Sensory cells in tunicates: insights into mechanoreceptor evolution.

Tunicates, the sister group of vertebrates, offer a unique perspective for evolutionary developmental studies (Evo-Devo) due to their simple anatomical organization. Moreover, the separation of tunicates from vertebrates predated the vertebrate-specific genome duplications. As adults, they include both sessile and pelagic species, with very limited mobility requirements related mainly to water filtration. In sessile species, larvae exhibit simple swimming behaviors that are required for the selection of a suitable substrate on which to metamorphose. Despite their apparent simplicity, tunicates display a variety of mechanoreceptor structures involving both primary and secondary sensory cells (i.e., coronal sensory cells). This review encapsulates two decades of research on tunicate mechanoreception focusing on the coronal organ's sensory cells as prime candidates for understanding the evolution of vertebrate hair cells of the inner ear and the lateral line organ. The review spans anatomical, cellular and molecular levels emphasizing both similarity and differences between tunicate and vertebrate mechanoreception strategies. The evolutionary significance of mechanoreception is discussed within the broader context of Evo-Devo studies, shedding light on the intricate pathways that have shaped the sensory system in chordates.

Comparative exploration of mammalian deafness gene homologues in the Drosophila auditory organ shows genetic correlation between insect and vertebrate hearing.

Johnston's organ, the Drosophila auditory organ, is anatomically very different from the mammalian organ of Corti. However, recent evidence indicates significant cellular and molecular similarities exist between vertebrate and invertebrate hearing, suggesting that Drosophila may be a useful platform to determine the function of the many mammalian deafness genes whose underlying biological mechanisms are poorly characterized. Our goal was a comprehensive screen of all known orthologues of mammalian deafness genes in the fruit fly to better understand conservation of hearing mechanisms between the insect and the fly and ultimately gain insight into human hereditary deafness. We used bioinformatic comparisons to screen previously reported human and mouse deafness genes and found that 156 of them have orthologues in Drosophila melanogaster. We used fluorescent imaging of T2A-GAL4 gene trap and GFP or YFP fluorescent protein trap lines for 54 of the Drosophila genes and found 38 to be expressed in different cell types in Johnston's organ. We phenotypically characterized the function of strong loss-of-function mutants in three genes expressed in Johnston's organ (Cad99C, Msp-300, and Koi) using a courtship assay and electrophysiological recordings of sound-evoked potentials. Cad99C and Koi were found to have significant courtship defects. However, when we tested these genes for electrophysiological defects in hearing response, we did not see a significant difference suggesting the courtship defects were not caused by hearing deficiencies. Furthermore, we used a UAS/RNAi approach to test the function of seven genes and found two additional genes, CG5921 and Myo10a, that gave a statistically significant delay in courtship but not in sound-evoked potentials. Our results suggest that many mammalian deafness genes have Drosophila homologues expressed in the Johnston's organ, but that their requirement for hearing may not necessarily be the same as in mammals.

Expression of Atoh1, Gfi1, and Pou4f3 in the mature cochlea reprograms nonsensory cells into hair cells.

Mechanosensory hair cells of the mature mammalian organ of Corti do not regenerate; consequently, loss of hair cells leads to permanent hearing loss. Although nonmammalian vertebrates can regenerate hair cells from neighboring supporting cells, many humans with severe hearing loss lack both hair cells and supporting cells, with the organ of Corti being replaced by a flat epithelium of nonsensory cells. To determine whether the mature cochlea can produce hair cells in vivo, we reprogrammed nonsensory cells adjacent to the organ of Corti with three hair cell transcription factors: Gfi1, Atoh1, and Pou4f3. We generated numerous hair cell-like cells in nonsensory regions of the cochlea and new hair cells continued to be added over a period of 9 wk. Significantly, cells adjacent to reprogrammed hair cells expressed markers of supporting cells, suggesting that transcription factor reprogramming of nonsensory cochlear cells in adult animals can generate mosaics of sensory cells like those seen in the organ of Corti. Generating such sensory mosaics by reprogramming may represent a potential strategy for hearing restoration in humans.

