopic HCLCs emerged in regions medial to inner HCs as well as in the stria vascularis. In vivo experiments were performed in mature Pou4f3DTR mice in which HCs were completely and specifically ablated by administration of diphtheria toxin. Adenoviral expression of Atoh1 or Atoh1 + Gfi1 in cochlear supporting cells induced appearance of HCLCs, with Atoh1 + Gfi1 expression leading to 6.2-fold increase of new HCLCs after 4 weeks compared to Atoh1 alone. New HCLCs were detected throughout the cochlea, exhibited immature stereocilia and survived for at least 8 weeks. Combinatorial Atoh1 and Gfi1 induction is thus a promising strategy to promote HC regeneration in the mature mammalian cochlea.

Developmental and Functional Hair Cell-Like Cells Induced by Atoh1 Overexpression in the Adult Mammalian Cochlea In Vitro.

Hair cells (HCs) in the mammalian cochleae cannot spontaneously regenerate once damaged, resulting in permanent hearing loss. It has been shown that Atoh1 overexpression induces hair cell-like cells (HCLCs) in the cochlea of newborn rodents, but this is hard to achieve in adult mammals. In this study, we used a three-dimensional cochlear culture system and an adenoviral-mediated delivery vector to overexpress Atoh1 in adult mouse cochleae. HCLCs were successfully induced from 3 days after virus infection (3 DVI) in vitro, and the number increased with time. HCLCs were myosin7a positive and distinguishable from remnant HCs in a culture environment. Meanwhile, patch-clamp results showed that noninactive outward potassium currents (sustained outward potassium currents) could be recorded in HCLCs and that their magnitude increased with time, similar to normal HCs. Furthermore, transient HCN currents were recorded in some HCLCs, indicating that the HCLCs experienced a developmental stage similar to normal HCs. We also compared the electrophysiological features of HCLCs from adult mice with native HCs and found the HCLCs gradually matured, similar to the normal HCs. Meanwhile, HCLCs from adult mice possessed the same bundles as developmental HCs. However, these HCLCs did not express prestin, which is a special marker for outer hair cells (OHCs), even at 13 DVI. These results demonstrate that Atoh1 overexpression induces HCLC formation in the adult mammalian cochlea and that these HCLCs were functional and experienced a developmental process similar to that of normal HCs.

Cellular origin and response of flat epithelium in the vestibular end organs of mice to Atoh1 overexpression

A flat epithelium (FE) may be found in the vestibular end organs of humans and mice with vestibular dysfunction. However, the pathogenesis of FE is unclear and inducing hair cell (HC) regeneration is challenging, as both HCs and supporting cells (SCs) in vestibular FE are damaged. To determine the cellular origin of vestibular FE and examine its response to Atoh1 overexpression, we fate-mapped vestibular epithelial cells in three transgenic mouse lines (vGlut3-iCreERT2:Rosa26tdTomato, GLAST-CreERT2:Rosa26tdTomato, and Plp-CreERT2:Rosa26tdTomato) after inducing a lesion by administering a high dose of streptomycin. Atoh1 overexpression in vestibular FE was mediated by an adeno-associated virus serotype 8 (AAV8) vector. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, was administered with AAV8 to enhance Atoh1 overexpression. The transduction efficiency and population of myosin VIIa-positive cells were analyzed. A small number of HCs were present in vestibular FE. FE did not show broad GLAST-Cre or Plp-Cre expression, unlike the original SCs. SAHA dramatically enhanced AAV8-mediated exogenous gene overexpression, and Atoh1 overexpression plus SAHA promoted myosin VIIa expression in FE cells. Our data provide insight into FE formation and will facilitate studies of gene therapy for vestibular FE.

