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.

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.

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.

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.

Examining Health Care Access for Refugee Children and Families in the North Carolina Triangle Area.

BACKGROUND Resettled refugees are at increased risk of poor health outcomes due to acculturation challenges, logistical barriers, experiences of trauma, and other barriers to care that are poorly understood. Refugee children may be particularly vulnerable due to disruptions in health, well-being, education, and nutrition during the resettlement process.METHOD To describe the health care barriers facing refugees in the North Carolina Triangle area (comprised of Durham, Chapel Hill, Raleigh, and their surrounding areas), we conducted three focus group interviews (in Arabic, French, and Swahili) with 25 refugee parents from Syria, Iraq, Central African Republic, the Democratic Republic of the Congo, and Chad. We also administered a survey to nine organizations that provide services for refugees.RESULTS Focus group responses highlighted the multidimensional nature of health care barriers for refugee families and children, encompassing challenges with acculturation, communication, transportation, finances, and health literacy. Organizations emphasized similar challenges and described their efforts to improve access to services through increased communication, coordination, and seeking new financial support for programs.LIMITATIONS Given the geographic focus of the study, results may not be generalizable to other populations and settings. Men spoke more than women in some focus groups, and participants may have been influenced by more vocal contributors. Furthermore, this study is limited by a lack of health outcomes data.CONCLUSIONS This study suggests that the health care needs of refugees living in the North Carolina Triangle area can be better met by providing comprehensive, coordinated, and culturally relevant care. This could include minimizing the number of visits by integrating multiple services under one roof, providing trauma-informed interpreters, and offering accessible transportation services.

Organ of Corti size is governed by Yap/Tead-mediated progenitor self-renewal.

Precise control of organ growth and patterning is executed through a balanced regulation of progenitor self-renewal and differentiation. In the auditory sensory epithelium-the organ of Corti-progenitor cells exit the cell cycle in a coordinated wave between E12.5 and E14.5 before the initiation of sensory receptor cell differentiation, making it a unique system for studying the molecular mechanisms controlling the switch between proliferation and differentiation. Here we identify the Yap/Tead complex as a key regulator of the self-renewal gene network in organ of Corti progenitor cells. We show that Tead transcription factors bind directly to the putative regulatory elements of many stemness- and cell cycle-related genes. We also show that the Tead coactivator protein, Yap, is degraded specifically in the Sox2-positive domain of the cochlear duct, resulting in down-regulation of Tead gene targets. Further, conditional loss of the Yap gene in the inner ear results in the formation of significantly smaller auditory and vestibular sensory epithelia, while conditional overexpression of a constitutively active version of Yap, Yap5SA, is sufficient to prevent cell cycle exit and to prolong sensory tissue growth. We also show that viral gene delivery of Yap5SA in the postnatal inner ear sensory epithelia in vivo drives cell cycle reentry after hair cell loss. Taken together, these data highlight the key role of the Yap/Tead transcription factor complex in maintaining inner ear progenitors during development, and suggest new strategies to induce sensory cell regeneration.

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

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

Spontaneous Hair Cell Regeneration Is Prevented by Increased Notch Signaling in Supporting Cells.

During embryonic development, differentiation of cochlear progenitor cells into hair cells (HCs) or supporting cells (SCs) is partially controlled through Notch signaling. Many studies have shown that inhibition of Notch signaling allows SCs to convert into HCs in both normal and drug damaged neonatal mouse cochleae. This mechanism is also implicated during HC regeneration in non-mammalian vertebrates; however, the mechanism of spontaneous HC regeneration in the neonatal mouse cochlea is less understood. While inhibition of Notch signaling can force SCs to convert into HCs and increase the number of regenerated HCs, it is currently unknown whether this pathway is involved in spontaneous HC regeneration observed in vivo. Therefore, we investigated the role of Notch signaling during the spontaneous HC regeneration process using Atoh1-CreERTM::Rosa26loxP-stop-loxP-DTA/+ mice injected with tamoxifen at postnatal day (P) 0 and P1 to ablate HCs and stimulate spontaneous HC regeneration. Expression changes of genes in the Notch pathway were measured using immunostaining and in situ hybridization, with most changes observed in the apical one-third of the cochlea where the majority of HC regeneration occurs. Expression of the Notch target genes Hes1, Hes5, Hey1, HeyL, and Jagged1 were decreased. To investigate whether reduction of Notch signaling is involved in the spontaneous HC regeneration process, we overexpressed the Notch1 intracellular fragment (N1ICD) in cochlear SCs and other non-sensory epithelial cells in the context of HC damage. Specifically, Atoh1-CreERTM::Rosa26loxP-stop-loxP-DTA/+::Sox10rtTA::TetO-LacZ::TetO-N1ICD mice were injected with tamoxifen at P0/P1 to stimulate spontaneous HC regeneration and given doxycycline from P0-P7 to induce expression of N1ICD as well as LacZ for fate-mapping. We observed a 92% reduction in the number of fate-mapped regenerated HCs in mice with N1ICD overexpression compared to controls with HC damage but no manipulation of Notch signaling. Therefore, we conclude that increased Notch signaling prevents spontaneous HC regeneration from occurring in the neonatal mouse cochlea. Understanding which components of the Notch pathway regulates regenerative plasticity in the neonatal mouse cochlea will inform investigations focused on stimulating HC regeneration in mature cochlea and eventually in humans to treat hearing loss.

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

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