Combinatorial Atoh1, Gfi1, Pou4f3, and Six1 gene transfer induces hair cell regeneration in the flat epithelium of mature guinea pigs.

Flat epithelium (FE) is a condition characterized by the loss of both hair cells (HCs) and supporting cells and the transformation of the organ of Corti into a simple flat or cuboidal epithelium, which can occur after severe cochlear insults. The transcription factors Gfi1, Atoh1, Pou4f3, and Six1 (GAPS) play key roles in HC differentiation and survival in normal ears. Previous work using a single transcription factor, Atoh1, to induce HC regeneration in mature ears in vivo usually produced very few cells and failed to produce HCs in severely damaged organs of Corti, especially those with FE. Studies in vitro suggested combinations of transcription factors may be more effective than any single factor, thus the current study aims to examine the effect of co-overexpressing GAPS genes in deafened mature guinea pig cochleae with FE. Deafening was achieved through the infusion of neomycin into the perilymph, leading to the formation of FE and substantial degeneration of nerve fibers. Seven days post neomycin treatment, adenovirus vectors carrying GAPS were injected into the scala media and successfully expressed in the FE. One or two months following GAPS inoculation, cells expressing Myosin VIIa were observed in regions under the FE (located at the scala tympani side of the basilar membrane), rather than within the FE. The number of cells, which we define as induced HCs (iHCs), was not significantly different between one and two months, but the larger N at two months made it more apparent that there were significantly more iHCs in GAPS treated animals than in controls. Additionally, qualitative observations indicated that ears with GAPS gene expression in the FE had more nerve fibers than FE without the treatment. In summary, our results showed that co-overexpression of GAPS enhances the potential for HC regeneration in a severe lesion model of FE.

The Foxi3 transcription factor is necessary for the fate restriction of placodal lineages at the neural plate border.

The Foxi3 transcription factor, expressed in the neural plate border at the end of gastrulation, is necessary for the formation of posterior placodes and is thus important for ectodermal patterning. We have created two knock-in mouse lines expressing GFP or a tamoxifen-inducible Cre recombinase to show that Foxi3 is one of the earliest genes to label the border between the neural tube and epidermis, and that Foxi3-expressing neural plate border progenitors contribute primarily to cranial placodes and epidermis from the onset of expression, but not to the neural crest or neural tube lineages. By simultaneously knocking out Foxi3 in neural plate border cells and following their fates, we show that neural plate border cells lacking Foxi3 contribute to all four lineages of the ectoderm - placodes, epidermis, crest and neural tube. We contrast Foxi3 with another neural plate border transcription factor, Zic5, the progenitors of which initially contribute broadly to all germ layers until gastrulation and gradually become restricted to the neural crest lineage and dorsal neural tube cells. Our study demonstrates that Foxi3 uniquely acts early at the neural plate border to restrict progenitors to a placodal and epidermal fate.

DNA methylation in the mouse cochlea promotes maturation of supporting cells and contributes to the failure of hair cell regeneration.

Mammalian hair cells do not functionally regenerate in adulthood but can regenerate at embryonic and neonatal stages in mice by direct transdifferentiation of neighboring supporting cells into new hair cells. Previous work showed loss of transdifferentiation potential of supporting cells is in part due to H3K4me1 enhancer decommissioning of the hair cell gene regulatory network during the first postnatal week. However, inhibiting this decommissioning only partially preserves transdifferentiation potential. Therefore, we explored other repressive epigenetic modifications that may be responsible for this loss of plasticity. We find supporting cells progressively accumulate DNA methylation at promoters of developmentally regulated hair cell genes. Specifically, DNA methylation overlaps with binding sites of Atoh1, a key transcription factor for hair cell fate. We further show that DNA hypermethylation replaces H3K27me3-mediated repression of hair cell genes in mature supporting cells, and is accompanied by progressive loss of chromatin accessibility, suggestive of facultative heterochromatin formation. Another subset of hair cell loci is hypermethylated in supporting cells, but not in hair cells. Ten-eleven translocation (TET) enzyme-mediated demethylation of these hypermethylated sites is necessary for neonatal supporting cells to transdifferentiate into hair cells. We also observe changes in chromatin accessibility of supporting cell subtypes at the single-cell level with increasing age: Gene programs promoting sensory epithelium development loses chromatin accessibility, in favor of gene programs that promote physiological maturation and function of the cochlea. We also find chromatin accessibility is partially recovered in a chronically deafened mouse model, which holds promise for future translational efforts in hearing restoration.