MCPyV Large T Antigen-Induced Atonal Homolog 1 Is a Lineage-Dependency Oncogene in Merkel Cell Carcinoma

Despite the fact that the transcription factor ATOH1 is a master regulator of Merkel cell development, its role in Merkel cell carcinoma (MCC) carcinogenesis remains controversial. Here, we provide several lines of evidence that ATOH1 is a lineage-dependent oncogene in MCC. Luciferase assays revealed binding of ATOH1 and subsequent activation to the promoter of miR-375, which is one of the most abundant microRNAs in MCCs. Overexpression of ATOH1 in variant MCC cell lines and fibroblasts induced miR-375 expression, whereas ATOH1 knockdown in classical MCC cell lines reduced miR-375 expression. Moreover, ATOH1 overexpression in these cells changed their growth characteristics from adherent to suspension and/orspheroidal growth, that is, resembling the neuroendocrine growth pattern of classical MCC cell lines. Notably, ectopic expression of different Merkel cell polyomavirus (MCPyV)-derived truncated large T antigens induced ATOH1 expression in fibroblasts, which was paralleled by miR-375 expression and similar morphologic changes. In summary, MCPyV-associated carcinogenesis is likely to induce the characteristic neuroendocrine features of MCC via induction of ATOH1; thus, ATOH1 can be regarded as a lineage-dependent oncogene in MCC.

Atoh1 Directs Regeneration and Functional Recovery of the Mature Mouse Vestibular System

SUMMARYUtricular hair cells (HCs) are mechanoreceptors required for vestibular function. After damage, regeneration of mammalian utricular HCs is limited and regenerated HCs appear immature. Thus, loss of vestibular function is presumed irreversible. Here, we found partial HC replacement and functional recovery in the mature mouse utricle, both enhanced by overexpressing the transcription factor Atoh1. Following damage, long-term fate mapping revealed that support cells non-mitotically and modestly regenerated HCs displaying no or immature bundles. By contrast, Atoh1 overexpression stimulated proliferation and widespread regeneration of HCs exhibiting elongated bundles, patent mechanotransduction channels, and synaptic connections. Finally, although damage without Atoh1 overexpression failed to initiate or sustain a spontaneous functional recovery, Atoh1 overexpression significantly enhanced both the degree and percentage of animals exhibiting sustained functional recovery. Therefore, the mature, damaged utricle has an Atoh1-responsive regenerative program leading to functional recovery, underscoring the potential of a reprogramming approach to sensory regeneration.

High-resolution transcriptional dissection of in vivo Atoh1-mediated hair cell conversion in mature cochleae identifies Isl1 as a co-reprogramming factor.

In vivo direct conversion of differentiated cells holds promise for regenerative medicine; however, improving the conversion efficiency and producing functional target cells remain challenging. Ectopic Atoh1 expression in non-sensory supporting cells (SCs) in mouse cochleae induces their partial conversion to hair cells (HCs) at low efficiency. Here, we performed single-cell RNA sequencing of whole mouse sensory epithelia harvested at multiple time points after conditional overexpression of Atoh1. Pseudotemporal ordering revealed that converted HCs (cHCs) are present along a conversion continuum that correlates with both endogenous and exogenous Atoh1 expression. Bulk sequencing of isolated cell populations and single-cell qPCR confirmed 51 transcription factors, including Isl1, are differentially expressed among cHCs, SCs and HCs. In transgenic mice, co-overexpression of Atoh1 and Isl1 enhanced the HC conversion efficiency. Together, our study shows how high-resolution transcriptional profiling of direct cell conversion can identify co-reprogramming factors required for efficient conversion.

Transcription factor induced conversion of human fibroblasts towards the hair cell lineage.

Hearing loss is the most common sensorineural disorder, affecting over 5% of the population worldwide. Its most frequent cause is the loss of hair cells (HCs), the mechanosensory receptors of the cochlea. HCs transduce incoming sounds into electrical signals that activate auditory neurons, which in turn send this information to the brain. Although some spontaneous HC regeneration has been observed in neonatal mammals, the very small pool of putative progenitor cells that have been identified in the adult mammalian cochlea is not able to replace the damaged HCs, making any hearing impairment permanent. To date, guided differentiation of human cells to HC-like cells has only been achieved using either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). However, use of such cell types suffers from a number of important disadvantages, such as the risk of tumourigenicity if transplanted into the host´s tissue. We have obtained cells expressing hair cell markers from cultures of human fibroblasts by overexpression of GFI1, Pou4f3 and ATOH1 (GPA), three genes that are known to play a critical role in the development of HCs. Immunocytochemical, qPCR and RNAseq analyses demonstrate the expression of genes typically expressed by HCs in the transdifferentiated cells. Our protocol represents a much faster approach than the methods applied to ESCs and iPSCs and validates the combination of GPA as a set of genes whose activation leads to the direct conversion of human somatic cells towards the hair cell lineage. Our observations are expected to contribute to the development of future therapies aimed at the regeneration of the auditory organ and the restoration of hearing.

Notch pathway inhibitor DAPT enhances Atoh1 activity to generate new hair cells in situ in rat cochleae.

Atoh1 overexpression in cochlear epithelium induces new hair cell formation. Use of adenovirus-mediated Atoh1 overexpression has mainly focused on the rat lesser epithelial ridge and induces ectopic hair cell regeneration. The sensory region of rat cochlea is difficult to transfect, thus new hair cells are rarely produced in situ in rat cochlear explants. After culturing rat cochleae in medium containing 10% fetal bovine serum, adenovirus successfully infected the sensory region as the width of the supporting cell area was significantly increased. Adenovirus encoding Atoh1 infected the sensory region and induced hair cell formation in situ. Combined application of the Notch inhibitor DAPT and Atoh1 increased the Atoh1 expression level and decreased hes1 and hes5 levels, further promoting hair cell generation. Our results demonstrate that DAPT enhances Atoh1 activity to promote hair cell regeneration in rat cochlear sensory epithelium in vitro.

Extensive Supporting Cell Proliferation and Mitotic Hair Cell Generation by In Vivo Genetic Reprogramming in the Neonatal Mouse Cochlea.

The generation of hair cells (HCs) from the differentiation of proliferating supporting cells (SCs) appears to be an ideal approach for replacing lost HCs in the cochlea and is promising for restoring hearing after damage to the organ of Corti. We show here that extensive proliferation of SCs followed by mitotic HC generation is achieved through a genetic reprogramming process involving the activation of β-catenin to upregulate Wnt signaling, the deletion of Notch1 to downregulate Notch signaling, and the overexpression of Atoh1 in Sox2(+) SCs in neonatal mouse cochleae. We used RNA sequencing to compare the transcripts of the cochleae from control mice and from mice with β-catenin activation, Notch1 deletion, and β-catenin activation combined with Notch1 deletion in Sox2(+) SCs. We identified the genes involved in the proliferation and transdifferentiation process that are either controlled by individual signaling pathways or by the combination of Wnt and Notch signaling. Moreover, the proliferation of SCs induced by Notch1 deletion disappears after deleting β-catenin in Notch1 knock-out Sox2(+) cells, which further demonstrates that Notch signaling is an upstream and negative regulator of Wnt signaling. We show here that the extensive proliferation of supporting cells (SCs) and the subsequent mitotic hair cell (HC) generation is achieved through a genetic reprogramming process involving activation of β-catenin to upregulate Wnt signaling, deletion of Notch1 to downregulate Notch signaling, and overexpression of Atoh1 in Sox2(+) SCs in neonatal mice cochleae. By comparing the transcripts of the cochleae among controls, β-catenin activation, Notch1 deletion, and β-catenin activation combined with Notch1 deletion group, we identified multiple genes involved in the proliferation and transdifferentiation process that are either controlled by individual signaling pathways or by the combination of Wnt and Notch signaling. This provides a better understanding of the mechanisms behind mitotic HC generation and might provide new approaches to stimulating mitotic HC regeneration.

Dual role for Sox2 in specification of sensory competence and regulation of Atoh1 function.