Foxi3GFP and Foxi3CreER mice allow identification and lineage labeling of pharyngeal arch ectoderm and endoderm, and tooth and hair placodes.

BACKGROUND: FOXI3 is a forkhead family transcription factor that is expressed in the progenitors of craniofacial placodes, epidermal placodes, and the ectoderm and endoderm of the pharyngeal arch region. Loss of Foxi3 in mice and pathogenic Foxi3 variants in dogs and humans cause a variety of craniofacial defects including absence of the inner ear, severe truncations of the jaw, loss or reduction in external and middle ear structures, and defects in teeth and hair. RESULTS: To allow for the identification, isolation, and lineage tracing of Foxi3-expressing cells in developing mice, we targeted the Foxi3 locus to create Foxi3GFP and Foxi3CreER mice. We show that Foxi3GFP mice faithfully recapitulate the expression pattern of Foxi3 mRNA at all ages examined, and Foxi3CreER mice can trace the derivatives of pharyngeal arch ectoderm and endoderm, the pharyngeal pouches and clefts that separate each arch, and the derivatives of hair and tooth placodes. CONCLUSIONS: Foxi3GFP and Foxi3CreER mice are new tools that will be of use in identifying and manipulating pharyngeal arch ectoderm and endoderm and hair and tooth placodes.

Transcriptional dynamics of delaminating neuroblasts in the mouse otic vesicle.

An abundance of research has recently highlighted the susceptibility of cochleovestibular ganglion (CVG) neurons to noise damage and aging in the adult cochlea, resulting in hearing deficits. Furthering our understanding of the transcriptional cascades that contribute to CVG development may provide insight into how these cells can be regenerated to treat inner ear dysfunction. Here we perform a high-depth single-cell RNA sequencing analysis of the E10.5 otic vesicle and its surrounding tissues, including CVG precursor neuroblasts and emerging CVG neurons. Clustering and trajectory analysis of otic-lineage cells reveals otic markers and the changes in gene expression that occur from neuroblast delamination toward the development of the CVG. This dataset provides a valuable resource for further identifying the mechanisms associated with CVG development from neurosensory competent cells within the otic vesicle.

FOXI3 pathogenic variants cause one form of craniofacial microsomia.

Craniofacial microsomia (CFM; also known as Goldenhar syndrome), is a craniofacial developmental disorder of variable expressivity and severity with a recognizable set of abnormalities. These birth defects are associated with structures derived from the first and second pharyngeal arches, can occur unilaterally and include ear dysplasia, microtia, preauricular tags and pits, facial asymmetry and other malformations. The inheritance pattern is controversial, and the molecular etiology of this syndrome is largely unknown. A total of 670 patients belonging to unrelated pedigrees with European and Chinese ancestry with CFM, are investigated. We identify 18 likely pathogenic variants in 21 probands (3.1%) in FOXI3. Biochemical experiments on transcriptional activity and subcellular localization of the likely pathogenic FOXI3 variants, and knock-in mouse studies strongly support the involvement of FOXI3 in CFM. Our findings indicate autosomal dominant inheritance with reduced penetrance, and/or autosomal recessive inheritance. The phenotypic expression of the FOXI3 variants is variable. The penetrance of the likely pathogenic variants in the seemingly dominant form is reduced, since a considerable number of such variants in affected individuals were inherited from non-affected parents. Here we provide suggestive evidence that common variation in the FOXI3 allele in trans with the pathogenic variant could modify the phenotypic severity and accounts for the incomplete penetrance.

Fbxo2CreERT2: A new model for targeting cells in the neonatal and mature inner ear.