The formation of inner ear sensory epithelia is believed to occur in two steps, initial specification of sensory competent (prosensory) regions followed by determination of specific cell-types, such as hair cells (HCs) and supporting cells. However, studies in which the HC determination factor Atoh1 was ectopically expressed in nonprosensory regions indicated that expression of Atoh1 alone is sufficient to induce HC formation suggesting that prosensory formation may not be a prerequisite for HC development. To test this hypothesis, interactions between Sox2 and Atoh1, which are required for prosensory and HC formation respectively, were examined. Forced expression of Atoh1 in nonprosensory cells resulted in transient expression of Sox2 prior to HC formation, suggesting that expression of Sox2 is required for formation of ectopic HCs. Moreover, Atoh1 overexpression failed to induce HC formation in Sox2 mutants, confirming that Sox2 is required for prosensory competence. To determine whether expression of Sox2 alone is sufficient to induce prosensory identity, Sox2 was transiently activated in a manner that mimicked endogenous expression. Following transient Sox2 activation, nonprosensory cells developed as HCs, a result that was never observed in response to persistent expression of Sox2. These results, suggest a dual role for Sox2 in inner ear formation. Initially, Sox2 is required to specify prosensory competence, but subsequent down-regulation of Sox2 must occur to allow Atoh1 expression, most likely through a direct interaction with the Atoh1 promoter. These results implicate Sox2-mediated changes in prosensory cells as an essential step in their ability to develop as HCs. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 3-13, 2017.

Abstract B30: Regulation of proliferation and differentiation by atonal homolog 1 phosphorylation

Abstract Atonal homolog 1 (Atoh1) is a basic helix-loop-helix (bHLH) transcription factor that acts as a tumor-suppressor as well as drives the differentiation of intestinal progenitor cells. Phosphorylation of Atoh1 is an important regulatory mechanism of its activity; proteins of the bHLH family can be phosphorylated by cyclin-dependent kinases during the cell cycle, which affects their differentiation-driving potential. We generated a colon cancer cell line model to investigate site-specific phosphoregulation of Atoh1. A mutant Atoh1, which has multiple serine/threonine residues changed to alanine at serine-proline (SP) and threonine-proline (TP) sites (9S/T-A Atoh1), was generated and expressed in a doxycycline-inducible model at a single copy level. The loss of phosphorylation induced hyper-activation of Atoh1 with respect to differentiation and proliferation arrest; 9S/T-A Atoh1 inhibited proliferation more than the wild-type protein. Overexpression of Atoh1 caused G1 cell-cycle arrest. Atoh1 induced expression of the secretory lineage markers, while absorptive lineage markers were down regulated. Phospho-mutant protein was able to not only inhibit proliferation but also drive the differentiation of previously undifferentiated colon cancer cell line more strongly than the wild type protein. Investigating the significance of phosphorylation of Atoh1 in the context of its tumor-suppressive role is of great importance in colon cancer. A better insight into the differentiation processes and their deregulation in cancer has the potential to open further avenues and introduce promising strategies in differentiation-targeted cancer therapies. Citation Format: Goran Tomic, Douglas J. Winton, Anna Philpott. Regulation of proliferation and differentiation by atonal homolog 1 phosphorylation. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B30.

Pseudo-immortalization of postnatal cochlear progenitor cells yields a scalable cell line capable of transcriptionally regulating mature hair cell genes.

The mammalian cochlea is a highly specialized organ within the inner ear. Sensory hair cells (HC) in the cochlea detect and transduce sound waves into electrical impulses that are sent to the brain. Studies of the molecular pathways regulating HC formation are hindered by the very sparse nature of HCs, where only ~3300 are found within an entire mouse cochlea. Current cell lines mimic certain aspects of HCs but lack terminal HC marker expression. Here we successfully “pseudo-immortalized” cochlear progenitor cells using the “conditional reprogramming” technique. These cells, termed “Conditionally Reprogrammed Otic Stem Cells” (CR-OSC), are able to bypass the senescence inherent to cochlear progenitor cells without genetic alterations, allowing for the generation of over 15 million cells from a single cochlea. These cells can be differentiated and up-regulate both early and terminal differentiation genes associated with HCs, including the terminal HC differentiation marker prestin. CR-OSCs also respond to known HC cues, including upregulation of HC genes in response to Atoh1 overexpression, and upregulation of prestin expression after thyroid hormone application. Overall, we describe the creation of a HC line capable of regulated expression of HC genes that can easily be recreated in any laboratory from any mouse of interest.