The mammalian inner ear contains six sensory patches that allow detection of auditory stimuli as well as movement and balance. Much research has focused on the organ of Corti, the sensory organ of the cochlea that detects sound. Unfortunately, these cells are difficult to access in vivo, especially in the mature animal, but the development of genetically modified mouse models, including Cre/Lox mice, has improved the ability to label, purify or manipulate these cells. Here, we describe a new tamoxifen-inducible CreER mouse line, the Fbxo2CreERT2 mouse, that can be used to specifically manipulate cells throughout the cochlear duct of the neonatal and mature cochlear epithelium. In vestibular sensory epithelia, Fbxo2CreERT2-mediated recombination occurs in many hair cells and more rarely in supporting cells of neonatal and adult mice, with a higher rate of Fbxo2CreERT2 induction in type 1 versus type 2 hair cells in adults. Fbxo2CreERT2 mice, therefore, are a new tool for the specific manipulation of epithelial cells of the inner ear and targeted manipulation of vestibular type 1 hair cells.

Early Wnt Signaling Activation Promotes Inner Ear Differentiation via Cell Caudalization in Mouse Stem Cell-Derived Organoids.

The inner ear is derived from the otic placode, one of the numerous cranial sensory placodes that emerges from the pre-placodal ectoderm (PPE) along its anterior-posterior axis. However, the molecular dynamics underlying how the PPE is regionalized are poorly resolved. We used stem cell-derived organoids to investigate the effects of Wnt signaling on early PPE differentiation and found that modulating Wnt signaling significantly increased inner ear organoid induction efficiency and reproducibility. Alongside single-cell RNA sequencing, our data reveal that the canonical Wnt signaling pathway leads to PPE regionalization and, more specifically, medium Wnt levels during the early stage induce (1) expansion of the caudal neural plate border (NPB), which serves as a precursor for the posterior PPE, and (2) a caudal microenvironment that is required for otic specification. Our data further demonstrate Wnt-mediated induction of rostral and caudal cells in organoids and more broadly suggest that Wnt signaling is critical for anterior-posterior patterning in the PPE.

Cellular reprogramming with ATOH1, GFI1, and POU4F3 implicate epigenetic changes and cell-cell signaling as obstacles to hair cell regeneration in mature mammals.

Reprogramming of the cochlea with hair-cell-specific transcription factors such as ATOH1 has been proposed as a potential therapeutic strategy for hearing loss. ATOH1 expression in the developing cochlea can efficiently induce hair cell regeneration but the efficiency of hair cell reprogramming declines rapidly as the cochlea matures. We developed Cre-inducible mice to compare hair cell reprogramming with ATOH1 alone or in combination with two other hair cell transcription factors, GFI1 and POU4F3. In newborn mice, all transcription factor combinations tested produced large numbers of cells with the morphology of hair cells and rudimentary mechanotransduction properties. However, 1 week later, only a combination of ATOH1, GFI1 and POU4F3 could reprogram non-sensory cells of the cochlea to a hair cell fate, and these new cells were less mature than cells generated by reprogramming 1 week earlier. We used scRNA-seq and combined scRNA-seq and ATAC-seq to suggest at least two impediments to hair cell reprogramming in older animals. First, hair cell gene loci become less epigenetically accessible in non-sensory cells of the cochlea with increasing age. Second, signaling from hair cells to supporting cells, including Notch signaling, can prevent reprogramming of many supporting cells to hair cells, even with three hair cell transcription factors. Our results shed light on the molecular barriers that must be overcome to promote hair cell regeneration in the adult cochlea.

FOXI3 haploinsufficiency contributes to low T-cell receptor excision circles and T-cell lymphopenia.

BACKGROUND: Newborn screening can identify neonatal T-cell lymphopenia through detection of a low number of copies of T-cell receptor excision circles in dried blood spots collected at birth. After a positive screening result, further diagnostic testing is required to determine whether the subject has severe combined immunodeficiency or other causes of T-cell lymphopenia. Even after thorough evaluation, approximately 15% of children with a positive result of newborn screening for T-cell receptor excision circles remain genetically undiagnosed. Identifying the underlying genetic etiology is necessary to guide subsequent clinical management and family planning. OBJECTIVE: We sought to elucidate the genetic basis of patients with T-cell lymphopenia without an apparent genetic diagnosis. METHODS: We used clinical genomic testing as well as functional and immunologic assays to identify and elucidate the genetic and mechanistic basis of T-cell lymphopenia. RESULTS: We report 2 unrelated individuals with nonsevere T-cell lymphopenia and abnormal T-cell receptor excision circles who harbor heterozygous loss-of-function variants in forkhead box I3 transcription factor (FOXI3). CONCLUSION: Our findings support the notion that haploinsufficiency of FOXI3 results in T-cell lymphopenia with variable expressivity and that FOXI3 may be a key modulator of thymus development.

GFI1 regulates hair cell differentiation by acting as an off-DNA transcriptional co-activator of ATOH1, and a DNA-binding repressor.

GFI1 is a zinc finger transcription factor that is necessary for the differentiation and survival of hair cells in the cochlea. Deletion of Gfi1 in mice significantly reduces the expression of hundreds of hair cell genes: this is a surprising result, as GFI1 normally acts as a transcriptional repressor by recruiting histone demethylases and methyltransferases to its targets. To understand the mechanisms by which GFI1 promotes hair cell differentiation, we used CUT&RUN to identify the direct targets of GFI1 and ATOH1 in hair cells. We found that GFI1 regulates hair cell differentiation in two distinct ways-first, GFI1 and ATOH1 can bind to the same regulatory elements in hair cell genes, but while ATOH1 directly binds its target DNA motifs in many of these regions, GFI1 does not. Instead, it appears to enhance ATOH1's transcriptional activity by acting as part of a complex in which it does not directly bind DNA. Second, GFI1 can act in its more typical role as a direct, DNA-binding transcriptional repressor in hair cells; here it represses non-hair cell genes, including many neuronal genes. Together, our results illuminate the function of GFI1 in hair cell development and hair cell reprogramming strategies.

Lunatic Fringe-GFP Marks Lamina-Specific Astrocytes That Regulate Sensory Processing.

Astrocytes are the most abundant glial cell in the brain and perform a wide range of tasks that support neuronal function and circuit activities. There is emerging evidence that astrocytes exhibit molecular and cellular heterogeneity; however, whether distinct subpopulations perform these diverse roles remains poorly defined. Here we show that the Lunatic Fringe-GFP (Lfng-GFP) bacteria artificial chromosome mouse line from both sexes specifically labels astrocyte populations within lamina III and IV of the dorsal spinal cord. Transcriptional profiling of Lfng-GFP+ astrocytes revealed unique molecular profiles, featuring an enriched expression of Notch- and Wnt- pathway components. Leveraging CRE-DOG viral tools, we ablated Lfng-GFP+ astrocytes, which decreased neuronal activity in lamina III and IV and impaired mechanosensation associated with light touch. Together, our findings identify Lfng-GFP+ astrocytes as a unique subpopulation that occupies a distinct anatomic location in the spinal cord and directly contributes to neuronal function and sensory responses.SIGNIFICANCE STATEMENT Astrocytes are the most abundant glial cell in the CNS, and their interactions with neurons are essential for brain function. However, understanding the functional diversity of astrocytes has been hindered because of the lack of reporters that mark subpopulations and genetic tools for accessing them. We discovered that the Lfng-GFP reporter mouse labels a laminae-specific subpopulation of astrocytes in the dorsal spinal cord and that ablation of these astrocytes reduces glutamatergic synapses. Further analysis revealed that these astrocytes have a role in maintaining sensory-processing circuity related to light touch.

Glial-Specific Deletion of Med12 Results in Rapid Hearing Loss via Degradation of the Stria Vascularis.

Mediator protein complex subunit 12 (Med12) is a core component of the basal transcriptional apparatus and plays a critical role in the development of many tissues. Mutations in Med12 are associated with X-linked intellectual disability syndromes and hearing loss; however, its role in nervous system function remains undefined. Here, we show that temporal conditional deletion of Med12 in astrocytes in the adult CNS results in region-specific alterations in astrocyte morphology. Surprisingly, behavioral studies revealed rapid hearing loss after adult deletion of Med12 that was confirmed by a complete abrogation of auditory brainstem responses. Cellular analysis of the cochlea revealed degeneration of the stria vascularis, in conjunction with disorganization of basal cells adjacent to the spiral ligament and downregulation of key cell adhesion proteins. Physiologic analysis revealed early changes in endocochlear potential, consistent with strial-specific defects. Together, our studies reveal that Med12 regulates auditory function in the adult by preserving the structural integrity of the stria vascularis.SIGNIFICANCE STATEMENT Mutations in Mediator protein complex subunit 12 (Med12) are associated with X-linked intellectual disability syndromes and hearing loss. Using temporal-conditional genetic approaches in CNS glia, we found that loss of Med12 results in severe hearing loss in adult animals through rapid degeneration of the stria vascularis. Our study describes the first animal model that recapitulates hearing loss identified in Med12-related disorders and provides a new system in which to examine the underlying cellular and molecular mechanisms of Med12 function in the adult nervous system.

Enhancer decommissioning imposes an epigenetic barrier to sensory hair cell regeneration.

Adult mammalian tissues such as heart, brain, retina, and the sensory structures of the inner ear do not effectively regenerate, although a latent capacity for regeneration exists at embryonic and perinatal times. We explored the epigenetic basis for this latent regenerative potential in the mouse inner ear and its rapid loss during maturation. In perinatal supporting cells, whose fate is maintained by Notch-mediated lateral inhibition, the hair cell enhancer network is epigenetically primed (H3K4me1) but silenced (active H3K27 de-acetylation and trimethylation). Blocking Notch signaling during the perinatal period of plasticity rapidly eliminates epigenetic silencing and allows supporting cells to transdifferentiate into hair cells. Importantly, H3K4me1 priming of the hair cell enhancers in supporting cells is removed during the first post-natal week, coinciding with the loss of transdifferentiation potential. We hypothesize that enhancer decommissioning during cochlear maturation contributes to the failure of hair cell regeneration in the mature organ of Corti.

Transcription Factor Reprogramming in the Inner Ear: Turning on Cell Fate Switches to Regenerate Sensory Hair Cells.

Non-mammalian vertebrates can restore their auditory and vestibular hair cells naturally by triggering the regeneration of adjacent supporting cells. The transcription factor ATOH1 is a key regulator of hair cell development and regeneration in the inner ear. Following the death of hair cells, supporting cells upregulate ATOH1 and give rise to new hair cells. However, in the mature mammalian cochlea, such natural regeneration of hair cells is largely absent. Transcription factor reprogramming has been used in many tissues to convert one cell type into another, with the long-term hope of achieving tissue regeneration. Reprogramming transcription factors work by altering the transcriptomic and epigenetic landscapes in a target cell, resulting in a fate change to the desired cell type. Several studies have shown that ATOH1 is capable of reprogramming cochlear non-sensory tissue into cells resembling hair cells in young animals. However, the reprogramming ability of ATOH1 is lost with age, implying that the potency of individual hair cell-specific transcription factors may be reduced or lost over time by mechanisms that are still not clear. To circumvent this, combinations of key hair cell transcription factors have been used to promote hair cell regeneration in older animals. In this review, we summarize recent findings that have identified and studied these reprogramming factor combinations for hair cell regeneration. Finally, we discuss the important questions that emerge from these findings, particularly the feasibility of therapeutic strategies using reprogramming factors to restore human hearing in the future.

Building the Border: Development of the Chordate Neural Plate Border Region and Its Derivatives.

The paired cranial sensory organs and peripheral nervous system of vertebrates arise from a thin strip of cells immediately adjacent to the developing neural plate. The neural plate border region comprises progenitors for four key populations of cells: neural plate cells, neural crest cells, the cranial placodes, and epidermis. Putative homologues of these neural plate border derivatives can be found in protochordates such as amphioxus and tunicates. In this review, we summarize key signaling pathways and transcription factors that regulate the inductive and patterning events at the neural plate border region that give rise to the neural crest and placodal lineages. Gene regulatory networks driven by signals from WNT, fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) signaling primarily dictate the formation of the crest and placodal lineages. We review these studies and discuss the potential of recent advances in spatio-temporal transcriptomic and epigenomic analyses that would allow a mechanistic understanding of how these signaling pathways and their downstream transcriptional cascades regulate the formation of the neural plate border region.

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.